[go: up one dir, main page]

WO2025046236A1 - Methods for nanopore-based analyte detection - Google Patents

Methods for nanopore-based analyte detection Download PDF

Info

Publication number
WO2025046236A1
WO2025046236A1 PCT/GB2024/052258 GB2024052258W WO2025046236A1 WO 2025046236 A1 WO2025046236 A1 WO 2025046236A1 GB 2024052258 W GB2024052258 W GB 2024052258W WO 2025046236 A1 WO2025046236 A1 WO 2025046236A1
Authority
WO
WIPO (PCT)
Prior art keywords
analyte
nanopore
change
sec
amino acids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/GB2024/052258
Other languages
French (fr)
Inventor
Andrew Heron
Timothy MASSINGHAM
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Portal Biotech Ltd
Original Assignee
Portal Biotech Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Portal Biotech Ltd filed Critical Portal Biotech Ltd
Publication of WO2025046236A1 publication Critical patent/WO2025046236A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides

Definitions

  • Nanopore sequencing is an approach to sequencing of nucleic acid molecules. Using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling of the sample. Nanopore sequencing can offer low-cost genotyping, high mobility for testing, and rapid processing of samples with the ability to display results in real-time. It has been used in the rapid identification of viral pathogens, epidemiological monitoring, environmental monitoring, food safety monitoring, human genome sequencing, plant genome sequencing, monitoring of antibiotic resistance, haplotyping, and other applications.
  • Nanopores can be detected and characterized by nanopores and nanopore sensors based on capture and modulation of ionic current.
  • Nanopores may identify and characterize many analytes, such as nucleic acid molecules, peptides, polypeptides, or proteins, or fragments thereof, or any combination thereof.
  • analytes such as nucleic acid molecules, peptides, polypeptides, or proteins, or fragments thereof, or any combination thereof.
  • the present disclosure provides a method for identifying a polypeptide characteristic, comprising (a) translocating a polypeptide through a nanopore disposed within a membrane; (b) detecting a current or change thereof while the polypeptide is translocating through the nanopore; and (c) using the current or change thereof detected in (b) to identify a characteristic of the polypeptide with an accuracy of at least 90%.
  • the characteristic comprises a sequence, a length, an identity, a secondary structure, a tertiary structure, a modification to the polypeptide, or combinations thereof.
  • the nanopore is a biological nanopore.
  • the biological nanopore is selected from FraC, a-hemolysin, CytK, Lysenin, MspA, CsgG, Aerolysin, or FhuA.
  • the biological nanopore comprises an outer membrane protein (OMP) such as OmpG or OmpF.
  • OmpG outer membrane protein
  • the biological nanopore comprises one or more point mutations.
  • the one or more point mutations affects a diameter of the biological nanopore.
  • the one or more mutations create smaller openings on a cis side of the biological nanopore.
  • the one or more mutations create smaller openings on a trans side of the biological nanopore.
  • the one or more mutations create smaller openings in the center of the biological nanopore.
  • the one or more mutations constrict multiple point throughout a channel of the biological nanopore.
  • the diameter of the biological nanopore is from about 0.5 nm to about 2 nm.
  • the one or more point mutations affects a charge of the biological nanopore.
  • the net charge of the biological nanopore is positive.
  • the charge of the channel of the biological nanopore comprises positively charged portions.
  • the net charge of the biological nanopore is negative.
  • the charge of the channel of the biological nanopore comprises negatively charged portions.
  • the one or more point mutations allow for conductance at a set pH.
  • the pH is from about 5 to about 10.
  • the one or more point mutations are one or more lumen facing mutations.
  • the nanopore is an artificial nanopore.
  • the polypeptide is unfolded prior to translocation.
  • the polypeptide is unfolded by one or more unfoldases selected from the group consisting of prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, and Valosin-containing protein-like ATPase.
  • the one or more of unfoldases are coupled to the nanopore.
  • the one or more of unfoldases are coupled to the nanopore by covalent or non-covalent forces.
  • the one or more of unfoldases are coupled to the nanopore by Pi bonding, Pi-Pi bonding, H-bonding, electrostatic interactions, or hydrophobic interfaces, or combinations thereof.
  • the one or more unfoldases are suspended in an electrolyte solution on one side of the membrane.
  • the one or more unfoldases are configured to position proximal to the nanopore upon a binding event with the polypeptide.
  • the nanopore comprises a proteasome and/or a translocase.
  • either the proteasome or the translocase are located on a cis-side of the nanopore.
  • either the proteasome or the translocase are located on a trans-side of the nanopore.
  • either the proteasome or the translocase are located on both a cis-side and/or a trans-side of the nanopore.
  • both the proteasome and translocase are located on a cis-side of the nanopore.
  • both the proteasome and the translocase are located on a trans-side of the nanopore.
  • both the proteasome and the translocase are located on both a cis-side and a trans-side of the nanopore.
  • the proteasome comprises one or more subunits. [0030] In some embodiments, the proteasome is fused with the nanopore. [0031] In some embodiments, the polypeptide is translocated from a cis to a trans side of the membrane. In some embodiments, the polypeptide is translocated from a trans to a cis side of the membrane. In some embodiments, a polypeptide bound to a translocase is partially translocated from a cis to trans side of the membrane, then the translocase pulls the partially translocated portion of the polypeptide back through the pore in a trans to cis direction.
  • the one or more of unfoldases are positioned on the cis side of the membrane. [0033] In some embodiments, the one or more of unfoldases are positioned on the trans side of the membrane. [0034] In some embodiments, the polypeptide is fragmented before translocation. [0035] In some embodiments, a polypeptide fragment comprises a portion comprising a non-natural amino acid, polyethylene glycol, PNA, DNA, or RNA, or combinations thereof. [0036] In some embodiments, the polypeptide is fragmented by one or more proteases comprising trypsin-type or chymotrypsin-type of activity.
  • the one or more proteases are coupled to the nanopore. [0038] In some embodiments, the one or more proteases are positioned on a cis side of the membrane [0039] In some embodiments, the polypeptide is translocated through the nanopore sequentially. [0040] In some embodiments, translocation is affected by a molecular motor. [0041] In some embodiments, the step size of translocation is determined by the molecular motor. [0042] In some embodiments, the molecular motor is ATP driven. In some embodiments, the molecular motor is NTP driven. [0043] In some embodiments, the step size of translocation is from about 0.2 to about 80 Angstroms.
  • the step size of translocation is from about 0.5 to about 20 Angstroms.
  • translocation occurs in the absence of a molecular motor. In some embodiments, translocation occurs with a molecular motor that is not being actively driven by ATP or NTP.
  • the rate of translocation is from about 0.1 nm/s to about 300 nm/s.
  • WSGR Docket Number: 64828-710.601 [0046]
  • the membrane is an insulating membrane.
  • the insulating membrane is a phospholipid bilayer.
  • the insulating membrane is a solid-state membrane.
  • the membrane has a thickness from about 4 nm to about 20 nm.
  • the polypeptide is unlabeled.
  • the polypeptide comprises a tag.
  • the polypeptide comprises an exogenous sequence.
  • the exogenous sequence is about 5 to about 50 amino acids in length.
  • the polypeptide is suspended in an electrolytic solution.
  • the electrolytic solution comprises water, potassium, lithium sodium, calcium, magnesium, phosphate, sulfate, or chloride, or any combination thereof.
  • the concentration of one or more electrolytes in the electrolytic solution is from about 0.1 M to about 1.5 M.
  • the pH of the electrolytic solution is from about 5 to about 10.
  • the pH of the electrolytic solution is different between a trans-side and a cis- side of a membrane.
  • a terminus of the polypeptide is chemically modified with a leader or a tail.
  • a machine learning algorithm is used to identify the characteristic of the polypeptide using the current signal.
  • the characteristic comprises a sequence.
  • identity of the characteristics is determined with an accuracy from about 90% to about 99.99%.
  • the multi pass accuracy is from about 90% to about 99.99%.
  • the single pass accuracy is from about 90% to about 99.99%.
  • the polypeptide comprises post-translational modifications.
  • the detected change in ionic current is from about 0.1 pA to about 150 pA.
  • the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane, wherein the step size of translocation is about 0.2 to about 80 Angstroms; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to characterize a property of the polypeptide or to identify a sequence of the polypeptide.
  • the step size is dependent on the polypeptide structure.
  • the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane, wherein the rate of translocation is about 0.1 nm/s to about 300 nm/s; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) characterize a property of the polypeptide or to identify a sequence of the polypeptide.
  • the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to identify a sequence of the polypeptide with a sensing throughput of at least 1 molecule / minute.
  • the present disclosure provides a method for proteome analysis, comprising: providing a cell comprising a plurality of polypeptides; translocating a polypeptide of the plurality of polypeptides through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide of the plurality of polypeptides is translocating through the nanopore; using the current or change thereof detected in (b) to identify a sequence of the polypeptide of the plurality of polypeptides; and characterizing one or more properties of a proteome using the sequence of the polypeptide identified in (d), wherein proteome coverage is at least 50%.
  • the present disclosure provides a method for proteome analysis, comprising: providing a cell comprising a plurality of polypeptides; translocating a polypeptide of the plurality of polypeptides through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide of the plurality of polypeptides is translocating through the nanopore; using the current or change thereof detected in (b) to identify a sequence of the polypeptide of the plurality of polypeptides; and characterizing one or more properties of a proteome using the sequence of the polypeptide identified in (d), wherein sequence coverage is at least 10%.
  • the present disclosure provides a device for identifying a polypeptide sequence, comprising: a first chamber configured to prepare a biological sample for polypeptide sequencing; a second chamber in fluidic communication with the first chamber, the second chamber comprising a support configured to connect and disconnect with a sensor array; a sensor array connected to the support, wherein the sensor array WSGR Docket Number: 64828-710.601 comprises: a plurality of membranes comprising a plurality of pores, the plurality of pores configured to translocate the plurality of polypeptides; and a plurality of electrodes configured to measure a plurality of electrical signals from the plurality of polypeptides translocating through the plurality of pores; and a recording device configured to receive and record a plurality of electrical signals from the plurality of electrodes.
  • the present disclosure provides a device for identifying a polypeptide sequence, comprising: a first chamber configured to prepare a biological sample for polypeptide sequencing by binding a plurality of polypeptides in the biological sample with a plurality of biomolecules; a second chamber in fluidic communication with the first chamber, the second chamber comprising a support configured to connect and disconnect with a sensor array; a sensor array connected to the support, wherein the sensor array comprises: a plurality of membranes comprising a plurality of pores, the plurality of pores configured to translocate the plurality of polypeptides, wherein the plurality of biomolecules are configured to facilitate translocating the plurality of polypeptides; and a plurality of electrodes configured to measure a plurality of electrical signals from the plurality of polypeptides translocating through the plurality of pores; and a recording device configured to receive and record a plurality of electrical signals from the plurality of electrodes.
  • the sensor array comprises a plurality of chambers, wherein the plurality of membranes form a plurality of surfaces of the plurality of chambers.
  • the plurality of chambers comprise a volume from about 0.1 ⁇ L to about 250 ⁇ L.
  • a chamber of the plurality of chambers comprises a volume from about 0.0001 ⁇ L to about 1.0 ⁇ L.
  • the plurality of chambers comprise a thickness of at most about 3 mm.
  • the plurality of surfaces each comprise an area of at most about 5 mm 2 .
  • a surface of the plurality of surfaces comprises an area of at most 100 ⁇ m2.
  • the device is configured to connect to a recording device.
  • the recording device comprises an analog-to-digital converter.
  • the recording device comprises an amplifier.
  • the plurality of electrodes are disposed on a second plurality of surfaces of the plurality of chambers.
  • the sensor array comprises an adhesive configured to adhere the plurality of membranes to the plurality of chambers.
  • the device comprises a display for displaying the electrical signal.
  • the device comprises a flow cell.
  • WSGR Docket Number: 64828-710.601 the present disclosure provides a microfluidic device for polypeptide characterization, comprising: one or more microfluidic channels for flowing fluid comprising a polypeptide therethrough; and one or more nanopores disposed within a membrane in fluid communication with the one or more microfluidic channels, the one or more nanopores configured to effect a change in a current applied across the membrane upon translocation of the polypeptide therethrough, wherein the changed effected in the current corresponds to a characteristic of the polypeptide.
  • the membrane comprises from about 10 to about 100,000 pores [0090] In some embodiments, the surface area of the membrane is at most about 5 mm2. [0091] In some embodiments, the surface area of the nanopores within the membrane is from about 50 to about 500 nm2. [0092] In some embodiments, the membrane is disposed within a fluidic chamber comprising an anode and a cathode. [0093] In some embodiments, each of the anode and cathode is independently positioned on either a cis side or a trans side of the membrane. [0094] In some embodiments, the device further comprises a potential generator for applying a potential difference across the anode and cathode.
  • the potential across the anode and cathode generated by the potential generator is from about 10 mV to about 1 V.
  • the microfluidic device further comprises a pump for flowing the fluid through the one or more microfluidic channels.
  • the present disclosure provides a kit for use with a device for identifying a polypeptide sequence, comprising: a chip comprising a sensor array, the sensor array comprising a plurality of chambers or wells comprising a plurality of lipids and pores; and a biomolecule configured to bind to a polypeptide to facilitate translocation of the polypeptide through a pore.
  • the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal and a database, the database comprising a plurality of reference signals for the polypeptide, proteoforms thereof, and post-translationally modified variants thereof.
  • the present disclosure provides a method for characterizing and/or identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and WSGR Docket Number: 64828-710.601 assigning an identification to the polypeptide based on the electrical signal and a machine learning algorithm, the machine learning algorithm configured to distinguish between a plurality of reference signals for the polypeptide, proteoforms thereof, and post-translationally modified variants thereof.
  • the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide from a biological sample through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal and a database, the database comprising a plurality of reference signals for a plurality of polypeptides, wherein the plurality of polypeptides comprise expressible polypeptides, or proteoforms thereof, or post-translationally modified variants thereof, or any combination thereof, based on genomic information of the biological sample.
  • the present disclosure provides a method for identifying a polypeptide sequence, comprising: contacting a composition comprising a biological sample with a nanopore, the biological sample comprising an initial volume of at most about 50 ⁇ L, wherein the composition comprises a polypeptide and a nucleic acid from the biological sample; translocating the polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal.
  • the present disclosure provides a method for generating a sample profile, comprising: receiving an electrical signal, wherein the electrical signal is derived at least from one analyte of a sample translocating across a nanopore; and generating the sample profile based on the electrical signal and a database, wherein the database comprises a plurality of reference electrical signals for a plurality of analytes; wherein the reference electrical signals for the plurality of analytes comprise expressible polypeptides, or proteoforms thereof, or post-translationally modified variants thereof, or combinations thereof.
  • the reference electrical signals further comprise small molecules, metabolites, DNA, or RNA, or combinations thereof.
  • the analyte is derived from a patient.
  • the sample profile comprises an assessment of patient health.
  • the analyte comprises a metabolite, a small molecule, a biopolymer, or a biomolecule.
  • the sample profile comprises an identification of a phenotype.
  • the sample profile comprises an identification of a metabolic state.
  • the sample profile comprises an identification of a disease.
  • the electrical signal is derived from a plurality of analytes of a sample translocating across a nanopore.
  • generating a sample profile comprises counting event instances of a subset of the plurality of analytes.
  • the sample profile comprises an environmental profile.
  • assigning an identification comprises determining a degree of similarity with a previously identified phenotypic profile.
  • the degree of similarity is measured by the proteomic coverage.
  • the present disclosure provides a method for generating a sample profile, comprising: receiving an electrical signal, wherein the electrical signal is derived from at least one analyte translocating across a nanopore; and generating a sample profile based on the electrical signal and a machine learning algorithm configured to distinguish between a plurality of reference signals for the analyte.
  • Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.
  • Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto.
  • the computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.
  • the present disclosure provides a method, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%.
  • the present disclosure provides a method for determining a characteristic of an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, or (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is WSGR Docket Number: 64828-710.601 translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage
  • the present disclosure provides a method for characterizing an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof.
  • the present disclosure provides a method for sample analysis, comprising: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or first fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or second fragments
  • FIG.1B illustrates a strong cis-to-trans EOF established in a system with positive voltage applied to the trans compartment across the membrane.
  • FIG.1C illustrates a strong cis-to-trans EOF established in a system with negative voltage applied to the trans compartment across the membrane.
  • FIG. 2 shows example nanopore-based systems for characterizing and/or translocating polymer analytes.
  • FIGs.3A-3D show CytK nanopores.
  • FIG.3A illustrates a cross-section of a surface representation of WT-CytK nanopores in 1 M KCl, pH 7.5. The nanopore was made by homology modelling from the alpha- hemolysin nanopore.
  • FIG. 3B shows a cartoon representation of WT-CytK ⁇ -barrel region.
  • FIG.3C shows a schematic of the residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK.
  • FIG. 3D shows a cross-section of a surface (left) and cartoon (right) representation of a high ion selectivity mutant, CytK-2E-4D.
  • FIG. 4. shows an amino acid sequence and corresponding schematic representation of the three designed unstructured model polypeptide analytes, in accordance with some embodiments (which can be referred to as S1, tzatziki and mujdei). Solid circles indicate negatively charged amino acids, and open circles indicate positively charged amino acids.
  • FIGs. 5A-5E illustrate translocation of analyte S1 through wildtype (WT)-CytK nanopores, in accordance with some embodiments.
  • FIG. 5A shows a schematic representation of the translocation of S1 through WT-CytK. The arrow denotes the analyte S1 translocating through the nanopore by an electrophoretic force (EF).
  • FIG.5B shows a voltage dependency of translocation rates for type 1 and type 2 blockades.
  • FIG. 5C shows a voltage dependency of the excluded current (I ex (%)) for type 1 and type 2 blockades.
  • type 1 blockades are shown as black squares and type 2 blockades are shown as light gray circles.
  • FIG. 5D shows representative traces at –160 mV bias, in accordance with some embodiments.
  • IO denotes the open pore current measurement and IB denotes the blocked pore current measurement.
  • FIG.5E shows dwell time versus current amplitude at –160 mV bias.
  • FIGs. 6A-6E illustrate translocation of S1 through 2E-4D-CytK nanopores, in accordance with some embodiments.
  • FIG. 6A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments.
  • FIGs. 7A-7E illustrate translocation of tzatziki through 2E-4D-CytK nanopores, in accordance with some embodiments.
  • FIG.7A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments.
  • the arrows denote the analyte tzatziki translocating through the WSGR Docket Number: 64828-710.601 nanopore by an electrophoretic force (EF; solid arrow) and electro-osmotic force (EOF; dotted arrow).
  • EF electrophoretic force
  • EEF electro-osmotic force
  • FIG.7B shows a voltage dependency of translocation rate, in accordance with some embodiments.
  • FIG.7C shows a voltage dependency of the excluded current (Iex%), in accordance with some embodiments.
  • FIG. 7D shows representative traces at –160 mV bias, in accordance with some embodiments.
  • FIG. 7E shows dwell time versus current amplitude at –160 mV bias, in accordance with some embodiments. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. [0133]
  • FIGs. 8A-8E illustrate translocation of mujdei through 2E-4D-CytK nanopores, in accordance with some embodiments.
  • FIGs.9A-9H illustrate translocation of model substrates through nanopores, in accordance with some embodiments. For each of FIGs.
  • FIG.9A shows representative current traces of substrates through WT-CytK, in accordance with some embodiments.
  • FIG. 9B shows representative current traces of substrates through K128D-CytK, in accordance with some embodiments.
  • FIG.9C shows representative current traces of substrates through K128-K155Q-CytK, in accordance with some embodiments.
  • FIG.9D shows representative current traces of K128D-K155D-CytK, in accordance with some embodiments.
  • FIG. 9E shows representative current traces of K128D-K155Q-Q122D-CytK, in accordance with some embodiments.
  • FIG. 9F shows representative current traces of K128D-K155D-Q145D-CytK, in accordance with some embodiments.
  • FIG.9G shows representative current traces of substrates through K128D-K155D- T147D-CytK, in accordance with some embodiments.
  • FIG.9H shows representative current traces of substrates through K128D-K155D-Q145D-S151D-CytK, in accordance with some embodiments.
  • FIGs. 10A-10G show translocation of model substrates through nanopores in accordance with some embodiments.
  • FIG. 10A shows a cut-through of a surface representation of the WT-CytK nanopore (left) and WSGR Docket Number: 64828-710.601 a cartoon representation of its ⁇ -barrel region (right).
  • FIG. 10B shows a cut-through of a surface representation of the K128D-K155Q-CytK nanopore (left) and a cartoon representation of its ⁇ -barrel region (right).
  • Plots depict voltage dependency of translocation rates (right) and voltage dependency of excluded current (I ex (%)) (left) for the translocation of substrates through K128D-K155Q-CytK.
  • FIG.10C shows a cut- through of a surface representation of the K128D-K155D-CytK nanopore (left) and a cartoon representation of its ⁇ -barrel region (right).
  • Plots depict voltage dependency of translocation rates for the two types of events, type 1 blockade (dark gray square) and type 2 blockade (light gray circle), and voltage dependency of excluded current (I ex (%)) of substrates through K128D-K155D-CytK.
  • FIG. 10D shows a cut-through of a surface representation of the K128D-K155Q-Q122D-CytK nanopore (left) and a cartoon representation of its ⁇ -barrel region (right).
  • FIG. 10E shows a cut- through of a surface representation of the K128D-K155D-Q145D-CytK nanopore (left) and a cartoon representation of its ⁇ -barrel region (right).
  • Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex(%)) (left) of analyte S1 and analyte tzatziki through K128D- K155D-Q145D-CytK.
  • FIG.10F shows a cut-through of a surface representation of the K128D-K155D-T147D- CytK nanopore (left) and a cartoon representation of its ⁇ -barrel region (right).
  • Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex%)(left) of analyte S1 and analyte tzatziki through K128D-K155D-T147D-CytK.
  • FIG. 10G shows a cut-through of a surface representation of the K128D-K155D-Q145D-S151D-CytK nanopore (left) and a cartoon representation of its ⁇ -barrel region (right).
  • FIGs. 11A-11F illustrate translocation of unfolded MalE219a across 2E-4D-CytK nanopores.
  • FIG. 11A shows representative traces of the translocation of MalE219a across 2E-4D-CytK in 2M urea.
  • FIG. 11B shows dwell time versus amplitude of current blockades under –100 mV.
  • FIG. 11C shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its ⁇ -barrel region (right).
  • FIG. 11D shows a cartoon representation of MalE219a.
  • FIG.11E shows a voltage dependency of the translocation speed.
  • FIG. 11F shows a voltage dependency of the excluded current. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. WSGR Docket Number: 64828-710.601 [0137]
  • FIGs.12A-12F show translocation of unfolded H152A-GBP across 2E-4D-CytK nanopores.
  • FIG.12A shows representative traces of the translocation of H152A-GBP across 2E-4D-CytK in 2.4 M urea showing the two levels, Level 1 (L1) and level 2 (L2), of the translocation blockades.
  • FIG. 12B shows dwell time versus amplitude of current blockades under –100 mV.
  • FIG.12C shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its ⁇ -barrel region (right).
  • FIG. 12D shows a cartoon representation of MalE219a.
  • FIG.12E shows a voltage dependency of the translocation speed.
  • FIG.12F shows a voltage dependency of the excluded current for L1 and L2 levels as indicated in panel A.
  • FIGs.13A-13E illustrate malE219a translocation through the 2E-2D CytK mutant in the presence of 1 M and 1.8 M GuHCl.
  • FIG. 13A shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its ⁇ -barrel region (right), in accordance with some embodiments.
  • FIG.13B shows a voltage dependency of the dwell time of malE219a in 1 M (squares) and 1.8 M (circles) GuHCl.
  • FIG. 13C shows a voltage dependency of the excluded current of malE219a in 1 M (squares) and 1.8 M (circles) GuHCl.
  • FIG. 13D shows a dwell time versus amplitude dependence at various voltages in 1M GuHCl as indicated.
  • FIG.13E shows a dwell time versus amplitude dependence at various voltages in 1M GuHCl as indicated.
  • FIGs.14A-14D show characterizations of the 2E-4D-CytK nanopore in the two denaturants. IV curves for 2E-4D-CytK nanopores in the urea is shown in FIG. 14A and GuHCl in FIG. 14B. Numerical values of the asymmetry using the ratio of the ionic current at -100 mV and +100 mV in different concentrations of urea (FIG.14C) and GuHCl (FIG.14D).
  • FIGs.15A-15C show MalE219a transport across WT-CytK.
  • FIG. 15A shows translocation events of 100 nM of malE219a-D10 unfolded by 2 M urea, in accordance with some embodiments.
  • FIG.15B shows 100 nM of malE219a added in the cis chamber did not induce events.
  • FIG.15C shows a sequence of malE219a- D10.
  • FIGs.16A-16C show actinoporins common sequence alignment and wild-type Fragaceatoxin C.
  • FIG. 16A shows common sequence alignment of some actinoporins, the dots represent the same amino acid as the common sequence, other amino acid differences between the pores are represented by their single-letter code.
  • FIG.18B shows representative trace of octameric Fragaceatoxin C (T1).
  • FIG. 18C shows representative trace of heptameric Fragaceatoxin C (T2).
  • FIG.18D shows representative trace of Fragaceatoxin C mutant (G13F).
  • FIGs. 19A-19E show event count and signal correlation of (mutant)’ Fragaceatoxin C with trypsin digested lysozyme.
  • FIG.19A-19D show observed excluded current (Iex%) spectra from tryptic digest of lysozyme.
  • FIG.19A shows octameric wild-type Fragaceatoxin C (T1).
  • FIG.19B shows heptameric wild-type Fragaceatoxin C (T2).
  • FIG. 19C shows Fragaceatoxin C mutant G13F.
  • FIG. 19D shows Fragaceatoxin C mutant G13N.
  • FIG.19E shows squared first derivative Euclidean cosine correlation of residual current spectra of (mutant) Fragaceatoxin C combined with equal units of trypsin digested lysozyme. Boxes 1-3 surrounding multiple mutants represent similar signals.
  • FIGs.20A-20C show peptide recognition of (mutant) Fragaceatoxin C.
  • FIG.20A shows the location of mutations in the lumen of Fragaceatoxin C (modeled on PDB: 4TSY) marked by arrows.
  • FIG. 20B shows Gaussian fits to histograms of the excluded currents from the clustered event blockade for the capture and detection of Angiotensin IV, Angiotensin III, Angiotensin I and Angiotensinogen recorded under an applied potential of -50 mV.
  • FIG.20C shows excluded current % (IEX%) versus dwell time scatter plots of the single- molecule peptide event blockades detected by the different pore types.
  • FIG.21 shows peptide recognition of (mutant) Fragaceatoxin C.
  • FIG. 22 shows an electrophysiology setup of an analytical system comprising a nanopore, in accordance with some embodiments.
  • FIGs. 23A-23D show bottom-up nanopore-based proteomics.
  • FIG. 23A shows an artistic representation of protease protein digestion to digest a protein into a mixture of peptide fragments.
  • FIG. 23B shows an artistic representation of the experimental setup of a nanopore system.
  • FIG. 23C shows an artistic representation of the resulting ionic current data for detected peptides from a nanopore-based electrophysiology experiment.
  • FIG.23D shows an artistic representation of a resulting residual current versus standard deviation spectrum obtained from analysis of the individual single-molecule event blockades.
  • FIGs. 24A-24B show excluded current - mass calibration using peptides and the spectrum obtained from tryptic lysozyme peptides.
  • FIG. 24A shows the mass of the synthetic model peptides (circles) plotted against the average measured excluded current (%) for each peptide when added to the G13F-FraC-T1 nanopore WSGR Docket Number: 64828-710.601 system.
  • FIG.24B shows excluded current spectrum (histogram of the excluded currents from event blockades) recorded from addition of a mixture of all the model peptides to a G13F-FraC-T1 pore.
  • FIGs. 25A-25B show nanopore experiments compared to electrospray ionization mass spectrometry.
  • FIG. 25A shows residual current spectrum as obtained by nanopore electrophysiology using G13F-FraC-T1 and a tryptic digest of Gallus-gallus lysozyme.
  • FIG.25B shows mass spectrometry results from the same tryptic digest, but measured with a mass spectrometer (ESI-MS).
  • FIGs.26A-26C show the reproducibility of nanopore protein spectra, using three independent repeats of the sensing of proteolytic digestions.
  • FIG.26A shows results from bovine serum albumin (BSA).
  • FIG.26B shows results from dihydrofolate reductase (DHFR).
  • FIG. 26C shows results from elongation factor P (EFP).
  • EFP elongation factor P
  • the left-side panel shows the excluded current histograms with a normalized area of 100%, which were obtained from the excluded current versus dwell time scatters of all event blockades shown in the respective right-side panels.
  • FIG. 27A-27B shows spectral matching using squared first difference correlation coefficient.
  • FIG. 27A shows example representative baseline corrected residual current spectra of the measurement of peptide fragment mixtures from 9 tryptic digested proteins. Unique spectra were observed for each protein type.
  • the right-side panel shows the excluded current histograms with a normalized area of 100%, which were obtained from the excluded current versus dwell time scatters of all event blockades shown in the respective left-side panel.
  • FIG. 27B shows leave-one-out spectral matching of the baseline corrected residual current spectra using Euclidean cosine cross-correlation. [0153]
  • FIG. 27A shows example representative baseline corrected residual current spectra of the measurement of peptide fragment mixtures from 9 tryptic digested proteins. Unique spectra were observed for each protein type.
  • the right-side panel shows the excluded current histograms with a normalized area of 100%, which were obtained from the excluded current versus dwell time scatters of all event blockades shown in
  • FIG. 28 shows the detection of proteins kemptide (LRRASLG) and phosphorylated kemptide (LRRA ⁇ pS ⁇ LG).
  • FIG.29 shows the detection of glycopeptides.
  • the peptides were unmodified peptide (ANVTLNTAG), peptide with one glycan (ANVT(Glc)LNTAG and peptide with two glycans (ANVT(Glc)LNTT(Glc)G).
  • FIGs.30A-30B show the detection of rhamnosylated proteins.
  • FIG.30A shows unmodified Elongation Factor P (EF-P), with residual current (IRes) plotted against dwell time.
  • FIG. 30B shows and rhamnosylated EF-P, with residual current (IRes) plotted against dwell time.
  • FIGs.31A-31C show discrimination between single amino changes.
  • FIG.31A shows detection of two forms of enkephalin with sequences added to the cis-chamber of G13F-FraC-T1 pores, in accordance with some embodiments: YGGFL, and YdAGFdL, wherein d represents a D-amino acid; all other amino acids are L- amino acid.
  • FIGs.32A-32D show detection of trypsinated lysozyme in Aerolysin nanopores, including WT-Aer at pH 7.5 (FIG. 32A), WT-Aer at pH 3.8 (FIG.32B), Aer-K238F at pH 3.8 (FIG.32C) and Aer-K238D-S264F at pH 3.0 (FIG. 32D).
  • FIGs. 33A-33I show detection of trypsinated lysozyme in Aerolysin nanopores.
  • FIG.33I shows measurement of 4 ⁇ g trypsinated lysozyme added to the cis compartment (final concentration l0ng/ ⁇ l) of nanopore system comprising Aer-K238W.
  • FIGs.34A-34C show detection of trypsinated lysozyme in Cytolysin K (CytK) nanopores.
  • the current traces show representative sections of ionic current data for selected pores, comprising either WT-CytK at pH 3.8 (FIG.34A), CytK-K128F at pH 3.8 (FIG.34B), or CytK-S126F-K128D at pH 3.8 (FIG.34C).
  • the open- pore current (I o ) and exemplary step-like current blockades (IB) from peptide captures are marked in each plot.
  • FIGs.35A-35H show detection of trypsinated lysozyme in Cytolysin K (CytK) nanopores.
  • FIG. 35A shows a homology model of CytK (left) mapped onto the structure of the alpha-hemolysin nanopore from Staphylococcus aureus, and predicted beta-strand showing inward water-facing amino acids for the beta-barrel lumen of the nanopore (right).
  • FIGS.35B-35G shows residual current versus dwell time scatter of individual peptide blockades provoked by 4 ⁇ g of trypsinated lysozyme added to the trans-chamber of a system comprising either (FIG.35B) wild type (WT-CytK) at pH 3.8, (FIG.35C) K128F CytK nanopore at pH 3.8, (FIG.35D) S126F- K128D CytK nanopore at pH 3.8, (FIG. 35E) S120F - K128D CytK nanopore at pH 3.0 (FIG.
  • FIG.35H shows measurement of two peptides (10 ⁇ M Lys4 and 10 ⁇ M Lys7) added to the trans compartment a system comprising K128W CytK nanopore.
  • FIGs.36A-36B show detection of Lys-C digested lysozyme in Lysenin nanopores.
  • FIG.36A shows a nanopore system comprising wildtype lysenin (Lys-WT).
  • FIG. 36B shows a nanopore system comprising mutant lysenin (Lys-E76F).
  • FIGs.36A shows a nanopore system comprising wildtype lysenin (Lys-WT).
  • FIG. 36B shows a nanopore system comprising mutant lysenin (Lys-E76F).
  • FIG. 37A-37F show detection of non-proteinaceous small molecules.
  • the system comprised heptameric wild-type FraC (FIG. 37A), mutant FraC_G13F nanopores with Thioflavin (FIG. 37B and FIG. 37C), octameric wild-type FraC (FIG. 37D), or mutant FraC_G13F nanopores with Vitamin B12 (FIG. 37E and FIG.37F).
  • FIG.38 shows the design of a transmembrane protein device for single-molecule protein analysis.
  • FIGs. 39A-39D show the fabrication and electrical optimization of a nanopore.
  • FIG.39A shows the effects of linker length on the nanopore expression in E. coli cells, insertion efficiency and nanopore stability.
  • FIG. 39B shows the electrical properties of ⁇ 4 mutant.
  • the left schematic of FIG. 39B shows the linker sequence of ⁇ 4 mutant.
  • the middle current representation of FIG.39B shows electrical recordings of a single nanopore at ⁇ 35 mV.
  • the right plot of FIG. 39B shows a histogram of the unitary conductance values of 59 nanopores at-35 mV.
  • FIG.39C shows the electrical properties of ⁇ 2 mutant.
  • the left schematic of FIG.39C shows the linker sequence of ⁇ 2 mutant.
  • FIG.39C shows a current trace and the current histogram corresponding the insertion of individual pore into a lipid membrane at +35 mV.
  • the right plot of FIG. 39C shows a histogram of the unitary conductance values of 59 artificial nanopores at-35 mV.
  • FIG. 39D shows interaction of DPhPC with the artificial transmembrane pore generated by molecular dynamics simulations.
  • FIGs.40A-40H show the electrical properties of optimized artificial pore ( ⁇ 2) and discrimination of substrates.
  • FIG. 40A shows the schematic of an ion-current measurement setup.
  • FIG. 40B shows a current trace recorded through an efficient single pore after optimization at ⁇ 35 mV.
  • FIG. 40A shows the schematic of optimized artificial pore ( ⁇ 2)
  • FIG. 40C shows averaged current—voltage (I— V) characteristics of three different nanopores.
  • the error bars represent a standard deviation from the mean curve of the ion selectivity of the nanopore (FIG.40D). Determination of the reversal potential shows that the pore is cation-selective, as expected from the electrostatic potentials at their constrictions.
  • FIG.40E shows the chemical structure of beta-cyclodextrin ( ⁇ -CD), scatter plots of Ires% versus dwell time, and representative trace.
  • FIG. 40F shows the chemical structure of gamma-cyclodextrin ( ⁇ -CD), scatter plots of Ires % versus dwell time, and representative trace.
  • FIGs.41A-41E show the design of the artificial proteasome-nanopore.
  • FIG.41A shows the structure of T. acidophilum proteasome-PA26. The C-terminal of PA26 (S231) is near L21 of the ⁇ subunit.
  • FIG. 41B shows the reconstitution of artificial proteasome-nanopore. To obtain subcomplex 3, two separate vectors were used to express the four proteins.
  • FIG.41C shows SDS-PAGE (left) and native PAGE (right) analyses of the purified complex 3.
  • SDS-PAGE revealed the presence of three unique bands of PAuA20 (top), ⁇ 12 (middle), and ⁇ (bottom) with molecular weights of 52.7, 25.8, and 22.3 kDa, respectively.
  • FIG.41D shows behavior of a single pore at ⁇ 35 mV in 1 M NaCl, 15 mM Tris, pH 7.5.
  • FIG. 41E depicts a cut-through of a surface representation of artificial transmembrane proteasome.
  • FIGs.42A-42C show SDS-PAGE analysis the hydrolyzing activity of subcomplex 3.
  • FIG.42A shows ⁇ -casein (1 mg/mL) that was incubated with subcomplex 3 at 53°C in buffer A (50 mM Tris, pH 7.5, 150 mM NaCl).
  • FIG. 42B shows ⁇ -casein (1 mg/mL) that was incubated with subcomplex 3 for 2 hours in buffer A.
  • FIG.42C shows ⁇ -casein (1 mg/mL) that was incubated with subcomplex 3 at 53°C for 0.5 hour in buffer B (50 mM Tris, pH 7.5, 0.3-1.0 M NaCl).
  • the ⁇ -casein/subcomplex 3 concentration ratio was 42.
  • FIGs.43A-43F show the discrimination of substrates with the proteasomal nanopore.
  • FIG.43A shows typical current trace provoked by the analyte substrate 1 (S1) using an inactive proteasome-nanopore.
  • FIG.43B shows translocation of S1 (20 ⁇ M) through an inactive proteasome-nanopore mediated by VAT (20.0 ⁇ M) and ATP (2.0 mM).
  • FIG.43C shows when an inactive proteasome is used in the presence of ATP and VAT, GFP- ssrA is unfolded and translocated intact through the proteasome chamber and nanopore.
  • FIG.43D shows typical current traces provoked by S1 using an active proteasome-nanopore.
  • FIG.43A shows typical current trace provoked by the analyte substrate 1 (S1) using an inactive proteasome-nanopore.
  • FIG.43B shows translocation of S1 (20 ⁇ M) through an inactive proteasome-nano
  • FIG. 43E shows when an active proteasome is used, in the presence of VAT and ATP, only rare and fast events may be observed suggesting that the active proteasome-nanopore can cleave S1 efficiently, producing small fragments.
  • FIG. 43F when an active proteasome is used in the presence of ATP and VAT, unfolded GFP-ssrA is cleaved in the proteasomal chamber and the degraded peptides may be too short to be detected by the nanopore.
  • FIGs. 44A-44B show discrimination of substrates with proteasomal nanopore.
  • FIG. 44A shows sequence comparison of substrate 1 (S1) and substrate 2 (S2).
  • FIG.44B shows scatter plots of fraction blockade versus time and representative blockades induced by cleaved S1 and S2.
  • FIGs.45A-45D show the design and membrane insertion of PA26 artificial nanopore.
  • FIG.45A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C).
  • FIG.45B shows a structure of PA26 (PDB ID: 1YA7).
  • FIG.45C shows the structure of artificial PA26-nanopore.
  • FIG.45D shows a current trace shows insertion of individual pore.
  • FIGs. 46A-46D show the design and insertion of ATPase artificial nanopores.
  • FIG. 46A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C). The transmembrane region is highlighted in blue.
  • FIG. 46B shows the structure of Aquifex aeolicus ATPase (PDB ID: 3M0E).
  • FIG. 46C shows the structure of artificial ATPase transmembrane pore.
  • FIG. 46D shows a current trace shows insertion and ATP hydrolysis of individual pore.
  • FIG.47 shows the design of a ClpP-artificial nanopore for single-molecule protein analysis. [0173] FIG.
  • FIG. 48 shows the current-voltage (I-V) characteristics of three different nanopores: opened ClpP- nanopore, closed ClpP-nanopore, and PA-nanopore.
  • FIG.49 shows the controlled translocation through the ClpP-nanopore.
  • FIGs. 50A-50G show the preparation and characterization of type I, type II, and type III FraC nanopores.
  • FIG. 50A shows a cut through of a surface representation of WT-FraC oligomer (PDB: 4TSY) colored according to the vacuum electrostatic potential as calculated by PyMOLPyMOL.
  • FIG.50B shows the percentage of the distribution of type I, type II, and type III for WT-FraC, W112S-FraC, W116S-FraC and W112S-W116S-FraC at pH 7.5 and 4.5.
  • FIG.50C shows IV curves of type II nanopores formed by WT-FraC, WSGR Docket Number: 64828-710.601 W116S-FraC and W112S-W116S-FraC at pH 7.5 (15 mM Tris-HCl, 1 M KCl).
  • FIG. 50D shows the single nanopore conductance of W116S-FraC in 1 M KC1 (0.1 M citric acid and 180 mM Tris base) at pH 4.5.
  • FIG. 50E shows typical current traces for the three nanopore types of W116S-FraC in 1 M KCl at pH 4.5 under -50 mV applied potential.
  • FIG.50F shows reversal potentials measured under asymmetric condition of KCl (1960 mM cis, 467 mM trans) at pH 4.5 for the three W116S-FraC nanopore types.
  • FIG.50G shows molecular models of three types of FraC nanopores constructed from the FraC crystals structure using the symmetrical docking function of Rosetta. [0176] FIGs.
  • FIG. 51A-51F show single channel conductance distributions of FraC nanopores at pH 7.5 and 4.5.
  • FIG. 51A shows a table reporting the average conductance values obtained by fitting Gaussian functions to conductance histograms, in accordance with some embodiments. S.D. represents the standard deviation of all single channels (number given as n).
  • FIG. 51B-51F each panel represents a different batch of FraC nanopores as indicated, in accordance with some embodiments.
  • FIGs. 52A-52B show discrimination of angiotensin peptides in mixture with type II W116S-FraC nanopores.
  • FIG.52A shows (i) sequences of angiotensin I (DRVYIHPFHL), II (DRVYIHPF), III (RVYIHPF) and IV (VYIHPF) with corresponding Ires% measured at -30 mV; (ii) blockades provoked by the four angiotensin peptides; (iii) density plot of the Ires% versus the standard deviation of the current amplitude for angiotensin I added to the cis compartment; and (iv) density plot after further addition of angiotensin II, angiotensin III, and angiotensin IV to the cis chamber.
  • FIG. 52A shows (i) sequences of angiotensin I (DRVYIHPFHL), II (DRVYIHPF), III (RVYIHPF) and IV (VYIHPF) with corresponding Ires% measured at -30 mV; (ii) blockades provoked by the four angiotens
  • FIGs. 53A-53C show an evaluation of biological peptides having different chemical compositions.
  • FIGs. 54A-54D show a nanopore peptide mass spectrometer at pH 3.8.
  • FIG. 54A shows amino acid sequences of four different peptides and their overall charge at different pH. The chargeable amino acids are underlined.
  • FIG.54B shows pH dependence of the Ires% for the four peptides (cis) shown in FIG.54A using type II W116S-FraC nanopores under -30 mV applied potential.
  • FIG. 54C shows comparison of the Ires% versus the mass of peptides at pH 4.5 and 3.8.
  • FIG.54D shows voltage dependence of c-Myc dwell times at different pHs.
  • FIGs.55A-55C show discrimination of short peptide mixture with type III FraC nanopores comprising mutant W112S-W116S-FraC.
  • FIG.55A-55C show discrimination of short peptide mixture with type III FraC nanopores comprising mutant W112S-W116S-FraC.
  • FIG.55A shows sequence, Ires% (-50 mV) and molecular weight (M.W) of angiotensin IV (VYIHPF), angiotensin 4-8 (YIHPF), endomorphin I (YPWF), and leucine enkephalin (Leu- enkephalin; YGGFL).
  • FIG.55B shows blockades provoked by the different peptides.
  • FIG.55C shows density plot showing the Ires% versus the standard deviation of the current blockade for the mixture of angiotensin IV, angiotensin 4-8, endomorphin I and leucine-enkephalin. [0181] FIGs.
  • FIGs. 56A-56B show characterization of type II FraC nanopores comprising an oxidized cysteine at position 10. Difference between the DOC/ W116S type II pore (FIG.56A) and the oxidized DIOC / W116S type II pore (FIG.56B).
  • FIGs. 57A-57D show wild type FraC (WtFraC) and D10R-K159E FraC (ReFraC) nanopores.
  • FIG. 57A shows a cross-section through octameric WtFraC showing coulombic surface coloring. Aspartate residue 10 (D10), located in the constriction zone of WtFraC, is indicated.
  • FIG.57B shows a top view on WtFraC (top) and ReFraC (bottom).
  • FIG. 57C shows single channel conductance histogram for ReFraC (left) and WtFraC (right) at +50 mV in IM NaCl, 15 mM Tris-HCl pH 7.5.
  • FIG.57D shows raw trace of WtFraC (top) and ReFraC (bottom).
  • FIGs.58A-58F show DNA discrimination with ReFraC.
  • FIG.58A shows representative blockades of a homopolymeric DNA strand in complex with NA using ReFraC.
  • the cartoon representations on the right of each current trace shows the interpretation of the current blockades.
  • FIG.58B shows representative distributions of residual currents obtained for A20, C20, T20 homopolymeric strands with ReFraC nanopores.
  • FIG.58C shows current blockades of a continuous trace induced by homopolymeric C20 and A20 nucleotides to the same ReFraC pore. Traces shown were digitally filtered with 100 Hz cut-off.
  • FIG. 58D shows a distribution of residual currents imposed by mixtures of C20 and A20 homopolymeric strands.
  • FIG.58E shows continuous trace of an experiment to resolve mixtures of homopolymeric C 20 and T 20 nucleotides and
  • FIG.58F shows the distribution of residual currents imposed by mixtures of C20 and T20 homopolymeric strands.
  • FIGs.59A-59B show unzipping/translocation of dsDNA by ReFraC.
  • FIG.59A shows a representative trace of ReFraC capturing a NA:A(dsDNA)C complex at +50 mV.
  • the open pore current is denoted as “1” and for comparison indicated after capture of the complex.
  • States 2 and 3 are indicative of the block.
  • the block Upon reversal of potential (“4”) the block is immediately released indicating that the double-stranded region NA:A(dsDNA)C complex was peeled off.
  • FIG.59A shows a representative trace of ReFraC capturing a NA:A(dsDNA)C complex at +50 mV.
  • the open pore current is denoted as “1” and for comparison indicated after capture of the complex.
  • States 2 and 3 are indicative of the block.
  • Upon reversal of potential (“4”) the block is immediately released indicating that the double-stranded region NA:A
  • FIG. 60A-60B show unitary channel conductance distribution and voltage current dependence determined for WtFraC and ReFraC nanopores.
  • FIG. 60A shows unitary channel conductance distribution measured for WtFraC (top) and ReFraC (bottom) pre-oligomerized pores reconstituted in planar lipid bilayers.
  • FIG.60B shows voltage current dependence measured for WtFraC and ReFraC nanopores.
  • FIG. 61 shows hemolytic activity of the WtFraC, D10R FraC and ReFraC. Hemolysis rate was calculated as inverse of the time elapsed till 50% decrease in turbidity (measured as optical density at 650 nm wavelength) observed in 1% of horse erythrocytes suspension in 15 mM Tris-HCl pH 7.5150 mM NaCl.
  • FIGs.62A-62B show translocation and immobilization of A(dsDNA)C DNA substrate recorded with ReFraC nanopore.
  • A(dsDNA)C substrate (depicted above the trace) was made by annealing of ohgo I (5’ biotinylated AAAAAAAAAAAAAAAAAAGTGCTACGACTCTCTGTGTGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC).
  • FIG.62A shows blockades provoked on ReFraC nanopore by 1 ⁇ M of A(dsDNA)C alone (left) and in complex with 0.25 ⁇ M of neutravidin (right), substrates were added in cis under +50 mV applied potential.
  • FIG. 62B shows blockades provoked on ReFraC nanopore by 1 ⁇ M of A(dsDNA)C alone (left) and in complex with 0.25 ⁇ M of neutravidin (right), substrates were added in cis under at + 70 mV.
  • FIG. 63 shows representative traces showing stepwise enhancements of the residual current within A(dsDNA)C-neutravidin blockades provoked on ReFraC nanopore. 1 ⁇ M of A(dsDNA)C and 0.25 ⁇ M of neutravidin were present in cis at +50 mV.
  • FIG. 63 shows representative traces showing stepwise enhancements of the residual current within A(dsDNA)C-neutravidin blockades provoked on ReFraC nanopore. 1 ⁇ M of A(dsDNA)C and 0.25 ⁇ M of neutravidin were present in cis at +50 mV.
  • FIG. 64 shows traces depicting rotaxane formation by A(dsDNA)C-neutravidin driven into ReFraC nanopore at +100 mV applied potential.1 ⁇ M of A(dsDNA)C and 0.250 ⁇ M of neutravidin were added in cis. Voltage stepping protocols are shown with the lines below each current trace diagram. Rotaxanes were dismantled by switching the applied potential to -40 mV. [0190]
  • FIGs. 65A-65B show representative traces showing pseudorotaxane and rotaxane formation by oligonucleotide I - neutravidin immobilized within the ReFraC nanopore.
  • FIG. 65A shows pseudorotaxane formation provoked by 1 ⁇ M of oligo I and 0.25 ⁇ M of neutravidin present in cis.
  • FIG. 65B shows rotaxane formation by 1 ⁇ M of oligonucleotide I and 0.25 ⁇ M neutravidin present in cis while 1 ⁇ M of oligonucleotide II was added in trans.
  • Rotaxanes were dismantled by switching the applied potential to -40 mV (two arrows above the trace indicate the dismantling of rotaxane).
  • FIGs.66A-66C show capture of an oligopeptide (Endothelin 1) and a protein (Chymotrypsin) with two FraC variants at two different pH conditions, in accordance with some embodiments.
  • FIG. 66A shows cross- sections of wild type FraC (WtFraC, PDB: 4TSY) and D10R-K159E-FraC (ReFraC).
  • FIGs. 66B-66C show representative traces induced by 1 ⁇ M endothelin 1 (FIG. 66B) and 200 nM chymotrypsin (FIG.
  • FIG. 67A-67B show electrostatic distribution and ion-selectivity of WtFraC and ReFraC.
  • FIG. 67A shows the monomer averaged simulated electrostatic potentials reveal the negatively and positively charged constrictions of WtFraC and ReFrac, respectively.
  • FIG. 67B shows determination of the reversal potential shows that WtFraC and ReFrac may be respectively cation- and anion-selective, as expected from the electrostatic potentials at their constrictions.
  • FIGs. 68A-68E show biomarker characterization with WtFraC at pH 4.5.
  • FIGs. 68A shows, from top to bottom: (i) surface representation with molecular surface and cartoon representations (PyMOL) of chymotrypsin (25 kDa, PDB: 5CHA), (ii) a representative trace obtained under -150 mV applied potential, (iii) a heatplot depicting the dwell time distribution versus Ires% at -150 mV, and (iv) the voltage dependence of Ires%, the voltage dependence of the dwell times, and the capture frequency.
  • FIGs. 68B-68E show the same information for ⁇ 2- macroglobulin (FIG.
  • FIGs. 69A-69E show discrimination of endothelin 1 and 2 with WtFraC at pH 4.5.
  • FIG. 69A shows molecular surface representation of endothelin 1 and endothelin 2 using electrostatic coloring (PyMOL).
  • FIG.69B shows amino acid sequences of endothelin 1 and 2 (top), and Ires% and dwell time for endothelin 1 and endothelin 2 blockades at -50 mV in pH 4.5 buffer (1 M KCl, 0.1 M citric acid, 180 mM Tris-Base (bottom). Lines (6901) indicate the disulfide bridges in each oligopeptide.
  • FIG.69C shows representative endothelin 1 and endothelin 2 blockades to the same FraC nanopore under -50 mV applied potential.
  • FIG. 70 shows a computer system for implementing a method, a system, or a device of the present disclosure, in accordance with some embodiments.
  • FIGS.71A-71B show a device comprising a nanopore as described herein.
  • FIG. 72 shows a method using devices, nanopores, systems, and sample preparation as described herein.
  • FIG.73 shows a kit as described herein.
  • FIG.74 shows an exemplary system for characterizing and translocating polymer analytes, for example mixed amino-acid composition protein analytes, through a nanopore in a membrane.
  • FIGs.75A-75B illustrate an example leader with potential components.
  • FIG.75A shows components of a (peptide based) “leader construct” (6) for attaching to a target protein substrate of interest, which can assist loading/binding of protein translocase motor(s) for unfolding and controlling translocation of the target protein substrate through a nanopore.
  • the construct comprises a number of possible elements: 1. Recognition motif, 2. Capture motif, 3.
  • FIG.75B shows an illustrative schematic of a leader construct (6) that is attached to a target protein substrate(s) of interest (7), e.g. a folded or structured protein.
  • FIG. 76 shows an exemplary process of a method of loading a protein translocase (8) onto a leader construct (6).
  • a translocase first binds to a leader construct (B) at or near the recognition motif, and then proceeds to translocate along the construct (C) in the direction of the subset arrow via NTP hydrolysis until encountering the stall and/or blocking motifs that stall/pause the progression of the translocase (D).
  • FIG. 77A-77B show an exemplary process of loading multiple protein translocases onto a leader construct.
  • FIG. 77A shows a schematic of a substrate designed to load and stall one protein translocase.
  • the capture (2) and/or stall (3) motifs in combination have a footprint long enough to accommodate a single translocase.
  • FIG.77B shows a schematic of a substrate designed to load and stall n multiple protein translocase, comprising a longer combination of capture (2) and/or stall (3) motifs that can effectively stall and accommodate the binding footprints of the n multiple translocases, such that the trailing translocase motor(s) cannot push the leading translocase(s) through the stall/block motifs.
  • FIGs. 77A shows a schematic of a substrate designed to load and stall one protein translocase.
  • the capture (2) and/or stall (3) motifs in combination have a footprint long enough to accommodate a single translocase.
  • FIG.77B shows
  • FIG.78A-78B show exemplary methods of loading a leader construct (6) with protein translocase(s) (8) and attaching the leader construct to a protein of interest (7).
  • FIG.78A shows leader constructs can first be coupled to target protein analytes, then loaded with translocases.
  • FIG.78B shows leader substrates can be pre- loaded with translocases, and then coupled to target protein analytes.
  • FIGs.79A-79D show electrical recordings of the capture of a Maltose Binding Protein substrate (MBP- 1) in wild-type alpha-hemolysin nanopores (WT ⁇ HL).
  • MBP-1 Maltose Binding Protein substrate
  • FIGs. 80A-80D show the same as detailed in FIGs. 79A-79D, but using wild-type CytK nanopores (WT CytK).
  • FIGs. 80A-80D show the same as detailed in FIGs. 79A-79D, but using wild-type CytK nanopores (WT CytK).
  • FIGs. 81A-81C show a representative example of electrical current vs time traces for testing of a Maltose Binding Protein substrate:ClpX translocase complexes (MBP-1:ClpX) in weak EOF wild-type alpha- WSGR Docket Number: 64828-710.601 hemolysin (FIGs. 81A-81B) or wild-type CytK (WT CytK) (FIG. 81C). Measurements were carried out in a system similar to that described in FIG.1 (except with low or zero EOF nanopores). [0207] FIGs.
  • FIGs.82A-82B show a representative examples of electrical current vs time traces for testing of a Maltose Binding Protein substrate:ClpX translocase complexes (MBP-1:ClpX) in strong EOF CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores according to the system described in FIG.74.
  • FIGs.82A-82B show representative sections at -80 mV from separate experiments. The characteristic ClpX controlled MBP-1 translocations are marked by numbered arrows.
  • FIG.83 shows a representative zoomed single example event of ClpX controlled MBP-1 translocation through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores.
  • Events start with a blockade (S1) from the open pore level (state i) to an almost 0 pA level (state ii), and terminate (S3) with a return to open pore current levels (state iv) when the ClpX reaches the end of the MBP-1.
  • FIGs.84A-84B show representative zoomed examples of ClpX controlled MBP-1 translocation events through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores.
  • FIGs.85A-85D show examples of ClpX controlled MBP-1 translocation events for selected high EOF nanopore systems using high ion-selectivity nanopores.
  • CytK_3D1F2E nanopore (CytK K128F_S120D_Q122D_K155D) system -80 mV FIG.
  • FIGs. 85B a CytK_4D2E_Alt nanopore (CytK K128D K155D S120D S151D) system at -80mV (FIG.85C), a CytK_2D1F2E nanopore (CytK K128F S120D Q122D) system at -120 mV (FIG.85D), all in cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, with preloaded MBP-1:ClpX in the cis compartment (to a final concentration 0.2 ⁇ M ClpX, 0.1 ⁇ M MBP-1 and 2.5 mM ATP). [0211] FIGs.
  • FIG. 86A-86B show representative zoomed exemplary events of ClpX controlled translocation of MBP-1 substrates (FIG.86A) compared to GFP-1 substrates (FIG.86B) through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores at -80 mV.
  • FIG. 87 shows exemplary electrical current vs. time traces for testing of a Maltose Binding Protein:ClpX translocase complexes (MBP-1:ClpX) in strong EOF CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores with non-hydrolyzable Gamma-S-ATP.
  • FIG. 88 shows a histogram of the translocation duration for 35 full-length ClpX controlled MBP-1 translocations through a CytK 4D2E nanopore at -80 mV (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5 containing 0.2 ⁇ M ClpX:0.1 ⁇ M MBP-1, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5).
  • FIGs.89A-89B show a comparison of ClpX controlled MBP-1 translocations at -80 mV through CytK 4D2E nanopore systems (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5) without (FIG.89A) and with (FIG.89B) pre-loading incubation to form the MBP-1:ClpX complexes.
  • FIG.89A shows a comparison of ClpX controlled MBP-1 translocations at -80 mV through CytK 4D2E nanopore systems (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5) without
  • FIG. 90 shows exemplary capture and ClpX controlled trans-to-cis translocation of MBP-1 through CytK 4D2E nanopores that were inserted from the cis compartment (cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2).
  • Preloaded MBP-1:ClpX complexes were added to the trans compartment (to a final concentration 0.2 ⁇ M ClpX, 0.1 ⁇ M MBP-1 and 2.5 mM ATP) and captured into the trans entrance of the nanopores at +80 mV, and translocated under ClpX control as a result of the strong trans- to-cis EOF created at positive applied voltage.
  • FIG. 91A-91B show gel results of ClpX activity assays.
  • FIG. 91A shows a gel showing the results of a ClpX/ClpP degradation assay of a GFP-ssrA substrate (GFP-0) under varying concentrations of KCl.
  • FIG. 91B shows gel showing the results of a degradation assay of GFP-0 under varying concentration of potassium glutamate (KGlu). (65 nM ClpX, 65nM ClpP, 2800 nM GFP) [0217]
  • FIG.92 shows representative ClpX controlled translocation of MBP-1 through a CytK 4D2E nanopore at -120 mV in a nanopore system. [0218] FIGs.
  • FIG. 94 shows a schematic of current vs time for a translocation event resulting from translocase controlled polypeptide translocation through a nanopore as described by the scheme in FIGs.93A-93E.
  • FIGs.95A-95D show structural models of CytK.
  • FIG.95A shows a structural model of the wild-type CytK nanopore from homology mapping onto the structure of the alpha-hemolysin nanopore.
  • FIG.95B shows a schematic of the residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK, marking water-facing residues of the down- and up- strands most suitable for mutagenesis.
  • FIG.95C shows a model of the CytK 4D2E nanopore (CytK K128D K155D S120D Q122D), showing very high net negative internal charge due to mutations.
  • FIG.95D shows a schematic location of mutations to negative residues in the barrel region of the CytK 4D2E nanopore.
  • FIGs. 96A-96E show recordings of different substrates in CytK nanopores.
  • FIG. 96A shows a schematic of the substrate design.
  • FIGs.96B-96E show electrophysiology reads acquired from ⁇ GFP ⁇ - ⁇ MBP- 1 ⁇ , ⁇ LIVBP ⁇ - ⁇ MBP-1 ⁇ , ⁇ SpuE ⁇ - ⁇ MBP-1 ⁇ , and ⁇ GBP ⁇ - ⁇ MBP-1 ⁇ substrates.
  • FIG.97 shows a single-molecule read of a 88 kDa MBP-MBP fusion protein.
  • FIGs.98A-98D show stalling of ClpX at 37°C using blocking domains.
  • FIG.98A shows unfolding of GFP-1 with (+ClpX) and without ClpX (-ClpX);
  • FIG.98B shows mNG with an alpha-helical blocking domain;
  • FIG. 99C shows mNG with a helix-turn-helix blocking domain
  • FIG. 98D shows mNG with a hairpin blocking domain.
  • FIGs. 99A-99B show depictions of maltose-binding protein (MBP) monomers (FIG. 99A) and maltose-binding protein dimers (FIG.99B) for analysis.
  • FIG.100 shows an electrophysiology recording of a MBP protein lacking the ssrA recognition motif, that was tagged on the C-terminus to allow binding of ClpX. ClpX controlled MBP translocations (marked by arrows) are evident from the characteristic pattern of changing ionic current signals.
  • FIGs.101A-101D show attachment of a single-stranded DNA to ClyA nanopore.
  • FIG.101A depicts a side view (left) and top view (right) of ClyA structure (PDB: 6mrt).
  • Serine at position 110 was genetically mutated to cysteine to enable site-specific chemical modification.
  • FIG. 101B shows a schematic model showing the conjugation strategy of attaching ssDNA to ClyA nanopore.
  • FIG. 101C shows a SDS- PAGE analysis of the conjugation efficiency.
  • FIG. 101D shows a native polyacrylamide gel analysis of the oligomerization of ClyA-f.
  • Lane 5 ClyA-f after oligomerization
  • lane 6 S110C mutated ClyA after oligomerization.
  • FIGs.102A-102F depict functionalization of ClyA nanopore with Spike nanobody Ty1 and electrical characterization of the nanopore.
  • FIG.102A shows a schematic model showing the strategy of functionalizing ClyA nanopore with Ty1 nanobody.
  • FIG. 102B shows I-V curves of ClyA-S110C (triangle), ClyA-f (square) and ClyA-f-Ty1 (circle) at applied potential ranging from -90 to 90 mV (three independent experiments).
  • FIG. 102C shows a histogram showing conductance distribution of ClyA-f nanopore with (white) and without (black) Ty1 nanobody.
  • FIG.102D depicts representative current traces of ClyA-f-Ty1 under an applied potential of - 20 mV.
  • FIG. 102E shows an all-point histogram of the current traces shown in FIG. 102D, which demonstrated well-defined distribution of the blockade signals.
  • FIG. 102F shows a schematic model interpreting the reversible conformation change between blocked (left) and open (right) states of ClyA-f-Ty1 at applied potential of -20 mV, which corresponded to the movement of one of the Ty1 nanobodies in and out of the vestibule of the pore.
  • FIGs. 103A-103F show single channel recording traces of ClyA-f-Ty1 and the analysis of residual current Ib/Io, tin and tout under different applied potentials.
  • FIG.103A shows representative current traces of ClyA-f-Ty1 under applied potentials ranging from -10 to -40 mV.
  • FIG. 103B shows an all-point histogram of current traces depicted in FIG.103A, showing Ty1 nanobodies tend to reside in ClyA nanopore with increasing applied potentials.
  • FIGs.103C-103D show a histograms of logarithmic time of Ty1 locating inside and outside of ClyA, respectively.
  • FIGs. 104A-104B depict nanobody attachment to ClyA through DNA oligo hybridization verified using DNase I.
  • FIG.104A shows current traces of ClyA-f-Ty1 before and after the addition of 5 U DNase I in the presence of 2.5 mM MgCl2 at an applied potential of -20 mV.
  • FIG. 104B shows enlarged representative current traces from FIG.104A, showing that the nanobodies attached to the ClyA nanopore were removed after the addition of DNase I after about 30 mins.
  • FIG.105 depicts detection of Spike protein by nanobody-functionalized nanopores.
  • the upper current trace shows ClyA-f-Ty1 before and after the sequential addition of 6 ⁇ M BSA and 2.3 nM Spike protein.
  • Below the upper current trace are enlarged representative current traces and all-point histograms of the current distribution. From left to right are: (i) representative traces before addition of BSA and spike protein; (ii) representative traces after addition of BSA and prior to spike protein; and (iii) after addition of BSA and spike protein.
  • FIGs. 106A-106G show the effect of BSA on nanobody internalization.
  • FIGs. 106A-106C show histograms distribution of tout before and after the addition of 3 ⁇ M BSA or 6 ⁇ M BSA to the first side (e.g., a cis side) of a ClyA-f-Ty1 nanopore system. The histograms were fitted with single exponential function.
  • FIGs. 106D-106G show the change of blockade percentage, open percentage, average time of the coupled Ty1 staying inside the ClyA nanopore (t in ), average time of Ty1 staying outside the ClyA nanopore (t out ) with increasing concentration of BSA, respectively.
  • FIGs.107A-107E show the effect of adding Spike protein to the ClyA-f-Ty1 pore.
  • FIG.107A depicts current traces showing the transition of the pore from a dynamic state (alternating between Ty1 in the pore and out the pore) to a fully open state (with the Ty1 trapped outside the pore through binding to Spike protein) in the period immediately following the addition of 2.3 nM Spike protein.
  • FIG. 107B shows current traces of ClyA-f-Ty1 in the period approximately 25 minutes post addition of 2.3 nM Spike protein.
  • FIG. 107C shows an all-point histogram of the current traces presented in FIG.107B.
  • FIGs.107D-107E show histograms of the WSGR Docket Number: 64828-710.601 logarithm of tin (FIG. 107D) and tout (FIG.107E) after the addition of 2.3 nM Spike protein in of the period about 25 minutes post addition of Spike protein. The histograms were fitted with the Gauss distribution function.
  • FIG.108A-108D show open probability of ClyA-f-Ty1 correlates positively with Spike trimer protein concentration.
  • FIG. 108A shows representative current traces of ClyA-f-Ty1 before and after the addition of increasing concentration of Spike trimer protein.
  • FIG. 108B shows an all-point histograms were displayed to show the current distribution before and after the addition of increasing concentration of Spike protein.
  • FIG.108D shows a schematic model depicting the dynamics of the interaction between ClyA-f-Ty1 and Spike protein. Ty1 nanobodies dynamically move in and out of ClyA nanopore under applied potential.
  • FIGs.109A-109J show the influence of Spike proteins concentration on binding kinetics to the ClyA- f-Ty1 pore.
  • FIGs. 110A-110D show histograms of log10(t out ) at Spike concentration of 115 pM, 230 pM, 345 pM, 460 pM, respectively. The data were fitted with Guass distribution.
  • FIGs.109E-109H show histograms of log10(tin) at Spike concentration of 115 pM, 230 pM, 345 pM, 460 pM, respectively. The data were fitted with Guass distribution.
  • FIGs.109I-109J show the concentration dependency of the logarithm of tout and tin.
  • FIGs. 110A-110H show the behavior of ClyA-f-Ty1 in the presence of blood.
  • FIG. 110A-110H show the behavior of ClyA-f-Ty1 in the presence of blood.
  • FIG. 110A shows a schematic model showing electrical measurement of ClyA-f-Ty1 in the presence of blood.
  • FIG. 110B shows current traces showing the current change before and after addition of 1 ⁇ L of blood to the ClyA-f-Ty1 nanopore presenting in 500 ⁇ L electrolyte buffer.
  • FIGs.110C and FIG. 110E show representative current traces in the presence of 6 ⁇ M BSA (FIG. 110C) and after addition of 1 ⁇ L of blood (FIG.110E).
  • FIGs.110D and FIG. 110F show all-point histograms of the current traces before (FIG.110D) and after (FIG.110F) addition of 1 ⁇ L of blood.
  • FIG.110G shows a histogram of the logarithm of dwell time in level0 before and after the addition of 1 ⁇ L of blood.
  • FIG. 110H shows a histogram of the logarithm of dwell time in level1 before and after the addition of 1 ⁇ L of blood.
  • FIGs. 111A-111B show detection of spike trimer in the presence of blood.
  • FIGs. 111A-111B show representative current traces before (FIG.111A) and after (FIG.111B) the addition of 2.3 nM Spike protein in the presence of 1 ⁇ L blood at a bias of -20 mV.
  • FIGs. 112A-112B show detection of Her2 with functionalized nanopores.
  • FIG. 112A-112B show detection of Her2 with functionalized nanopores.
  • FIG. 112A shows representative current traces of ClyA attached by 2Rs15d nanobody (ClyA-f-15d) before and after the addition of 32.8 nM Her2 protein under an applied potential of -20 mV.
  • FIG.112B shows representative current traces of ClyA attached by 2Rb17c nanobody (ClyA-f-17c) before and after the addition of 20.8 nM Her2 protein at the same applied potential.
  • FIGs.113A-113E show functionalized ClyA nanopore for the detection of muPA.
  • FIG. 113A shows the crystal structure of muPA in complex with nb22 nanobody (PDB: 5LHR).
  • FIG. 113B shows representative current traces of ClyA-f-nb22 before and after adding 3 nM muPA under -15 mV applied potential.
  • FIG. 113C shows enlarged representative current traces after adding 3 nM muPA at -15 mV.
  • FIG. 113D shows a heatmap of the blockade events observed after the addition of 3 nM muPA with the logarithm of the dwell time against current blockade percentage.
  • FIGs.114A-114C shows schematic illustrations of some of the options for coupling targeting moieties R to nanopores N via the hybridization of duplexed oligonucleotide (e.g. dsDNA) linkers L (where one oligonucleotide strand of the duplex linker L is coupled to the nanopore N, and the other complementary strand is coupled to the binding moiety R).
  • FIG. 114A-114C illustrates 3 possible options for coupling the components.
  • FIG.114A shows the nanopore N and binding moiety R components are located at opposite ends of the duplex linker L.
  • FIG. 114B shows the nanopore N and binding moiety R components are located at the same end of the hybridized duplex linker L.
  • FIG.114C shows one or both the nanopore N and binding moiety R components are coupled to the oligonucleotide strands of the hybridized duplex linker L at an internal position along the strand.
  • FIGs. 115A-115D show schematic illustrations of a nanopore N with a linker L that is initially in a protected state (FIG.115A), comprising a hybridized protecting polynucleotide strand (i) that can be removed by applying voltage to the nanopore in a membrane system to capture and strip the protecting strand from the linker L (FIG.115B).
  • FIG.115C shows a depiction of a computer system that is programmed or otherwise configured to implement the methods provided herein.
  • FIGs. 117A-117B show example workflow methods for signal extraction and processing with representative current signals (e.g., electrophysiology traces or waveforms).
  • FIG. 117A shows a schematic illustrating a method for extracting a signal or portion thereof or change thereof and processing the signal.
  • FIG. 117A shows a schematic illustrating a method for extracting a signal or portion thereof or change thereof and processing the signal.
  • FIG.117B shows the representative signals for each step shown in the method schematic of FIG.117A, beginning with raw signal (step 1), extracted and denoised portions of signal (reads, putative reads and other event types, step 2), filtering (e.g., based on read metrics) to remove events that do not meet the criteria of good reads (step 3), segmenting the reads and performing merging and other resegmentation steps to produce a segmented read (squiggle) (steps 4 and 5), and finally performing further filtering to remove reads based on the metrics of the segmented reads.
  • WSGR Docket Number: 64828-710.601 [0243]
  • FIG.118 shows a representative schematic of a model architecture of a model described herein.
  • FIG.119 shows a representative workflow for scaling a reference squiggle to read as described herein.
  • FIG. 120 shows a representative workflow for polypeptide identification. The polypeptide identification can be for a single molecule.
  • FIG.121 shows a representative workflow for polypeptide quantification.
  • FIG.122 shows a representative workflow for alignment and consensus of signal reads.
  • FIGs.123A-123D show alignment and generation of consensus sequence from data.
  • FIG.123A shows representative current signal reads for protein translocation events of data produced by a nanopore system described herein.
  • FIG.123B shows representative reference signals (e.g., generated by machine learning from trained models as described herein).
  • FIG.123C shows an example of an alignment schematic mapping of one of the signal reads of the detected current signals of FIG.123A and the reference signals of FIG.123B.
  • FIG. 123D shows an example of the output consensus of multiple signal reads of FIG.123A that all aligned to one of the reference signals of FIG. 123B all piled up into a consensus plot with shared x-axis sequence position (also called index position) and scaled and normalized current (performed during scaling as described herein).
  • FIG. 124A-124E show representative raw current signal (specifically portions of signal reads corresponding to protein translocations through a nanopore)showing how point mutations and other small amino acid motif 2-mer or 3-mer changes in the analytes alters the ionic current signal where indicated by the dotted box.
  • FIG. 124A shows the current signal from an analyte with the KNK motif, with point mutations relative to KWK and ENK motifs.
  • FIG.124B shows the current signal from an analyte with the WWW motif, with 2-mer and 3-mer changes relative to analytes with the other motifs.
  • FIG. 124C shows the current signal from the ENE motif, with a single point mutation relative to the ENK motif.
  • FIGs. 125A-125B show the consensus analysis of point mutation sequencing of maltose protein containing analyte MBP-1. Circles represent wildtype MBP (MBP-MBP1), triangles represent MBP1 with ENK mutation, squares represent MBP1 with KWK mutation, and downward-facing triangles represent MBP1 with WWW mutation. FIGs.
  • 125A-125B show the consensus squiggle along index positions of the analyte (determined as described herein by aligning many reads to a reference squiggle of MBP-1 and adjusted the reference squiggle based on the deviations in the reads) as the lines and markers.
  • Overlaid over the consensus lines and markers are the points corresponding to the average current for the given segments aligned the sequence index position for all the reads used in the consensus analysis.
  • the spread of points are tightly clustered around the mean of the consensus squiggle at each index position, showing that all the single molecule WSGR Docket Number: 64828-710.601 reads closely match the consensus.
  • FIG.125B shows the zoom region of FIG.125A containing the mutation, illustrating the clear deviations in current that are observed in the reads and the consensus from the point mutations.
  • FIGs.126A-126D show consensus squiggles (overlaid with their corresponding aligned reads, plotting the 1-standard deviation spread of the difference in the current of the corresponding segment at each index position as the width of the lines) from samples containing protein analytes with and without post-translational modifications (PTMs).
  • FIG. 126A shows detection of phosphorylation, specifically a phosphorylated serine residue, from the sequence index.
  • FIG. 126B shows detection of glycosylation, specifically a glycosylated cysteine residue, from the sequence index.
  • FIG.126C shows detection of acetylation, specifically an acetylated lysine residue, from the sequence index.
  • FIG.126D shows detection of deamidation from the sequence index. Resides Q255, N203, N175, and Q174 were deaminated.
  • FIGs.127A-127B show a poly-PTM measurements from an analyte.
  • FIG.127A shows the consensus squiggle plots for both treated (phosphorylated) and untreated samples, obtained from alignment of the reads for the samples as described herein. The figure plots current vs index position for the two consensus squiggles obtained (lines), overlaying points corresponding to the average current for the given segments aligned the sequence index position for all the reads used in the consensus.
  • FIG. 127B shows the zoom of FIG.127A in the region containing two deviations that arise from two phosphorylation sites along the proteins. Both Site 1 and Site 2 showed clear deviations in current for the modified reads and consensus squiggle vs the unmodified.
  • FIGs. 128A-128C show detection of post-translational modifications and variants of analytes in a mixed population.
  • FIG. 128A shows a multiple aligned reads (aligned as described herein to a reference squiggle to align the segments in sequence index space) overlaid into a pile-up of reads for the sample measured at 0 hours of mixing (e.g., treatment).
  • FIG. 128B shows the similar multiple aligned reads pile-up from the sample measured at 16 hours after mixing (e.g., treatment).
  • the degraded sub-population can be viewed as a new population of reads containing a clear downward deflection in current at the position indicated by the arrow.
  • FIG. 128C shows quantification of the percentage of reads aligning to the degraded sub-population in the mixture versus the sample time points.
  • FIGs.129A-129B show bi-directional current signal reads of protein analyte nanopore translocations.
  • FIG. 129A shows reading of the MBP1 analyte in C-terminus to N-terminus direction when fed through a nanopore cis-to-trans as described herein.
  • FIG.129B shows reading of the MBP1 analyte in N-terminus to C- terminus direction when fed through a nanopore cis-to-trans as described herein.
  • FIG.130 shows a representative current waveform for a MBP-1 analyte translocated through an MspA nanopore with VAT (VAT- ⁇ N unfoldase).
  • VAT- ⁇ N unfoldase VAT- ⁇ N unfoldase
  • S. and E. mark the start and end of the reads respectively, and IO the open-pore current.
  • the applied voltage is -80 mV.
  • FIG. 131 shows a representative portion of the electrophysiology current signal of a substrate with truncation of a C-terminal peptide tag.
  • the substrate had no poly-glycine region .
  • the applied voltage is -80 mV.
  • FIG.132 shows translocation read of a C-terminus to C-terminus linked protein created by chemical conjugation of a tag to the substrate. The location of recognizable and repeatable features in the reads are indicated by arrows. Measurements obtained with a MspA_D90N nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV.
  • FIGs. 133A-133F show representative portions of current signal reads for different proteins.
  • FIG. 133A shows the current signal for translocation of alpha-synuclein through a nanopore, displayed as current (pA) versus time (s).
  • FIG.133B shows the current signal for translocation of p53 through a nanopore, displayed as current (pA) versus time (s).
  • FIG. 133C shows the current signal for translocation of TauF through a nanopore, displayed as current (pA) versus time (s).
  • FIG.133D shows the current signal for translocation of a nanobody through a nanopore, displayed as current (pA) versus time (s).
  • FIG. 133E shows the current signal for translocation of a light chain of an antibody through a nanopore, displayed as current (pA) versus time (s).
  • FIG.133F shows the current signal for translocation of Titin I27 through a nanopore, displayed as current (pA) versus time (s).
  • FIGs. 134A-134C show representative current signal reads for the translocation of long peptides (peptides with a length of 900 amino acids or more).
  • FIG. 134A shows a representative current signal for translocation of analyte DNAK-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s).
  • FIG. 134B shows a representative current signal for translocation of analyte CH60-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s).
  • FIG. 134C shows a representative current signal for translocation of analyte ODP2-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s).
  • FIGs.136A-136D show schematic current signal reads of current versus time that illustrate differences in the kinetics or speed of translocation as a function of the position through the read/sequence.
  • FIG. 136A illustrates longer lived segments or pauses in the middle of translocations (marked by arrows), after which the read continues to translocate at the normal average speed.
  • FIG 136B illustrates stalls (marked by arrows) that terminate the translocation of the reads (e.g.
  • FIG.136C shows representative current signal reads for CH60 protein translocations, marking pauses and changes in kinetics by arrows.
  • FIG. 136D shows representative current signal reads for antibody protein translocations, marking pauses and changes in kinetics by arrows. Pauses and other changes in kinetics can be used for determination of polypeptide characteristics.
  • FIGs. 137A-137E show schematic representations of electrophysiology systems as described herein.
  • FIG. 137A shows an enclosed trans compartment fluidically connected to an open cis compartment by a membrane.
  • FIG. 137B shows an enclosed cis compartment directly connected to an enclosed trans compartment, via a membrane, both compartments within a solid substrate.
  • FIG. 137A shows an enclosed trans compartment fluidically connected to an open cis compartment by a membrane.
  • FIG. 137B shows an enclosed cis compartment directly connected to an enclosed trans compartment, via a membrane, both compartments within a solid substrate.
  • FIG. 137A shows an enclosed trans compartment fluidically connected
  • FIGs. 138A-138E show a schematic of a system for translocating a protein substrate through a nanopore which is initially captured from the trans, then translocated with a motor protein trans-to-cis against a net cis-to-trans EOF (after a reversal of voltage and EOF in step D and E).
  • FIG.139 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.138A-138E.
  • FIGs.140A-140E show a schematic of a system for translocating a protein substrate, initially captured from the trans, then translocated with a motor protein cis-to-trans with a net cis-to-trans EOF, (after a reversal of voltage and EOF in step D and E).
  • FIG. 141 shows shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.140A-140E.
  • FIGs. 140A-140E shows shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.140A-140E.
  • FIG.143 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.142A-142E.
  • FIG.145 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.144A-144D.
  • compositions and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 80%, 85%, 90%, 95% identical or higher to the sequence specified.
  • amino acid sequence in the context of an amino acid sequence, the term “substantially identical” can be used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are (i) identical to, or (ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity.
  • amino acid sequences that contain a common structural domain having at least about 80%, 85%, 90%.91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein.
  • nucleotide sequence the term “substantially identical” can be used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.
  • the term “variant” can refer to a polypeptide and/or at least a portion of a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or can be encoded by a substantially identical nucleotide sequence. In some embodiments, the variant can be a functional variant.
  • the term “functional variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or can be encoded by a substantially identical nucleotide sequence, and can be capable of having one or more activities of the reference amino acid sequence.
  • Calculations of homology or sequence identity between sequences can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, and at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions can then be compared.
  • a position in the first sequence can be occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” can be WSGR Docket Number: 64828-710.601 equivalent to amino acid or nucleic acid “homology”).
  • a nanopore described herein may comprise one or more components.
  • the one or more components may be of a family of binary toxin, or a mutant thereof, or a functional homolog thereof, or a functional ortholog thereof, or a functional paralog thereof.
  • “Homologs” can refer to proteins, peptides, oligopeptides, polypeptides having amino acid substitutions, deletions, or insertions, or any combination thereof relative to an unmodified (e.g., wild-type) protein and having similar biological and/or functional activity as the unmodified protein from which they are derived.
  • “Ortholog” can refer to a gene or protein from different organisms (e.g., different species) that are derived from a common ancestral gene.
  • “Paralog” can refer to a gene or protein from the same organism (e.g., same species) that can be a product of gene duplication of a common ancestral gene.
  • the percent identity between the two sequences may be a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol.
  • Biol.48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a set of parameters can be a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences.
  • Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res.
  • amino acid can embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids.
  • Amino acids can include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing.
  • amino acid can comprise both the D- or L- optical isomers and peptidomimetics.
  • a “conservative amino acid substitution” can be one in which the amino acid residue can be replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), or aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), or any combination thereof.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspart
  • the term “mutation” can refer to an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA.
  • the mutation may be a large-scale mutation, such as amplifications (or gene duplications) or repetitions of a chromosomal segment, deletions of large chromosomal regions, chromosomal rearrangements (e.g., chromosomal translocations, chromosomal inversions, non-homologous chromosomal crossover, and interstitial deletions), and loss of heterozygosity.
  • the mutation may be a small-scale mutation, such as insertions, deletions, and substitution mutations.
  • substitution mutation can refer to the transition that exchange a single nucleotide for another.
  • a mutation herein may comprise a chemical conjugation to a non-natural amino acid.
  • analytes may be unfolded and translocated through a nanopore.
  • analyte domains may be recognized, a complex and inconsistent current signal obtained from measurement of an analyte-translocase complex and arising from the unfolding process that the analyte undergoes in the system may prevent the recognition of protein, polypeptides, or peptide sequences or subtle characteristics thereof.
  • analytes might be cleaved at specific sites and nanopore currents may be used to identify the released polypeptide or peptides. Therefore, the present disclosure may be aimed at designing and engineering new, analyte-based (e.g., protein-based) nanopores and nanopore systems that may be capable (for example, as part of a multi-protein sensor complex) of unfolding proteins, controlling processive and unidirectional transit across the nanopore, and producing highly consistent and reproducible signals that enable the analytes to be accurately characterized.
  • analyte-based e.g., protein-based
  • nanopore systems e.g., protein-based nanopore systems that may be capable (for example, as part of a multi-protein sensor complex) of unfolding proteins, controlling processive and unidirectional transit across the nanopore, and producing highly consistent and reproducible signals that enable the analytes to be accurately characterized.
  • the alignment methods disclosed herein can work incredibly well because the movement control is achieved to yield consistent high quality data signals.
  • the ability to accurately characterize peptides/polypeptides/proteins (or the properties of the samples from which they are derived) can be highly dependent on the consistency (e.g., between runs, systems, nanopores, membranes) and quality of the signal, which in turn depends on the translocation properties as the molecules move through the nanopore.
  • the methods and systems disclosed herein allow for translocation movement of single reads of non-nucleic acid based polymer analytes (e.g., one or more peptides, polypeptides, or proteins) through nanopores with high quality movement characteristics from which the step size can be obtained from reads.
  • non-nucleic acid based polymer analytes e.g., one or more peptides, polypeptides, or proteins
  • One of the goals of the present invention is to improve the accuracy of characterizing individually captured peptides by a nanopore sensor.
  • engineered proteinaceous nanopores can be developed to improve the capture of unlabeled peptides and/or labeled analytes (e.g., unlabeled or labeled non-nucleic acid based polymer analytes), to increase a residence (dwell time) of analytes in a nanopore sensor, and to improve the discrimination between analyte species.
  • the present disclosure provides engineered nanopores to improve analyte sensing under about physiological conditions as well as at low pH conditions that may be optimized for analyte detection. [0290] It was previously not thought possible to push/feed analytes into pores from, for example, the cis side in their native form (e.g.
  • EEF electro-osmotic flow
  • translocase on the cis side of a nanopore that can controllably feed and pass a wide range of analytes from cis to trans through the nanopore against the direction of the electrophoretic forces (EPFs).
  • the present disclosure further demonstrates that the portions of the signals that result from translocation of peptide/polypeptide/protein analytes may be highly consistent between different measurements of the same type of analyte.
  • the present disclosure further demonstrates that the high signal consistency and reproducibility between different measurements of single analyte molecules (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, etc.) can enable signals from analyte molecules to be combined and compared in analytical bioinformatics methods disclosed herein.
  • the high reproducibility may enable forming databases that are generated from a plurality of the same type of molecule (where multiple reads of the same molecule improves the precision of the database information).
  • the high reproducibility can mean molecules can be compared with high accuracy on a single molecule basis, either to each other or to an artificial reference signal that is generated by training from a plurality of training molecules.
  • the high reproducibility enables characteristics (e.g. identification, variants, modifications, length, speed, etc.) to be determined with high accuracy by comparison to reference information.
  • High reproducibility may mean molecules can be combined into sets and analysed in aggregate (e.g. to achieve higher accuracy by averaging or to determine properties of a sample that require a plurality of molecules).
  • the high reproducibility can mean that cleaner (less noisy and variable) signal data may be measured on a molecule by molecule basis, which can provide more detail of the analytes. For example, very small changes in the analytes (e.g.
  • the present disclosure in combination with the analytical methods shown herein (including comparisons to references and databases) also can demonstrate that the EOF exerts a powerful force that stretches and/or pulls on the portions of the peptide/polypeptide/protein analyte within or near the nanopore while the analyte resides in the nanopore.
  • An advantage of the EOF may be that this force also keeps the motor WSGR Docket Number: 64828-710.601 bound to the analyte held against the nanopore entrance during motor controlled analyte translocation.
  • Another advantage of the EOF force acting on the portions of the peptide/polypeptide/protein in the nanopore may be improved signal quality, for example, resulting from more consistent signal molecule-to-molecule, and/or by limiting variable changes in current signal that can result from the random stochastic movements of the portion of the peptide/polypeptide/protein in the nanopore (e.g. crumpling, folding, wobbling, shifting up and down of portions of the peptide/polypeptide/protein in the nanopore).
  • the present disclosure demonstrates that the EOF in the systems and methods of the invention can further improve the consistency and reproducibility of portions of the signal, enabling signals obtained from multiple sources (e.g.
  • the EOF exerts a stretching force on the peptide/polypeptide/protein that can reduce the length of the portion of the peptide/polypeptide/protein that is in the “reader” region of the nanopore (for example the one or more constrictions of the nanopore that give rise to the majority of the changes in current).
  • the reduction in the portion within the reader can have several benefits, including simplifying the signal, increasing the magnitude of current ranges as the analyte moves, provides higher resolution of closely separated features along the analyte, and/or simplifies the analysis of the sequence-to-signal (due to reduced sequence contributing to the signal).
  • the present disclosure provides novel approaches that yield highly reproducible signals from one or more analytes (e.g., one or more peptides, polypeptides, proteins, or any combinations thereof).
  • the present disclosure further demonstrates that the portions of the signals that can result from translocation of non-nucleic acid based polymer analytes can be highly consistent between different measurements of the same type of analyte.
  • the present disclosure demonstrates that the high signal consistency and reproducibility between different measurements of single analyte molecules (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combinations thereof) can enable signals from analyte molecules to be combined and/or compared in analytical bioinformatics methods disclosed herein.
  • the high reproducibility enables forming databases that can be generated from a plurality of the same type of molecule (e.g., where multiple reads of the same molecule can improve the precision of the database information).
  • the high reproducibility can demonstrate that molecules can be compared with high accuracy on a single molecule basis, to each other and/or to an artificial reference signal that can be generated by training from a plurality of training molecules.
  • the high reproducibility can enable one or more characteristics (e.g. identification, variants, modifications, length, speed, or any combinations thereof) to be WSGR Docket Number: 64828-710.601 determined with high accuracy by comparison to reference information.
  • High reproducibility means molecules can be combined into sets and analyzed in aggregate.
  • one or more analytes may be combined and/or analyzed in aggregate to achieve higher accuracy by averaging and/or to determine properties of a sample that require a plurality of analytes.
  • the high reproducibility can mean that cleaner (e.g., less noisy and variable) signal data may be measured on a molecule by molecule basis, which can provide more detail of the analytes. For example, enabling very small changes in the analytes (e.g.
  • the present disclosure in combination with the analytical methods shown herein (for example, including comparisons to references and databases) can also demonstrate that the electro-osmotic force (EOF) can exert a powerful force that stretches and pulls on the portions of the one or more analytes (e.g., one or more non-nucleic acid based polymer analytes) within or near a nanopore while the one or more analytes reside in the nanopore.
  • EEF electro-osmotic force
  • An advantage of the EOF is that this force can also keep a motor protein as described herein bound to the analyte held against the nanopore entrance during motor controlled analyte translocation.
  • An advantage of the EOF force acting on the portions of the one or more analytes (e.g., one or more non-nucleic acid based polymer analytes) in the nanopore can be improved signal quality.
  • the improved signal quality may result from more consistent signal molecule-to-molecule, for example by limiting variable changes in current signal that can result from random stochastic movements of the portion of the one or more non-nucleic acid based polymer analytes in the nanopore.
  • Random stochastic movements of the one or more analytes may comprise crumpling, folding, wobbling, shifting up and down, or any combination thereof of portions of the one or more analytes in the nanopore.
  • the present disclosure demonstrates that the EOF in the systems and methods of the invention can further improve the consistency and reproducibility of portions of the signal. This may enable signals obtained from multiple sources (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combinations thereof) to be combined and/or measured in aggregated analyses, and may be compared to each other and/or to signals in one or more databases generated from other analytes. These comparisons may accurately determine characteristics of the analytes and/or samples of a plurality of analytes (e.g.
  • the EOF can exert a stretching force on the one or more non-nucleic acid based polymer analytes that may reduce the length of the portion of the one or more non-nucleic acid based polymer analytes that may be in the “reader” region of the nanopore.
  • the “reader” region can comprise one or more constrictions (e.g., constriction regions) of a nanopore that may give rise to one or more changes in current.
  • the reduction in the portion within the constriction regions can have several benefits, including, but not limited to: (i) simplifying a signal, (ii) increasing a magnitude of current ranges as one or more analytes move, (iii) providing higher WSGR Docket Number: 64828-710.601 resolution of closely separated features along the one or more analytes, (iv) simplifying an analysis of the sequence-to-signal (e.g., due to reduced sequence contributing to the signal), or (v) any combination thereof.
  • the present disclosure provides a membrane comprising a pore.
  • a pore can be inserted into a membrane such as a lipid bilayer.
  • the pore can be a biological pore.
  • a pore can be engineered to bind with specific analytes.
  • a pore can be engineered to bind with a class of analytes (e.g., a peptide).
  • a pore can be engineered to not to bind with a class of analytes.
  • a pore can be engineered to permit certain ionic species to traverse through the pore.
  • a pore can be engineered to prevent certain ionic species from traversing through the pore.
  • the array of sensors can comprise at least about 1, 2, 3, 4, 8, 16, 32, 64, 96, 100, 500, 1000, 2000, or greater than about 2000 sensors.
  • the array of sensors can comprise, at most about 2000, 1000, 500, 100, 96, 64, 32, 16, 8, 4, 3, 2, 1, or less than about 1 sensor.
  • an array of sensors can comprise between about 1 sensor to about 1,000 sensors.
  • an array of sensors can comprise between about 1 sensor to about 2 sensors, about 1 sensor to about 3 sensors, about 1 sensor to about 4 sensors, about 1 sensor to about 8 sensors, about 1 sensor to about 16 sensors, about 1 sensor to about 32 sensors, about 1 sensor to about 64 sensors, about 1 sensor to about 96 sensors, about 1 sensor to about 400 sensors, about 1 sensor to about 800 sensors, about 1 sensor to about 1,000 sensors, about 2 sensors to about 3 sensors, about 2 sensors to about 4 sensors, about 2 sensors to about 8 sensors, about 2 sensors to about 16 sensors, about 2 sensors to about 32 sensors, about 2 sensors to about 64 sensors, about 2 sensors to about 96 sensors, about 2 sensors to about 400 sensors, about 2 sensors to about 800 sensors, about 2 sensors to about 1,000 sensors, about 3 sensors to about 4 sensors, about 3 sensors to about 8 sensors, about 3 sensors to about 16 sensors, about 3 sensors to about 32 sensors, about 3 sensors to about 64 sensors, about 3 sensors to about 96 sensors, about 3 sensors to about 400 sensors, about 3 sensors to about 800 sensors, about 2 sensors to about 1,000 sensors, about 3 sensors to
  • the array of sensors can comprise the same membrane, the same pore, and the same electrolyte conditions. At least two sensors in the array of sensors can comprise a different membrane, a different pore, and/or different electrolyte conditions.
  • the array of sensors can provide signals in parallel, which can increase the throughput of analyte detection and/or identification. Sensors in the array may each analyze the same sample. In some embodiments, one fraction of sensors in the array can analyze one sample, and another fraction of sensors in the array can analyze a different sample.
  • Various configurations and embodiments for arrays of sensors are provided herein. [0300] In some aspects, the present disclosure provides a device.
  • the device can comprise a sensor or an array of sensors.
  • the device can comprise an electrical energy source and two electrodes.
  • One of the two electrodes may be disposed on one side of the membrane of a sensor, and another electrode may be disposed on the other side.
  • the electrical energy source can apply a potential between the two electrodes, which can cause ions in an electrolyte to conduct through the fluid, and through the pore of the sensor.
  • the potential can also cause an analyte, if charged, to translocate through the pore.
  • the potential can create an electrophoretic force (EPF), described further in detail elsewhere in this application, which can provide a driving force for an analyte to translocate through the pore in order to generate a change in signal.
  • the device may further comprise two or more additional electrodes.
  • these electrodes can be configured to measure the electrical potential across the nanopore and/or membrane that changes when an analyte translocates through a pore. These electrodes can be configured to measure the current across a membrane as an analyte translocates through a pore.
  • the device can be in electrical communication with a recording device to record measured signals.
  • the device can be in electrical communication with a computer or a processor (e.g., a circuit, or an integrated circuit, or any combination thereof), which can receive a signal from the sensor or the array of sensors, store the signals in digital form, and/or process the signal.
  • the device can comprise a flow cell.
  • the flow cell can comprise or be fluidically coupled to the sensor or the sensor array.
  • the sensor or the sensor array can be integrated with the flow cell into a single piece, or they can be separate.
  • the device can comprise or be fluidically coupled to a fluidic control system (e.g., pumps, or controllers, or any combination thereof).
  • the fluidic control system may comprise a pump, a pressure based flow controller, a pressurized reservoir, a pressure sensor, a vacuum control system, one or more valves, a bubble trap, or fluidic channels that can generate a capillary-force, or combinations thereof.
  • a pump may be a syringe pump, a peristaltic pump, or piezoelectric pump.
  • the device can be a handheld device or a tabletop device.
  • the device can be configured to detect a single analytes (e.g., chemical species) (e.g., detecting the presence of a particular WSGR Docket Number: 64828-710.601 pathogen like coronavirus).
  • the device can be configured to detect a variety of analytes (e.g., chemical species).
  • the device can be configured to identify any analytes (e.g., chemical species) in a sample.
  • Various forms of devices and methods of using devices are disclosed herein.
  • the nanopores, methods, and system provided herein comprise detecting and/or characterizing one or more characteristics of an analyte.
  • Characteristics of the analyte may comprise a shape of the non-nucleic acid based polymer analyte, a structure of the non-nucleic acid based polymer analyte, one or more mutations of the non-nucleic acid based polymer analyte, a sequence of the non-nucleic acid polymer analyte, a surface charge of the non-nucleic acid based polymer analyte, one or more post-translation modifications of the non-nucleic acid based polymer analyte, or one or more ligands coupled to the non-nucleic acid based polymer analyte, or any combination thereof.
  • the present disclosure provides methods for processing signals.
  • the methods can be implemented on a computer.
  • the methods can be written as a set of instructions, which can be stored in a non- transitory storage medium.
  • the methods can be executable by a computer processor.
  • the methods and algorithms can be configured to store or process one or more signals and determine one or more identifications and/or characteristics of analytes associated with the one or more signals.
  • a computer or processor implementing the methods can be in electrical (wireless or wired) communication with the device.
  • Various methods for processing signals to identify analytes are disclosed herein.
  • analyte e.g., a non-nucleic acid based polymer analyte
  • the methods may comprise translocating one or more analytes (e.g., a plurality of analytes, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more analytes).
  • the analyte may be translocated through a pore described herein.
  • the pore e.g., nanopore
  • the pore may be disposed within a membrane.
  • the pore may be part of a nanopore system described herein.
  • an analyte e.g., a non-nucleic acid based polymer analyte
  • the at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or any combination thereof.
  • the terms “polypeptide” and “peptide” generally to refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond.
  • a polypeptide is a protein.
  • the amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid (i.e., amino acid analogue).
  • the polymer can be linear or branched and can include modified amino acids, and/or may be interrupted by non-amino acids. Polypeptides can occur as single chains or associated chains.
  • the polymer may include a plurality of amino acids and may have a secondary and tertiary structure (i.e., protein).
  • the polymer comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1000, at least about WSGR Docket Number: 64828-710.601 10,000, or more amino acids.
  • the at least a portion of the analyte translocated through a pore may comprise at least a portion of an analyte described herein.
  • a method may comprise detecting a current or change thereof.
  • a method may comprise detecting a voltage or change thereof.
  • a signal or change thereof can comprise a measure of an ionic current, voltage, or any combination thereof.
  • the method may comprise detecting a signal or change thereof.
  • the method may comprise detecting a current or change thereof while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore).
  • the method may comprise detecting a current or change thereof while at least a portion of an analyte translocates through a pore.
  • the method may comprise detecting a current or change thereof while at least a portion of an analyte (e.g., one or more analytes) resides in a pore.
  • the method may comprise using a current or change thereof.
  • the method may comprise using a voltage or change thereof.
  • the method may comprise using a current or change thereof to identify one or more characteristics of an analyte (e.g., a non- nucleic acid based polymer analyte).
  • the method may comprise using a current or change thereof to determine one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte). Determining a characteristic can comprise measuring a characteristic of an analyte, or quantitating a characteristic of an analyte, or any combination thereof.
  • the current or change thereof and/or voltage or change thereof may be used to identify a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics). In some embodiments, the current or change thereof and/or voltage or change thereof may be used to determine a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics).
  • the characteristics of at least a portion of the analyte may comprise characteristics of an analyte described herein (e.g., a shape of the analyte, a structure of the analyte, one or more mutations of the analyte, a sequence of the non-nucleic acid polymer analyte, a surface charge of the analyte, one or more post- translation modifications of the analyte, or one or more ligands coupled to the analyte, or any combination thereof).
  • the current or change thereof may be used to identify one or more characteristics of an analyte with an accuracy.
  • the accuracy of identifying the one or more characteristics of the analyte may be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.
  • the accuracy of identifying the one or more characteristics of the analyte may be at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or less than about 20%. In some embodiments, the accuracy of identifying the one or more characteristics of the analyte may be between about 20% to about 100%.
  • the accuracy of identifying the one or more characteristics of the analyte may be between about 20% to about 30%, about 20% to about 40%, about 20% to WSGR Docket Number: 64828-710.601 about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%
  • a method provided herein can comprise: (a) translocating at least a portion of an analyte through a nanopore disposed within a mem-brane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%.
  • translocating of at least a portion of an analyte can comprise translocating in a C-terminus to N-terminus (C-to-N) direction, or a N-terminus to C-terminus (N-to-C) direction, or any combination thereof.
  • C-to-N and/or N-to-C directions can be relative to at least a portion of the analyte sequence.
  • Determining one or more characteristics can comprise using the current or change thereof and/or the voltage or change thereof associated with a C-to-N direction, N-to-C direction, or any combination thereof.
  • Determining one or more characteristics can comprise using an electrical signal or change thereof associated with a C-to-N direction, or N-to-C direction, or any combination thereof.
  • an analyte e.g., a non-nucleic acid based polymer analyte
  • the method may comprise translocating at least a portion of an analyte.
  • the method may comprise translocating one or more analytes (e.g., a plurality of analytes, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more analytes).
  • the analyte may be translocated through a pore described herein (e.g., a nanopore disposed within a membrane).
  • the pore may be part of a nanopore system described herein.
  • at least a portion of analyte e.g., at least a portion of a protein, at least a portion WSGR Docket Number: 64828-710.601 of a polypeptide, at least a portion of a peptide, or a combination thereof
  • the rate can comprise an average rate of translocation through the pore.
  • the average rate of translocation may comprise an average of an analyte (e.g., a non-nucleic acid based polymer analyte) translocating through a pore one or more times.
  • the average rate of translocation may comprise an average of two or more analytes translocating through a pore one or more times.
  • a rate of translocation of an analyte through a pore may be expressed as amino acids per second (amino acids/sec) and/or nanometers/sec (nm/s).
  • a rate of translocation (e.g., an average rate of translocation) may comprise translocation of an analyte with a motor protein (e.g., a translocase) or without a motor protein.
  • an average rate of translocation can comprise at least about 0.1 amino acids/sec, at least about 0.5 amino acids/sec, at least about 0.6 amino acids/sec, at least about 0.7 amino acids/sec, at least about 0.8 amino acids/sec, at least about 0.9 amino acids/sec, at least about 1 amino acid/sec, at least about 2 amino acids/sec, at least about 3 amino acids/sec, at least about 4 amino acids/sec, at least about 5 amino acids/sec, at least about 6 amino acids/sec, at least about 7 amino acids/sec, at least about 8 amino acids/sec, at least about 9 amino acids/sec, at least about 10 amino acids/sec, at least about 11 amino acids/sec, at least about 12 amino acids/sec, at least about 13 amino acids/sec, at least about 14 amino acids/sec, at least about 15 amino acids/sec, at least about 16 amino acids/sec, at least about 17 amino acids/sec, at least about 18 amino acids/sec, at least about 19 amino acids/sec, at least about 20 amino acids/
  • an average rate of translocation can comprise at most about 50000 amino acids/sec, at most about 45000 amino acids/sec, at most about 40000 amino acids/sec, at most about 35000 amino acids/sec, at most about 30000 amino acids/sec, at most about 25000 amino acids/sec, at most about 20000 amino acids/sec, at most about 15000 amino acids/sec, at most about 10000 amino acids/sec, at most about 5000 amino acids/sec, at most about 1000 amino acids/sec, at most about 900 amino acids/sec, at most about 800 amino acids/sec, at most about 700 amino acids/sec, at most about 600 amino acids/sec, at most about 500 amino acids/sec, at most about 400 amino acids/sec, at most about 300 amino acids/sec, at most about 200 amino acids/sec, at most about WSGR Docket Number: 64828-710.601 100 amino acids/sec, at most about 90 amino acids/sec, at most about 80 amino acids/sec, at most about 70 amino acids/sec, at most about 60 amino acids/sec,
  • an average rate of translocation can be between about 0.1 amino acids/sec to about 100 amino acids/sec. In some embodiments, an average rate of translocation can comprise between about 0.1 amino acids/sec to about 0.5 amino acids/sec, about 0.1 amino acids/sec to about 1 amino acid/sec, about 0.1 amino acids/sec to about 10 amino acids/sec, about 0.1 amino acids/sec to about 20 amino acids/sec, about 0.1 amino acids/sec to about 30 amino acids/sec, about 0.1 amino acids/sec to about 40 amino acids/sec, about 0.1 amino acids/sec to about 50 amino acids/sec, about 0.1 amino acids/sec to about 60 amino acids/sec, about 0.1 amino acids/sec to about 70 amino acids/sec, about 0.1 amino acids/sec to about 80 amino acids/sec, about 0.1 amino acids/sec to about 100 amino acids/sec, about 0.5 amino acids/sec to about 1 amino acid/sec, about 0.5 amino acids/sec to about 10 amino acids/sec, about 0.5 amino acids/sec to about 20 amino acids/sec. In some embodiments,
  • an average rate of translocation can be between about 100 amino acids/sec to about 50,000 amino acids/sec. In some embodiments, an average rate of translocation can comprise between about 100 amino acids/sec to about 500 amino acids/sec, about 100 amino acids/sec to about 1,000 amino acids/sec, about 100 amino acids/sec to about 5,000 amino acids/sec, about 100 amino acids/sec to about 10,000 amino acids/sec, about 100 amino acids/sec to about 15,000 amino acids/sec, about 100 amino acids/sec to about 20,000 amino acids/sec, about 100 amino acids/sec to about 25,000 amino acids/sec, about 100 amino acids/sec to about 30,000 amino acids/sec, about 100 amino acids/sec to about 40,000 amino acids/sec, about 100 amino acids/sec to about 50,000 amino acids/sec, about 500 amino acids/sec to about 1,000 amino acids/sec, about 500 amino acids/sec to about 5,000 amino acids/sec, about 500 amino acids/sec to about 10,000 amino acids/sec, about 500 amino acids/sec to about 15,000 amino acids/sec, about 500 amino acids/sec, about 500 amino acids
  • an average rate of translocation can comprise at least about 0.01 nm/s, at least about 0.1 nm/s, at least about 0.5 nm/s, at least about 0.6 nm/s, at least about 0.7 nm/s, at least about 0.8 nm/s, at least about 0.9 nm/s, at least about 1 amino acid/sec, at least about 2 nm/s, at least about 3 nm/s, at least about 4 nm/s, at least about 5 nm/s, at least about 6 nm/s, at least about 7 nm/s, at least about 8 nm/s, at least about 9 nm/s, at least about 10 nm/s, at least about 11 nm/s, at least about 12 nm/s, at least about 13 nm/s, at least about 14 nm/s, at least about 15 nm/s, at least about 16 nm/s, at least
  • an average rate of translocation can comprise at most about 20000 nm/s, at most about 15000 nm/s, at most about 10000 nm/s, at most about 5000 nm/s, at most about 1000 nm/s, at most about 900 nm/s, at most about 800 nm/s, at most about 700 nm/s, at most about 600 nm/s, at most about 500 nm/s, at most about 400 nm/s, at most about 300 nm/s, at most about 200 nm/s, at most about 100 nm/s, at most about 90 nm/s, at most about 80 nm/s, at most about 70 nm/s, at most about 60 nm/s, at most about 50 nm/s, at most about 40 nm/s, at most about 30 nm/s, at most about 20 nm/s, at most about 19 nm/s, at most about 18 nm
  • an average rate of translocation can be between about 0.1 nm/s to about 10000 nm/s. In some embodiments, an average rate of translocation can be between about 0.1 nm/s to about 100 nm/s.
  • an average rate of translocation can comprise between about 0.1 nm/s to about 0.5 nm/s, about 0.1 nm/s to about 1 amino acid/sec, about 0.1 nm/s to about 10 nm/s, about 0.1 nm/s to about 20 nm/s, about 0.1 nm/s to about 30 nm/s, about 0.1 nm/s to about 40 nm/s, about 0.1 nm/s to about 50 nm/s, about 0.1 nm/s to about 60 nm/s, about 0.1 nm/s to about 70 nm/s, about 0.1 nm/s to about 80 nm/s, about 0.1 nm/s to about 100 nm/s, about 0.5 nm/s to about 1 amino acid/sec, about 0.5 nm/s to about 10 nm/s, about 0.5 nm/s to about 20 nm/s, about 0.5 nm/s
  • an average rate of translocation can comprise between about 100 nm/sec to about 20,000 nm/sec. In some embodiments, an average rate of translocation can comprise between about 100 nm/sec to about 200 nm/sec, about 100 nm/sec to about 300 nm/sec, about 100 nm/sec to about 400 nm/sec, about 100 nm/sec to about 500 nm/sec, about 100 nm/sec to about 1,000 nm/sec, about 100 nm/sec to about 2,500 nm/sec, about 100 nm/sec to about 5,000 nm/sec, about 100 nm/sec to about 7,500 nm/sec, about 100 nm/sec to about 10,000 nm/sec, about 100 nm/sec to about 15,000 nm/sec, about 100 nm/sec to about 20,000 WSGR Docket Number: 64828-710.601 nm/sec, about 200 nm/sec to about 300
  • a method may comprise detecting a current or change thereof.
  • a current or change thereof may be detected while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore).
  • a current or change thereof may be detected while at least a portion of an analyte translocates through a pore.
  • the method may comprise using a current or change thereof to determine one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte) and/or at least a portion of an analyte.
  • the current or change thereof may be used to determine a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics).
  • the characteristics of at least a portion of the analyte may comprise characteristics of an analyte described herein.
  • a method for determining a characteristic of an analyte comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a mem-brane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, at WSGR Docket Number: 64828-710.601 least a portion of a peptide, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino ac-ids per second to about 35000 amino acids per second or (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s; (b) detecting (1) a current or change thereof, or (2) voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the
  • the methods described herein may comprise translocating an additional analyte (e.g., an additional non-nucleic acid based polymer analyte) through a pore (e.g., a nanopore).
  • the additional analyte may be translocated through a same pore as a first analyte.
  • the additional analyte may be translocated through a different pore (e.g., an additional pore) as a first analyte.
  • an additional analyte comprises at least a portion of the additional analyte.
  • the at least a portion of the additional analyte can comprise a non-nucleic acid based polymer analyte (e.g., at least a portion of an additional protein, at least a portion of an additional polypeptide, at least a portion of an additional peptide, or a combination thereof).
  • at least a portion of the additional analyte may translocate through a pore described herein.
  • the additional analyte may translocate through a pore described herein with an average rate of translocation described herein.
  • an additional current or change thereof and/or an additional voltage or change thereof may be detected.
  • a characteristic of the additional analyte can comprise a shape of the additional analyte, a structure of the additional analyte, one or more mutations of the additional analyte, a sequence of the additional analyte, a surface charge of the additional analyte, one or more post-translation modifications of the additional analyte, or one or more ligands coupled to the additional analyte, or any combination thereof.
  • an analyte e.g., a non-nucleic acid based polymer analyte.
  • the analyte may be translocated through a pore described herein.
  • the pore may be disposed within a membrane.
  • the pore may be part of a nanopore system described herein.
  • at least a portion of an analyte e.g., a non-nucleic acid based polymer analyte
  • the at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or any combination thereof.
  • a signal or change WSGR Docket Number: 64828-710.601 thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected.
  • a signal or change thereof e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof
  • a signal or change thereof e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof
  • variants of the analyte may comprise one or more post-translational modifications (PTMs) and/or one or more conjugations to the analyte (e.g., drug conjugate, barcode, polynucleotide, or leader construct, or any combination thereof).
  • PTMs post-translational modifications
  • conjugations to the analyte e.g., drug conjugate, barcode, polynucleotide, or leader construct, or any combination thereof.
  • the one or more post-translational modifications may comprise naturally occurring PTMs, or non-naturally occurring PTMs, or any combination thereof.
  • a method for characterizing an analyte comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more fragments thereof, or one or more proteoforms thereof, or one or more variants thereof, or a combination thereof.
  • assigning can comprise measuring one or more characteristics, or quantitating one or more characteristics, or any combination thereof.
  • the one or more characteristics WSGR Docket Number: 64828-710.601 may be assigned by scoring a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof).
  • the signal or change thereof may be scored against the one or more reference signals and/or at least a portion of the one or more reference signals.
  • scoring may comprise aligning at least a portion of the signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof).
  • the signal or change thereof may be aligned with the one or more reference signals and/or at least a portion of the one or more reference signals.
  • the alignment may comprise soft alignment.
  • the methods provided herein may be repeated to generate a plurality of signals or changes thereof (e.g., electrical signals or changes thereof, currents or changes thereof, or any combination thereof).
  • a plurality of analytes may translocate through a plurality of nanopores.
  • the plurality of analytes may translocate through a plurality of nanopores disposed in membranes.
  • a plurality of signals (e.g., electrical signals, current signals, or any combination thereof) may be generated.
  • the plurality of signals (e.g., electrical signals, current signals, or any combination thereof) may be detected.
  • One or more characteristics may be assigned to the plurality of analytes (e.g., the plurality of non-nucleic acid based polymer analytes).
  • the one or more characteristics may be assigned based on the plurality of signals (e.g., electrical signals, current signals, or any combination thereof).
  • the one or more characteristics may be assigned based on the plurality of signals, a database, or any combination thereof.
  • a sample may be provided.
  • the sample can comprise an analyte.
  • the sample can comprise a plurality of analytes (e.g., a plurality of non-nucleic acid based polymer analytes). At least a subset of the plurality of analytes may undergo any of the methods and/or system disclosed herein.
  • the plurality of analytes can comprise a first analyte and a second analyte. In some embodiments, the plurality of analytes may be translocated.
  • the plurality of analytes may be translocated through a pore described herein (e.g., a pore disposed within a membrane).
  • a pore described herein e.g., a pore disposed within a membrane.
  • at least a portion of the analyte e.g., a first analyte and/or a second analyte
  • the at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or any combination thereof.
  • translocation of the analyte (e.g., the first analyte and/or the second analyte) through a pore may generate a signal or change thereof (e.g., a first current or change thereof and/or a second current or change thereof).
  • the first current or change thereof and/or the second current or change thereof may be detected.
  • the first current or change thereof and/or the second current or change thereof may be used to determine a characteristic (e.g., a first characteristic and/or a second characteristic).
  • the first current or change thereof and/or the second current or change thereof may be used to determine a WSGR Docket Number: 64828-710.601 plurality of characteristics.
  • At least a plurality of characteristics may be determined using the methods and/or system disclosed herein.
  • a sample may be analyzed by using the first characteristic and/or a second characteristic.
  • one or more properties of the sample may be characterized by using the first characteristic and/or a second characteristic.
  • a method for sample analysis can comprise: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, at least a portion of a first peptide, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, at least a portion of a second peptide, or a combination thereof; (c) detecting (i) (1) a
  • Characterizing one or more properties of the sample may comprise determining a feature.
  • the feature may be a feature of a proteome.
  • the proteome may be associated with the sample.
  • the proteome may refer to a set of proteins expressed by the organism from which the same may be derived.
  • Characterizing one or more properties of the sample may comprise a proteome coverage of a proteome.
  • Sequence coverage can refer to a number of sequencing reads that may be uniquely mapped to a reference sequence and may be applied to a known part of the genome.
  • a first nanopore and a second nanopore may be the same nanopore.
  • a first nanopore and a second nanopore may be different nanopores.
  • a first membrane and a second membrane may be the same membrane.
  • a first membrane and a second membrane may be different membranes.
  • One or more properties of a sample can comprise identification of a type of a sample (e.g., a type associated with the sample).
  • the sample type can comprise any sample type described herein.
  • One or more properties of a sample can comprise identification of an origin of a sample.
  • the sample origin can comprise any sample origin described herein.
  • one or more properties may comprise one or more dynamic changes in a sample.
  • Dynamic changes may be determined through continuous measurement of a sample (e.g., one or more analytes in a sample). Sensors of a nanopore system described herein may be exposed to the sample and detect changes as the sample changes over time in situ. Dynamic changes may be observed via continuous measurement of a sample. In some embodiments, dynamic changes may be observed via repeated measurements of a sample.
  • An interaction may be determined. The interaction can comprise an interaction of a first analyte and/or second analyte of a sample.
  • the interaction can comprise an interaction of a first analyte and/or second analyte with one or more molecules.
  • a sample e.g., a cell of a biological sample
  • a first analyte and/or a second analyte e.g., a first non-nucleic acid based polymer analyte and a second non-nucleic acid based polymer analyte
  • a plurality of analytes may have one or more interactions with one or more molecules (e.g., one or more polypeptides, one or more proteins, one or more peptides, one or more nucleic acids, or one or more small molecules, or any combination thereof).
  • a characterized property may comprise an interaction between the analyte and a bound entity (e.g., a leader construct, a recognition element, a PTM, or a conjugation, or any combination thereof).
  • the characterized property can provide information on the analyte (e.g., the bound entity) and the sample (e.g., the sample containing binding partners to at least the analyte).
  • characterizing properties of the sample may be performed through binding partners (e.g., binding molecules) to bind to and/or isolate an analyte from a sample.
  • the binding partners e.g., binding molecules
  • the binding partners may bind to an analyte via affinity binding.
  • the binding may be measured by extracting the analyte from the sample and translocating through a nanopore described herein.
  • the present disclosure provides pores for analyzing analytes.
  • a pore can be a biological pore.
  • a biological pore comprises a biological molecule.
  • a pore comprises a protein.
  • a pore comprises an assembly of proteins.
  • a pore comprises an assembly of subunits.
  • a pore comprises an assembly of fused proteins.
  • a pore comprises DNA.
  • a pore comprises a DNA origami structure.
  • a pore comprises a hybrid of DNA and peptides.
  • a pore comprises a G-quadruplex.
  • a pore comprises a polymer or a covalent organic framework.
  • a pore comprises a helical self-assembling pore or a chemically synthesized pore.
  • a pore comprises silicon, carbon, metal, metallic oxide, a metal-organic-framework, or a MXene.
  • a DNA based pore carries a large surface charge that can be used to create strong ion-selective electro-osmotic gradients.
  • a pore can be comprised in a membrane.
  • a pore comprises a toroidal protein.
  • a pore comprises a non- transmembrane protein.
  • a pore comprises cyclic peptides.
  • a pore comprises an assembly of cell penetrating molecules. In some embodiments, a pore comprises cell penetrating peptides. In some embodiments, a pore comprises portions of phage portal complexes. In some embodiments, a pore comprises portions of cellular transmembrane transport complexes. [0329] In some aspects, the present disclosure provides pores for detecting and/or characterizing an analyte (e.g., a biopolymer). A pore may be a wild-type pore and/or a pore may be an engineered pore. In some embodiments, the pore (e.g., nanopore) comprises a transmembrane region.
  • the pore comprises a hydrophilic portion. In some embodiments, the pore comprises a hydrophobic portion. In some embodiments, the pore comprises a hydrophilic and a hydrophobic portion. In some embodiments, a pore comprises an opening (e.g., an entrance). In some embodiments, a pore comprises at least one opening. In some embodiments, a pore can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) openings.
  • An entrance to a nanopore may be defined by a widest dimension (e.g., a measure from a first edge of an entrance to a second edge of the entrance). A pore may be measured by a diameter, or a circumference, or any combination thereof.
  • a pore can comprise a channel through which an analyte may enter.
  • the channel may be of the wild-type biological nanopore or the engineered biological nanopore.
  • an analyte may be trapped in the channel of the nanopore.
  • an analyte may translocate through the channel of the nanopore.
  • an analyte may partially translocate through the channel of the nanopore.
  • the channel may be a same width through the entire channel or a channel may have two or more different widths through the entire channel.
  • the channel may comprise at least one region.
  • the channel of the pore may comprise a first region, a second region, and/or a third region.
  • the channel of the nanopore comprises a constriction (e.g., a constriction region).
  • the constriction region may be a region of the channel different in size (e.g., width, length, diameter, circumference, or a widest dimension, or any combination thereof) than one or more other regions of the channel.
  • the second region of the channel may have the constriction region.
  • the first region and/or third region of the channel and the second region of the channel (e.g., comprising the constriction region) may be adjacent (e.g., immediately adjacent) to one another.
  • the first region and/or third region and second region of the channel may be separated by a distance of at most about 4.0 nm, at most about 3.0 nm, at most about 2.0 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, at most about 0.1 nm, or less than about 0.1 nm.
  • a first region and/or third region of the channel and a second region of the channel are separated by a distance of at least about 0.001 nm, at least about 0.01 nm, at least about 0.05 nm, at least about 0.1 nm, at least about 0.5 nm, at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm.
  • a first region and/or third region of the channel and a second region of the channel are separated by a distance of at most about 15 nm, at most about 10 nm, at most about 5 nm, at most about 4 nm, at most about 3 nm, at most about 2 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.1 nm, at most about 0.05 nm, at most about 0.01 nm, at most about 0.001 nm, or less than about 0.001 nm.
  • a first region and/or third region of the channel and a second region of the channel are separated by a distance from about 0.001 nm to about 15 nm. In some embodiments, a first region and/or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance from at least about 0.001 nm.
  • a first region and/or third region of the channel and a second region of the channel are separated by a distance from about 0.001 nm to about 0.01 nm, about 0.001 nm to about 0.05 nm, about 0.001 nm to about 0.1 nm, about 0.001 nm to about 0.5 nm, about 0.001 nm to about 1 nm, about 0.001 nm to about 2 nm, about 0.001 nm to about 3 nm, about 0.001 nm to about 4 nm, about 0.001 nm to about 5 nm, about 0.001 nm to about 10 nm, about 0.001 nm to about 15 nm, about 0.01 nm to about 0.05 nm, about 0.01 nm to about 0.1 nm, about 0.01 nm to about 0.5 nm, about 0.01 nm to about 1
  • the constriction region of the nanopore may be a narrower region of the channel than another region of the channel.
  • the constriction region of the nanopore can contribute to the electrical resistance of the nanopore.
  • a modulation of electrical resistance may allow the nanopore to differentiate between analytes in a complex sample. Therefore, modifying a constriction region of a nanopore to shift an electrical resistance may modulate the electro-osmotic force and/or may improve the ability of the nanopore to characterize an analyte. Characterization of an analyte may occur at the constriction region.
  • the current flow may be modulated most by the composition (e.g., local composition, e.g., amino acid composition) of the analyte within.
  • the electro-osmotic flow (EOF) may be maximally created at a narrow region (e.g., a constriction region).
  • the EOF may be maximally created at a constriction region due to a maximal electrostatic effect on cation or anion flux in the constrained dimensions of the constriction.
  • the nanopore comprises a shape (e.g., a geometry).
  • a nanopore may be cylindrical.
  • the nanopore can be conical shape.
  • the nanopore can be globular shape.
  • the nanopore can be hourglass shape. In some embodiments, the nanopore can be a toroidal shape, comprising a ring and a channel. In some embodiments, a nanopore comprises a biological nanopore or a solid state nanopore.
  • the toroidal shape may comprise a toroidal polyhedral shape comprising a ring and a channel.
  • the ring may comprise the protein or proteins that form the nanopore.
  • the ring may comprise a cross sectional geometry similar to the protein or proteins that form the nanopore.
  • the ring may be wider at a first side (e.g., a cis side) than a second side (e.g., a trans side), or wider at the second side (e.g., the trans side) than the first side (e.g., the cis side).
  • the ring can comprise a portion comprising a conical geometry, a cylindrical geometry, or an amorphous geometry, or combinations thereof.
  • the channel can comprise the central portion of the nanopore geometry that does not comprise the proteins or peptides of the nanopore.
  • the channel may allow molecules to translocate through the nanopore (i.e. through the channel). [0334]
  • a channel may restrict molecules from translocating through the nanopore.
  • the restriction may be based on a width of the channel or a charge of the channel.
  • the channel can comprise a channel length.
  • the channel length can be the length of the channel as measured along a longitudinal axis of the channel. This longitudinal axis may run perpendicular to a membrane.
  • the length may be measured perpendicular to the ring of the shape (e.g., the toroidal shape) of the geometry of the nanopore.
  • the channel length can be measured as the distance along the longitudinal axis of the channel between the most distant points of the nanopore along the longitudinal axis of the channel.
  • a channel may have a start point on a first side (e.g., WSGR Docket Number: 64828-710.601 a cis side) of a nanopore, and an end point on a second side (e.g., a trans side) of a nanopore, or a start point on a second side (e.g., a trans side) of a nanopore, and an end point on a first side (e.g., a cis side) of a nanopore.
  • a channel length can be less than a linear length or a contour length of an analyte. In some embodiments a channel length can be greater than a linear length or a contour length of an analyte.
  • a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from an alpha-carbon position of an amino acid backbone.
  • the dimension of the constriction region may be measured from a first alpha-carbon position to a second alpha-carbon position.
  • a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position of at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about 1.0 nm, at least about 1.1 nm, at least about 1.2 nm, at least about 1.3 nm, at least about 1.4 nm, at least about 1.5 nm, at least about 1.6 nm, at least about 1.7 nm, at least about 1.8 nm, at least about 1.9 nm, at least about 2.0 nm, at least about 2.1 nm, at least about 2.2 nm, at least about 2.3
  • a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position of at most about 4.0 nm, at most about 3.9 nm, at most about 3.8 nm, at most about 3.7 nm, at most about 3.6 nm, at most about 3.5 nm, at most about 3.4 nm, at most about 3.3 nm, at most about 3.2 nm, at most about 3.1 nm, at most about 3.0 nm, at most about 2.9 nm, at most about 2.8 nm, at most about 2.7 nm, at most about 2.6 nm, at most about 2.5 nm, at most about 2.4 nm, at most about 2.3 nm, at most about 2.2 nm, at most about 2.1 nm, at most about 2.0 nm, at most about 1.9
  • a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position from about 0.2 nm to about 4 nm.
  • a dimension e.g., diameter, circumference, and/or widest dimension
  • a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position from about 0.2 nm WSGR Docket Number: 64828-710.601 to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1.5 nm, about 0.2 nm to about 2 nm, about 0.2 nm to about 2.5 nm, about 0.2 nm to about 3 nm, about 0.2 nm to about 3.5 nm, about 0.2 nm to about 4 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 0.2 nm
  • a distance or dimension may be measured from an atom to a nearest atom of the side chain of the amino acid residue.
  • the side chain e.g., atom of the side chain
  • the side chain may protrude into the constriction region of the channel and/or constriction-forming portion of the monomer.
  • a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be at least about 0.0001 nm, at least about 0.0005 nm, at least about 0.001 nm, at least about 0.005 nm, at least about 0.01 nm, at least about 0.02nm, at least about 0.03 nm, at least about 0.04 nm, at least about 0.05 nm, at least about 0.06 nm, at least about 0.07 nm, at least about 0.08 nm, at least about 0.09 nm, at least about 0.1 nm, at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about
  • a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be at most about 2.0 nm, at most about 1.9 nm, at most about 1.8 nm, at most about 1.7 nm, at most about 1.6 nm, at most about 1.5 nm, at most about 1.4 nm, at most about 1.3 nm, at most about 1.2 nm, at most about 1.1 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, at most about 0.1 nm, at most about 0.09 nm, at most WSGR Docket Number: 64828-710.601 about
  • a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be from about 0.0001 nm to about 2 nm.
  • a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be from about 0.0001 nm to about 0.001 nm, about 0.0001 nm to about 0.005 nm, about 0.0001 nm to about 0.01 nm, about 0.0001 nm to about 0.05 nm, about 0.0001 nm to about 0.1 nm, about 0.0001 nm to about 0.2 nm, about 0.0001 nm to about 0.3 nm, about 0.0001 nm to about 0.4 nm, about 0.0001 nm to about 0.5 nm, about 0.0001 nm to about 1 nm, about 0.0001 nm to about 2 nm, about 0.001 nm to about 0.005 nm, about 0.001 nm to about 0.01 nm, about 0.001 nm to
  • a pore can be a non-biological pore. In some embodiments, a pore can be a solid state pore. [0340] In some embodiments, a pore can be a nanopore. In some embodiments, a pore comprises a width of at least about 0.5 nanometers (nm), 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.
  • nm nanometers
  • a pore comprises a width of at most about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, WSGR Docket Number: 64828-710.601 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.
  • a pore comprises a diameter of at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.
  • a pore comprises a diameter of at most about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.
  • a pore comprises an outer diameter of at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises an outer diameter of at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm.
  • a pore comprises an inner diameter of at least about 0.1 nm, 0.2, nm, 0.3 nm, 0.4 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.
  • a pore comprises an inner diameter of at most about 0.1 nm, 0.2, nm, 0.3 nm, 0.4 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.
  • an inner diameter can be a diameter of one or more constrictions (e.g., constriction regions) of a pore.
  • a pore can function at a temperature of at least about 4, 10, 20, 30, 40, 50, 60, 70, 80, or about 90 degrees Celsius. In some embodiments, a pore can function at a temperature of at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 degrees Celsius. [0342] In some embodiments, a pore can be disposed in a membrane. In some embodiments, a pore comprises transmembrane region. In some embodiments, a transmembrane region can be formed upon assembly of multiple transmembrane region sequences present in a plurality of subunits that together form a pore. In some embodiments the transmembrane region can be partially or fully composed of beta-strands.
  • the beta strands can partially or fully comprise amphipathic surfaces that can interface with an amphipathic membrane.
  • the transmembrane region can be partially or fully composed of alpha-helicases.
  • the alpha- helicases can partially or fully comprise amphipathic surfaces that can interface with the amphipathic membranes.
  • a transmembrane region sequence comprises an alternation of hydrophobic residues and hydrophilic residues.
  • a pore comprises a hydrophobic portion and a hydrophilic portion.
  • a transmembrane region of the pore comprises a hydrophobic portion.
  • a ring portion of a pore comprises a hydrophilic portion.
  • a pore comprises a protein that controls the translocation of an analyte (e.g. polypeptide or polynucleotide) across the pore.
  • a molecular motor can cause the translocation of an analyte across the pore.
  • the translocation of an analyte across the pore can be NTP-driven or ATP-driven.
  • the translocation of an analyte across the pore does not depend on NTP or ATP.
  • the translocation of an analyte across the pore does not depend on a molecular motor.
  • the pore can be a monomer. In some embodiments the pore can be formed from multiple monomeric units. In some embodiments, the monomers that comprise the oligomeric pore may be identical. In some embodiments the monomers that comprise the oligomeric pore may be different.
  • the nanopore may be an artificial biological nanopore.
  • the monomers of the nanopore may comprise proteins designed de novo (e.g., designed using machine learning algorithms). In some embodiments, portions of the nanopore (e.g., portions of the one or more monomers of the nanopore) may comprise proteins designed de novo (e.g., designed using machine learning algorithms).
  • the nanopore may comprise an assembly of monomers.
  • the nanopore may comprise a number of monomers. Monomers may be arranged vertically, horizontally, and/or layered as rings to form a nanopore described herein.
  • a nanopore e.g., biological nanopore
  • a nanopore comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, or greater than 50 monomers.
  • a nanopore e.g., biological nanopore
  • a nanopore comprises at most about 50, 40, 30, 25, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 monomers.
  • a nanopore may comprise 1 monomer.
  • a nanopore comprises from about 3 monomers to about 40 monomers.
  • a nanopore comprises from about 3 monomers to about 4 monomers, about 3 monomers to about 5 monomers, about 3 monomers to about 6 monomers, about 3 monomers to about 7 monomers, about 3 monomers to about 8 monomers, about 3 monomers to about 9 monomers, about 3 monomers to about 10 monomers, about 3 monomers to about 15 monomers, about 3 monomers to about 20 monomers, about 3 monomers to about 30 monomers, about 3 monomers to about 40 monomers, about 4 monomers to about 5 monomers, about 4 monomers to about 6 monomers, about 4 monomers to about 7 monomers, about 4 monomers to about 8 monomers, about 4 monomers to about 9 monomers, about 4 monomers to about 10 monomers, about 4 monomers to about 15 monomers, about 4 monomers to about 20 monomers, about 4 monomers to about 30 monomers, about 4 monomers to about 4 monomers to about 5 monomers, about 4 monomers to about 6 monomers, about 4 monomers to
  • the amino acid residues may form one or more rings of charges.
  • a pore may be engineered to contain regions of separate rings of charges along the longitudinal length of the channel.
  • a nanopore may be engineered to contain regions of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or greater than about 20 separate rings of charges along the longitudinal length of the channel.
  • a nanopore may be engineered to contain regions of at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or less than about 2 separate rings of charges along the longitudinal length of the channel.
  • a nanopore may be engineered to contain regions from about 2 to about 20 separate rings of charges along the longitudinal length of the channel.
  • a nanopore may be engineered to contain regions from about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 3 to about 15, about 3 to about 20, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 15, about 6 to about 20, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 15, about 7 to about 20, about 8 to about 9, about 8 to about 10, about 8 to about 15, about 8 to
  • a polypeptide can translocate through a pore at a rate of about 10 amino acids per second (aa/sec), 20 aa/sec, 30 aa/sec, 40 aa/sec, 50 aa/sec, 60 aa/sec, 70 aa/sec, 80 aa/sec, 90 aa/sec, 100 aa/sec, 110 aa/sec, 120 aa/sec, 130 aa/sec, 140 aa/sec, 150 aa/sec, 200 aa/sec, 250 aa/sec, 300 aa/sec, 350 aa/sec, 400 aa/sec, 450 aa/sec, or about 500 aa/sec.
  • a polypeptide can translocate through a pore at a rate greater than about 10 aa/sec, 20 aa/sec, 30 aa/sec, 40 aa/sec, 50 aa/sec, 60 aa/sec, 70 aa/sec, 80 aa/sec, 90 aa/sec, 100 aa/sec, 110 aa/sec, 120 aa/sec, 130 aa/sec, 140 aa/sec, 150 aa/sec, 200 aa/sec, 250 aa/sec, 300 aa/sec, 350 aa/sec, 400 aa/sec, 450 aa/sec, or about 500 aa/sec.
  • a polypeptide can translocate through a pore at a rate less than about 10 aa/sec, 20 aa/sec, 30 aa/sec, 40 aa/sec, 50 aa/sec, 60 aa/sec, 70 aa/sec, 80 aa/sec, 90 aa/sec, 100 aa/sec, 110 aa/sec, 120 aa/sec, 130 aa/sec, 140 aa/sec, 150 aa/sec, 200 aa/sec, 250 aa/sec, 300 aa/sec, 350 aa/sec, 400 aa/sec, 450 aa/sec, or about 500 aa/sec.
  • a pore can be configured to provide a condition for dominant electro-osmotic capture of an analyte.
  • a nanopore system e.g., an Aerolysin nanopore system or another nanopore system described herein
  • low pH conditions may increase the net positive charge inside the pore channel, resulting in (i) increased anion selectivity, (ii) a strong net anion-selective pore, or (iii) an increased electrostatic repulsion of mostly positively charged analytes, or (iv) any combination thereof.
  • Low pH can comprise a pH of at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, or less than about 1.
  • the resulting strong electro-osmotic flux through the pore can be exploited to capture analytes against the direction of the electrophoretic forces (EPF) acting upon them (e.g. with a positive applied potential at the trans electrode for a system with mostly positively charged peptides in the cis solution). It can be advantageous to exploit electro-osmotic forces to capture analytes since it can be less sensitive to charge composition. It can be advantageous for capturing and/or detecting a diverse composition of unlabeled peptides (e.g. neutral, net positive, net negative).
  • EPF electrophoretic forces
  • an electro-osmotic force may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an electro-osmotic force may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an electrophoretic force may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an electrophoretic force may be in a second side (e.g., trans side) to first side (e.g., cis side) direction.
  • an EOF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction and an EPF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction.
  • an EOF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction and an EPF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction.
  • an EOF may be in a second side (e.g., trans side) to WSGR Docket Number: 64828-710.601 first side (e.g., cis side) direction and an EPF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction.
  • an EOF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction and an EPF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction.
  • a pore (e.g., nanopore) described herein can comprise one or more mutations.
  • the mutation may be a substitution, an insertion, a deletion, or a chemical modification, or any combination thereof.
  • the mutation may comprise a positively-charged amino acid residue, or a negatively-charged amino acid reside, or any combination thereof.
  • the electro-osmotic force can be reduced to increase the duration for which the analytes may be retained in the pore.
  • the anion ion-selectivity bias and resulting net anionic electro-osmotic flux that results from low pH conditions can be reduced by introducing acidic residues (e.g., by substitution adjacent to the aromatic mutations). Acidic mutation substitutions that reduce net positive charge can reduce electrostatic repulsion of mostly positively charge analytes. Net positive charge can also be reduced by replacing basic residues with neutral or acidic residues.
  • one or more mutations of negatively charged amino acid residues may increase a negative charge of a pore described herein. The increased negative charge may increase a duration that a positively-charged analyte resides in the pore.
  • one or more mutations of positively charged amino acid residues may increase a positively charge of a pore described herein.
  • the increased positive charge may increase a duration that a negatively- charged analyte resides in the pore.
  • one or more aromatic mutations e.g., insertion of one or more aromatic amino acid residues and/or substitution of one or more aromatic amino acid residues
  • the one or more aromatic mutations may decrease a diameter of a nanopore described herein due to the bulky side chain configuration of aromatic amino acid residues.
  • Mutations of one or more amino acid residues in a constriction region and/or lumen-facing region of a nanopore described herein may provide for greater accuracy of determining one or more characteristics of an analyte.
  • one or more aromatic mutations e.g., insertion of one or more aromatic amino acid residues and/or substitution of one or more aromatic amino acid residues
  • a membrane can provide a partition for providing a voltage difference between a first side (e.g., cis side) and a second side (e.g., trans side) of a pore.
  • an EOF can result from a net ionic current flow cis to trans.
  • a cis to trans EOF results from a net ionic current flow cis to trans over a total ionic current flow, also referred to as a relative net current flow cis to trans, of greater than about 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, or about 0.99.
  • a cis to trans EOF results from a net ionic current flow trans to cis over a total ionic current flow, also referred WSGR Docket Number: 64828-710.601 to as a relative net current flow cis to trans, of less than about 0.0, -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, - 0.9, -0.95, or about -0.99.
  • the electro-osmotic force comprises a net ionic current flow from the first side (e.g., cis side) to the second side (e.g., trans side).
  • the electro-osmotic force can be modulated by a pH, a type of a salt, a concentration of a salt, an osmotic pressure across the membrane of the system, or a modification of the nanopore, or any combinations thereof.
  • the electro- osmotic force can be modulated by a modification of a charge of the nanopore.
  • the electro-osmotic force can be modulated by an asymmetric salt distribution between the first side (e.g., cis side) and second side (e.g., trans side) of the membrane.
  • the electro-osmotic force can be modulated by modification of a charge of the nanopore.
  • a pore can be configured to provide a dominant EOF in the direction cis to trans across the membrane of a pore system. In some embodiments, a pore can be configured to provide a dominant EOF in the direction trans to cis across the membrane of a pore system. In some embodiments, a pore can be configured to provide an EOF that acts against an electrophoretic force (EPF) across the membrane of a pore system. As shown in FIGs. 1A-1C, nanopores can have a strong net Electro-Osmotic Force (EOF) in the direction cis-to-trans across a membrane as indicated by the arrow.
  • EEF Electro-Osmotic Force
  • the Electrophoretic Forces (EPF) acting on the analyte can depend on the composition of charges on the analyte in the sections in and near the nanopore channel, and therefore can sometimes act in the net direction cis-to-trans or trans-to-cis.
  • a strong and dominant cis-to-trans EOF can cause the capture, stretching, and efficient translocation of long polymer analytes from the cis compartment to the trans compartment regardless of the net direction of the EPF.
  • the net flow can arise from a large cis-to-trans ion flow dominating over any trans-to-cis ion flows (e.g., a lower flow or counter- charged ions under an applied potential).
  • a positive charge may be applied to a compartment of the nanopore system (e.g., a trans compartment). With a positive voltage applied to a compartment (e.g., a trans compartment) across the membrane, nanopores with net positive internal charge may be used to limit the flow of cations from trans to cis.
  • a negative charge may be applied to a compartment of the nanopore system (e.g., a trans compartment). With a negative voltage applied to a compartment of the nanopore system (e.g., a trans compartment) across the membrane, nanopores with net negative internal charge may be used to limit the flow of anions from trans to cis.
  • a pore can be configured to provide an EOF that acts with an EPF across the membrane of a pore system.
  • a pore can comprise a relative ion selectivity P(+)/P(-) of greater than about 5.0 or less than about 0.1.
  • a pore can comprise a relative ion selectivity P(+)/P(-) of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0.
  • P(+)/P(-) of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0.
  • a pore can comprise a WSGR Docket Number: 64828-710.601 relative ion selectivity P(+)/P(-) of less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0.
  • a pore can comprise a relative ion selectivity P (+) /P (-) of at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or greater than about 5 under an applied voltage difference across the membrane.
  • P (+) /P (-) of at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or greater than about 5 under an applied voltage difference across the membrane.
  • a pore can comprise a relative ion selectivity P (+) /P (-) of at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, at most about 0.5, at most about 0.4, at most about 0.3, at most about 0.2, at most about 0.1, or less than about 0.1 under an applied voltage difference across the membrane.
  • a pore can comprise a relative ion selectivity P(+)/P(-) from about 0.1 to about 5 under an applied voltage difference across the membrane.
  • a pore can comprise a relative ion selectivity P(+)/P(-) from about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 to about 0.5, about 0.1 to about 1, about 0.1 to about 1.5, about 0.1 to about 2, about 0.1 to about 2.5, about 0.1 to about 3, about 0.1 to about 4, about 0.1 to about 5, about 0.2 to about 0.3, about 0.2 to about 0.4, about 0.2 to about 0.5, about 0.2 to about 1, about 0.2 to about 1.5, about 0.2 to about 2, about 0.2 to about 2.5, about 0.2 to about 3, about 0.2 to about 4, about 0.2 to about 5, about 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 1, about 0.3 to about 1.5, about 0.3 to about 2, about 0.3 to about 2.5, about 0.3 to about 3, about 0.3 to about 4, about 0.3 to about 5, about 0.4 to about 0.5, about 0.3 to about 1, about 0.3 to about 1.5, about 0.3 to about
  • a pore can comprise a relative ion selectivity P (+) /P (-) of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5 under an applied voltage difference across the membrane.
  • a pore comprises a lumen.
  • a nanopore lumen comprises a net charge of at least about 2 coulombs, at least about 3 coulombs, at least about 4 coulombs, at least about 5 coulombs, at least about 10 coulombs, at least about 15 coulombs, at least about 20 coulombs, at least about 25 coulombs, at least about 30 coulombs, at least about 35 coulombs, at least about 40 coulombs, at least about 45 WSGR Docket Number: 64828-710.601 coulombs, at least about 50 coulombs, at least about 55 coulombs, at least about 60 coulombs, at least about 70 coulombs, at least about 80 coulombs, at least about 90 coulombs, at least about 100 coulombs, at least about 150 coulombs, at least about 200 coulombs, or greater than about 200 coulombs.
  • a nanopore lumen comprises a net charge of at most about 200 coulombs, at most about 150 coulombs, at most about 100 coulombs, at most about 90 coulombs, at most about 80 coulombs, at most about 70 coulombs, at most about 60 coulombs, at most about 55 coulombs, at most about 50 coulombs, at most about 45 coulombs, at most about 40 coulombs, at most about 35 coulombs, at most about 30 coulombs, at most about 25 coulombs, at most about 20 coulombs, at most about 15 coulombs, at most about 10 coulombs, at most about 5 coulombs, at most about 4 coulombs, at most about 3 coulombs, at most about 2 coulombs, or less than about 2 coulombs.
  • a nanopore lumen comprises a net charge from about 2 to about 200 coulombs. In some embodiments, a nanopore lumen comprises a net charge from at most about 200. In some embodiments, a nanopore lumen comprises a net charge from about 2 to about 5, about 2 to about 10, about 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 75, about 2 to about 100, about 2 to about 125, about 2 to about 150, about 2 to about 200, about 5 to about 10, about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 5 to about 75, about 5 to about 100, about 5 to about 125, about 5 to about 150, about 5 to about 200, about 10 to about 20, about 10 to about 30, about 10 to about 40, about 10 to about 50, about 10 to about 75, about 10 to about 100, about 10 to about 125, about 10 to about 150, about 10 to about 200, about 20 to about 30, about 20 to about 40, about 20 to about 20 to about
  • a pore lumen comprises a net positive charge. In some embodiments, a pore lumen comprises a net negative charge.
  • a pore comprises a recognition region.
  • a recognition region can be identified structurally by the dimensions of the central channel, e.g., X-ray diffraction structures, electron-microscopy structures, and/or computer modeling (e.g., molecular modeling, or homology modeling, or any combination thereof).
  • a recognition region may be a region where electric field lines concentrate.
  • a recognition region may be where a presence of an analyte disrupts the most the ionic current flowing through a pore, e.g., under an applied potential.
  • a recognition region may comprise one or more narrow cross-sections of a pore channel, WSGR Docket Number: 64828-710.601 which can be, e.g., a diameter of less than 2 nanometers or 1 nanometer.
  • a pore can be engineered to have one or more narrow sections of an internal diameters (constrictions) within the recognition region, which can provide higher sensitivity/ionic current deflection to analytes.
  • a pore can be engineered to provide longer or shorter residence time of analytes.
  • a lumen-facing recognition region of a pore can be engineered (by one or more natural or non-natural amino acid substitutions, deletions, or modifications) to manipulate the internal dimensions, hydrophobicity, or aromaticity, or combinations thereof, of the pore.
  • engineering the lumen-facing recognition region of the pore increases the dwell time and/or resolution for peptides traversing the pore.
  • a lumen-facing recognition region of a pore can be engineered to decrease a translocation speed of an analyte through a pore.
  • a lumen- facing region of the pore can be engineered to reduce the analyte interacting or binding to the pore lumen.
  • a lumen-facing region of the pore can be engineered to increase the analyte interacting or binding to the pore lumen.
  • a lumen-facing recognition region of a pore can be engineered by modifying outwards facing residues to perturb the nearby lumen-facing residues.
  • a charge in a channel of a pore can adapted to alter the selectivity of the pore.
  • a pore may be modified by one or more mutations.
  • a mutation comprises one or more point mutations.
  • a point mutation can be at a non-conserved position.
  • a point mutation can be a lumen-facing mutation.
  • a point mutation can be a membrane-facing mutation. In some embodiments, a point mutation can alter a characteristic of a pore. In some embodiments, a point mutation can alter a pore channel charge, conductance at a set pH, ion selectivity, electro-osmotic flux, conductivity, shape, or structure, or combinations thereof. In some embodiments, a point mutation can allow for a conductance or analyte translation at a pH of less than about 1, 2, 3, 3.8, 4, 4.5, 6, 7, 8, 9, 1011, 12, 13, or about 14.
  • a point mutation can allow for a conductance or analyte translation at a pH of greater than about 1, 2, 3, 3.8, 4, 4.5, 6, 7, 8, 9, 1011, 12, 13, or about 14.
  • one or more point mutations may affect a diameter of a pore described herein.
  • the one or more point mutations may modulate (e.g., widen or narrow) a diameter of a constriction region of a pore described herein.
  • the constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from an alpha-carbon position of an amino acid backbone.
  • the dimension of the constriction region may be measured from a first alpha-carbon position to a second alpha-carbon position.
  • one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter of at least about 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 1.0 nm, 1.5 nm, 2.0 nm, 2.5 nm, 3.0 nm, 3.5 nm, 4.0 nm, 4.5 nm, 5.0 nm, or greater than about 5.0 nm.
  • one or more point mutations may modulate a constriction region of a nanopore to comprise a WSGR Docket Number: 64828-710.601 diameter of at most about 5.0 nm, at most about 4.5 nm, at most about 4.0 nm, at most about 3.5 nm, at most about 3.0 nm, at most about 2.5 nm, at most about 2.0 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, or less than about 0.2 nm.
  • one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter between about 0.2 nm to about 5 nm. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter between about 0.2 nm to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1.5 nm, about 0.2 nm to about 2 nm, about 0.2 nm to about 2.5 nm, about 0.2 nm to about 3 nm, about 0.2 nm to about 3.5 nm, about 0.2 nm to about 4 nm, about 0.2 nm to about 5 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 5
  • a charge in a channel of a pore can be adapted to alter an EOF.
  • a charge in a channel of a pore can be altered by changing the type of charged residue, the location of a charge, or the dimensions of the pore, or combinations thereof.
  • a pore can be modified to create a high overlap between Debye layers (alternatively termed Stern layers, the Gouy-Chapman diffuse layer or the electric double layer) or double layers to create energy barriers to limit the flow of a specific ion.
  • increased positive charge in the pore channel can increase transport of anionic species and/or decrease the transport of cationic species.
  • increased negative charge in the pore channel can increase transport of cationic species and/or decreases the transport of anionic species. This can, in turn, alter the net electro-osmotic flux of hydrated ions flowing through the pore under an applied WSGR Docket Number: 64828-710.601 potential.
  • electro-osmotic forces may act against an electrophoretic force during analyte capture.
  • electro-osmotic forces may dominate an electrophoretic force during analyte capture.
  • the anion ion-selectivity bias and resulting net anionic electro-osmotic flux can be reduced by introducing acidic residues by substitution adjacent to the aromatic mutations.
  • net positive charge can be also reduced by replacing basic residues with neutral or acidic residue(s), optionally by substitution with aromatic residue(s) that also separately and additively improve peptide capture and/or discrimination (e.g. CytK-K128F and Aer-K238F).
  • a pore may be engineered to contain regions of 1, 2, 3, 4, 5, 6, or more separate rings of charges along the longitudinal length of the channel.
  • a pore comprising a point mutation described herein can have an open pore current of at least about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA.
  • a pore comprising a point mutation described herein can have an open pore current of at least about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA at a pH of less than about 2, 3, 3.8, 4, 4.5, 7, 7.5, or about 8.
  • a pore comprising a point mutation described herein can have an open pore current of at most about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA.
  • a pore comprising a point mutation described herein can have an open pore current of at most about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA at a pH of less than about 2, 3, 3.8, 4, 4.5, 7, 7.5, or 8.
  • a pore comprises an aromatic amino acid within a lumen of the pore.
  • a pore comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 aromatic amino acids within a lumen of the pore. In some embodiments, a pore comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 aromatic amino acids within a lumen of the pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases an analyte interaction with a lumen of a pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases an analyte residence time withing a pore.
  • a pore comprises a monomer.
  • a pore comprises a dimer, a trimer, a tetramer, pentamer, a hexamer, a heptamer, an octamer, a nonamer, a decamer, an undecamer, or a dodecamer .
  • a pore comprises an oligomer.
  • a pore comprise a homo-oligomer or a hetero-oligomer.
  • a pore can comprise a plurality of subunits.
  • a pore comprises several repeating subunits, such as at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • a pore comprises several repeating subunits, such as at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • a plurality of subunits of a pore may be axially aligned.
  • a plurality of subunits of a pore comprise an approximately central axis.
  • a pore comprises a channel through which an ion can flow.
  • a pore comprises a plurality of subunits. In some embodiments, a pore comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 subunits. In some embodiments, a pore comprises at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 subunits. In some embodiments, a pore comprises a ring of multiple identical mutations. In some embodiments, a pore comprises a ring of multiple identical mutations in a recognition region that can be co-planar with a membrane and orthogonal to the direction of analyte passage. In some embodiments, a pore comprises different mutations for its subunits to comprise a hetero-oligomeric assembly.
  • a pore comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more mutant monomers.
  • the number of mutated units can be adapted to modulate the extent/magnitude of the change to the recognition region.
  • a pore comprises mutations on one or more beta-strands.
  • beta-strands can comprise mutations either in the down strand or in the up strand of a beta strand, or in both.
  • the pore comprises mutations to one or more alpha-helices.
  • a mutation can be inward or lumen facing.
  • a mutation can be outward or membrane facing.
  • a pore comprises a plurality of protomers.
  • a plurality of protomers can be comprised in an assembly that forms at least a portion of the pore, wherein the protomers form a channel region of the pore.
  • a plurality of protomers can comprise identical sequences or different sequences.
  • a pore comprises a plurality of mixed protomers.
  • a plurality of protomers may be separated from a pore.
  • a plurality of protomers may be fused to a pore.
  • a pore lumen comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 charged amino acids pointing towards the lumen of the pore per protomer.
  • a pore lumen comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 charged amino acids pointing towards the lumen of the pore per protomer.
  • a pore lumen comprises a plurality of negatively charged amino acids.
  • a pore lumen comprises a plurality of positively charged amino acids.
  • a pore can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, or about 100 individual proteins.
  • a pore can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, or about 100 individual proteins.
  • a pore can comprise charged residues either in rings around the pore in plane with the membrane, and/or vertically up the pore channel perpendicular to plane of the membrane.
  • a pore comprises charged residues at a pore entry.
  • a pore comprises charged residues at a pore exit.
  • a pore comprises charged residues at a pore constriction.
  • charged residues can comprise Asp, Glu, Arg, Lys, His, or non-natural amino acids.
  • a pore can be configured to detect analytes larger than 40 kDa.
  • the size of a pore opening, channel, or constriction region, or any combination thereof, may be large enough to accommodate a large analyte (e.g., an analyte larger than 40 kDa).
  • a pore can be configured to detect analytes larger than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or about 10000 kDa.
  • a pore can be configured to detect analytes smaller than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or about 10000 kDa.
  • a pore comprises a cylindrically-shaped region.
  • a pore comprises a cone-shaped region.
  • a pore comprises a cylindrical chamber on a second side (e.g., trans side). In some embodiments, a pore comprises a cone chamber on a first side (e.g., cis side). In some embodiments, a pore comprises a cylindrical chamber on a second side (e.g., trans side) and a cone chamber on a first side (e.g., cis side) separated by an inner constriction. In some embodiments a pore comprises an hourglass shape. In some embodiments a pore comprises a cone shape on a first side (e.g., cis side) and a cone shape on a second side (e.g., trans side), separated by an inner constriction.
  • a pore comprises an affinity tag, e.g., a His-tag or Strep-tag.
  • an affinity tag can be appended to a N- or C-terminus of a pore or a subunit thereof.
  • an affinity tag can be appended to a pore or a subunit thereof via a linker, e.g., a GSA linker.
  • An affinity tag may comprise a Glutathione-S-transferase (GST) tag, a Maltose-Binding Protein (MBP) tag, a FLAG tag, a c-myc tag, a hemagglutinin (HA) tag, a T7 tag, a calmodulin-binding peptide (CBP) tag, a biotinylation tag, or any combination thereof.
  • GST Glutathione-S-transferase
  • MBP Maltose-Binding Protein
  • FLAG tag e.g., a Maltose-Binding Protein
  • HA hemagglutinin
  • T7 a calmodulin-binding peptide
  • CBP calmodulin-binding peptide
  • biotinylation tag or any combination thereof.
  • a linker can comprise (GGGGS)n and/or (SG)n, where n may comprise any integer (e.g., 1, 2,
  • a linker described herein may comprise (GGGGS)3, (Gly)8, (Gly)6, (EAAAK)3, (EAAAK)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE, WSGR Docket Number: 64828-710.601 AGNRVRRSVG, RRRRRRR, GFLG, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, disulfide bond, or cysteine linkages, or any combination thereof.
  • Gated nanopores can comprise nanopores described herein that may act as rapid, closable channels to gate transport of one or more analytes across a membrane.
  • a nanopore may exhibit spontaneous gating, in which the channel may not be open for an analyte to enter, translocate through, or any combination thereof.
  • a pore may exhibit no spontaneous gating for a period of at least about 10, 20, 30, or 60 s at an applied potential of less than about -50 mV or greater than about 50mV.
  • a pore may exhibit no spontaneous gating for a period of at most about 10, 20, 30, or 60 s at an applied potential of -50 mV.
  • a pore may exhibit no spontaneous gating for a period of at least about 5, 10, 15, or 20s at an applied potential of -150 mV. In some embodiments, a pore may exhibit no spontaneous gating for a period of at most about 5, 10, 15, or 20s at an applied potential of -150 mV. [0370] In some embodiments, a pore comprises residues configured to anchor the pore to a membrane. In some embodiments, a pore comprises a mutation that increases the solubility of the pore or a subunit thereof in a lipid bilayer. In some embodiments, a pore comprises a mutation that increases the solubility of the pore or a subunit thereof in a lipid bilayer in an external water environment.
  • a pore comprises residues configured to improve insertion efficiency into a membrane. In some embodiments, a pore comprises residues configured to reduce spontaneous gating. In some embodiments, a pore comprises residues configured to reduce signal noise. The signal noise can be noise in a signal comprising ionic current, impedance, current rectification, potential, tunnelling, conductivity, light, or mechanical deformation. [0371] In some embodiments, a pore comprises a protease. In some embodiments, a protease can be configured to degrade a polypeptide into a fragment. In some embodiments, one or more proteases degrade a polypeptide into one or more fragments.
  • a fragment enters a channel of a pore.
  • a polypeptide fragment comprises a portion comprising a non-natural amino acid, polyethylene glycol, PNA, DNA, or RNA, or combinations thereof.
  • a protease can be active.
  • a protease can be inactive.
  • a polypeptide can be degraded into a fragment before translocation through a pore.
  • a pore comprises an unfoldase.
  • an unfoldase linearizes a polymer which enters the pore.
  • an unfoldase comprises a prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, or Valosin-containing protein-like ATPase.
  • an unfoldase may be modulated by an adaptor protein or other accessory proteins or chaperones.
  • the AAA+ enzyme (e.g., AAA+ unfoldase) is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, WSGR Docket Number: 64828-710.601 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, orthologs, paralogs thereof.
  • a pore comprises a ring-forming protein.
  • a ring-forming protein can be configured to transport a polymer, e.g.
  • a ring-forming protein comprises a toroidal or donut-shaped multi-subunit protein.
  • a ring-forming protein comprises a ring-forming multimeric protein, such as an octameric, heptameric or hexameric protein.
  • a ring-forming protein comprises a heptameric protein.
  • a heptameric protein include those submitted to the Protein Data Bank (PDB) under one of the following unique accession or identification code codes: lg31, 1h64, 1hx5, 1i4k, 1i5l, 1i8f, 1i81, 1iok, 1j2p, 1jri, 1lep, 1lnx, 1loj, 1mgq, 1n9s, 1ny6, 1p3h, 1tzo, 1wnr, 1xck, 2cb4, 2cby, 2yf2, 3bpd, 3cf0, 3j83, 3ktj, 3m0e, 3st9, 4b0f, 4emg, 4gm2, 4hnk, 4hw9, 4jcq, 4ki8, 4owk, 4qhs, 4xq3, 5jzh, 5msj, 5msk, 5mx5 and 5uw8e.
  • PDB Protein Data Bank
  • a transmembrane portion of a pore comprises a linker.
  • the linker may be a helical linker, a non-helical linker, a flexible linker, or a cleavable linker, or any combination thereof.
  • a linker can by hydrophilic or mostly hydrophilic.
  • a transmembrane portion of a pore comprises a flexible hydrophilic linker.
  • a flexible linker can comprise a linker rich in small and/or polar amino acids (e.g., glycine, serine, or threonine, or any combination thereof), which can provide good flexibility and solubility.
  • a transmembrane portion can comprise on the N-and/or C-terminal side a flexible hydrophilic linker of at least about 3, 4, 5, 10, 15, or 20 amino acids.
  • a transmembrane portion can comprise on the N-and/or C-terminal side a flexible hydrophilic linker of at most about 3, 4, 5, 10, 15, or 20 amino acids.
  • a hydrophilic linker can interact with the charged head groups of membrane (phospho)lipids.
  • hydrophilic residues include serine, threonine, asparagine, glutamine, aspartate, glutamate, lysine and arginine.
  • a hydrophilic linker comprises at least a portion comprising non-hydrophilic or non-natural amino acids to tune the linker’s properties.
  • a pore comprises a protease.
  • a pore comprises a protease- unfoldase pair.
  • a protease-unfoldase pair can be attached on a first side (e.g., cis side) of a FraC pore. Then, cleaved peptides can be sequentially recognized and translocated across the pore.
  • a protease-unfoldase pair can comprise a barrel-shaped ATP-dependent ClpXP protease.
  • a protease can encase digested peptides, preventing their release in solution.
  • a pore can be configured to proteolytically cleave analytes.
  • a pore can be configured to not to proteolytically cleave analytes.
  • the conditions of a pore and/or nanopore system may comprise ATP concentration, or buffer types, or any combination thereof. The conditions of the pore and/or nanopore system can be configured to cleave or not to cleave analytes.
  • VAT WSGR Docket Number: 64828-710.601 can be capable of feeding the polypeptide through the pore at a speed that can be tuned by the concentration of ATP.
  • a transmembrane proteasome can simultaneously process and identify different analytes.
  • translocated peptides may be proteolytically degraded.
  • the pore can be employed with an inactivated protease/proteasome which recognizes proteins as they are linearized and transported across the pore at a controlled speed.
  • the activity of the protease/proteasome can be monitored at the single molecule level.
  • translocated peptides may not be proteolytically degraded.
  • a proteasome and/or a portion of a proteasome may comprise a protease domain, or a translocase domain, or any combination thereof.
  • a pore comprises natural or non-natural aromatic amino acid residues.
  • a non-natural aromatic amino acid can be selected from the group consisting of 3,4-dihydroxy- L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr).
  • a non-natural aromatic amino acid can be a D-amino acid, a Homo-amino acid (methylene), a Beta-homo-amino acid, a N-methyl amino acid, or an Alpha-methyl amino acid.
  • a non-natural aromatic amino acid can be a derivatized Phe/Tyr/Trp amino acid, e.g., a ring- substituted Phe/Tyr/Trp amino acids.
  • a non-natural aromatic amino acid can be a derivative of Phe, Tyr or Trp, substituted by, e.g., a halogen, -CH 3 , OH, -CH 2 NH 3 , -C(O)H, -CH 2 CH 3 ,-CN, - CH2CH2CH3, -SH, or another group.
  • Non-natural aromatic amino acids include, but are not limited to, O- methyl-L-tyrosine; 3-methyl- phenylalanine; a p-acetyl-L-phenylalanine; O-4-allyl-L-tyrosine; 4-propyl-L- tyrosine; fluorinated phenylalanine; isopropyl-L-phenylalanine; ap-azido-L- phenylalanine; a p-acyl-L- phenylalanine; a p-benzoyl-L-phenylalanine; a phosphonotyrosine; a p-iodo-phenylalanine; p- bromophenylalanine; p- amino-L-phenylalanine; an isopropyl-L-phenylalanine; an amino-, isopropyl-, or O- allyl-containing phenylalanine analogue; a
  • a non-naturally-occurring amino acids may be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express a mutant monomer.
  • a non- naturally-occurring amino acids may be introduced by expressing a mutant monomer in E. coli that may be auxotrophic for specific amino acids in the presence of synthetic (i.e. non-naturally-occurring) analogues of those specific amino acids.
  • a non-naturally-occurring amino acids may be introduced using synthetic peptide chemistry methods. Monomeric units of the pores may be formed entirely from synthetic peptides constructed using conjugation methods, e.g., chemical ligation, or cysteine coupling.
  • monomers of the pore may comprise partially synthetic units coupled to naturally expressed peptide units using coupling methods.
  • WSGR Docket Number: 64828-710.601 [0379]
  • a pore comprises an alpha-helical or beta-barrel oligomeric pore forming toxin or porin.
  • a pore can comprise a beta-barrel pore forming protein and/or peptide and/or an alpha helical pore forming protein and/or peptide.
  • a pore can be selected from the group consisting of Aerolysin (Aer), Cytolysin K (CytK), Mycobacterium smegmatis porin A (MspA), alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, phage derived portal proteins (Phi29, or G20c, or any combination thereof) or a mutant thereof.
  • a pore comprises a phage portal complex, or a cellular transmembrane transport complex.
  • a pore comprises an alpha-helix bundle or channel.
  • a pore comprises a transmembrane protein pore derived from beta-barrel pores or alpha- helix bundle pores.
  • a beta-barrel pore or beta-barrel pore forming protein and/or peptide comprises a barrel or channel structure comprising beta-strands.
  • a pore comprises charged residues on both the “up” strands and the “down” of a beta-strand.
  • charged residues can be located at sequence positions that co-locate them approximately co-planar in a beta-barrel.
  • the nanopore comprises a pore-forming toxin.
  • the nanopore can comprise an ⁇ - pore-forming toxin, or a ⁇ -pore-forming toxin, or any combination thereof.
  • the nanopore can comprise a pore- forming toxin derived from a bacterium.
  • the bacterium can be of a genus of bacteria including, but not limited to, Xenorhabdus, Yersinia, Providencia, Pseudomonas, Proteus, Morganella, or Photorhabdus.
  • the nanopore comprises a pore-forming toxin derived from a bacterial species selected from the group consisting of Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus, Salmonella typhi, P.
  • aeruginosa A. baumanii, Klebsiella oxytoca, Bacillus cereus, A. hydrophila, S. marcescens, V. cholerae, P. entomophila, C. perfringens, and Y. enterocolitica.
  • a nanopore described herein may comprise one or more monomers of a T7 pore, a PN pore, a stable protein 1 (SP1) pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin ( ⁇ -HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a cytolysin A (ClyA) pore, a FhuA pore, a PFO pore, a TMH4C4 pore, any combination thereof, or homolog, paralog, ortholog, or any combination thereof.
  • SP1 stable protein 1
  • ⁇ -HL alpha-hemolysin
  • the nanopore (e.g., the biological nanopore) can comprise a conical geometry or a semi-conical geometry.
  • a conical geometry can comprise a shape in which a nanopore tapers over a longitudinal axis, wherein a first entrance of a nanopore is larger (e.g., comprises a wider dimension) than a second entrance.
  • the nanopore (e.g., the biological nanopore) can comprise a straight geometry (e.g., a cylindrical geometry).
  • a straight geometry may comprise a shape in which a channel of a nanopore can be the same width (e.g., diameter) over its longitudinal axis.
  • the nanopore can be a T7 pore, a PN pore, a stable protein 1 (SP1) pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin ( ⁇ - HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a cytolysin A (ClyA) pore, a FhuA pore, a PFO pore, or a TMH4C4 pore, or any combination thereof.
  • SP1 stable protein 1
  • ⁇ - HL alpha-hemolysin
  • SPP1 alpha-hemolysin
  • FraC pore a MspA pore
  • CsgG pore a CsgG pore
  • OmpG pore an aerolysin pore
  • ClyA cytolysin A
  • the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, ⁇ -HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or any combination thereof.
  • an engineered biological nanopore described herein may comprise one or monomers from a T7 pore, a PN pore, a SP1 pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin ( ⁇ - HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a ClyA pore, a FhuA pore, a PFO pore, or a TMH4C4 pore, or any combination thereof.
  • the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, ⁇ -HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or any combination thereof.
  • the nanopore e.g., the biological nanopore
  • can comprise a vestibule geometry e.g., a globular geometry or goblet geometry.
  • the nanopore may comprise an alpha-hemolysin nanopore, or a curli specific gene G (CsgG) nanopore, or any combination thereof.
  • a pore comprises comprise beta-toxins, such as alpha-hemolysin, anthrax toxin and leukocidins.
  • a pore comprises outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, CsgG.
  • Msp Mycobacterium smegmatis porin
  • a pore comprises outer membrane porin F (OmpF).
  • a pore comprises outer membrane porin G (OmpG).
  • a pore comprises outer membrane phospholipase A.
  • a pore comprises outer membrane protein FhuA.
  • a pore comprises outer membrane protein A (OmpA). In some embodiments, a pore comprises Neisseria autotransporter lipoprotein (NalP). In some embodiments, a pore comprises lysenin. In some embodiments, a pore comprises bacterial nucleoside transporter Tsx. In some embodiments, a pore comprises inner membrane proteins and outer membrane proteins, such as WZA and FraC. In some embodiments, a pore comprises Aer, CytK, MspA, aHL, CsgG, or FraC or an engineered mutant thereof. In some embodiments, a pore comprises a transmembrane pore derived from or based on Msp, e.g.
  • a pore comprises an oligomeric accessory protein coupled to a transmembrane domain of the pore. In some embodiments, a pore comprises alpha-helical or beta- barrel transmembrane regions.
  • a beta-barrel pore or beta-barrel pore forming protein and/or peptide can include, but may not be limited to, beta-toxins, such as alpha-hemolysins, aerolysins, lysenin, cytolysins, cytolysin K, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), ferric hydroxamate uptake component A (FhuA), Curli production transport component CsgG, and Neisseria autotransporter lipoprotein (NalP).
  • beta-toxins such as alpha-hemolysins, aerolysins, lysenin, cytoly
  • an alpha-helix pore or alpha-helical forming protein and/or peptide can include, but may not be limited to, inner membrane proteins and outer membrane proteins, such as WSGR Docket Number: 64828-710.601 Actinoporins, the outer membrane core complex (OMCC) of H. pylori Cag T4SS particles, and the transmembrane domain of the E. coli polysaccharide transporter Wza.
  • a pore comprises CytK or a genetically engineered mutant thereof.
  • a pore can comprise a porin.
  • a pore can comprise OmpF, OmpG, or FhuA.
  • a pore comprises a pore forming protein (PFP).
  • a PFP comprises an ⁇ -PFP.
  • a PFP comprises a ⁇ -PFPs.
  • a PFP comprises a bundle of ⁇ -helices.
  • a PFP comprises a transmembrane ⁇ -barrel.
  • the nanopore comprises CytK or a genetically engineered mutant thereof.
  • the mutant CytK comprises one or more of the amino acid substitutions selected from the group consisting of K128D, K128F, K155D, S120D, Q122D, G122D and S151D.
  • FIG. 3A-3D depict representations of a CytK nanopore and one or more mutations.
  • FIG. 3A shows a surface representation of a pore (e.g., a CytK pore) and FIG. 3B depicts a cartoon representation of a ⁇ -barrel region, with N-terminal strands depicted as dark gray and C-terminal strands depicted as light gray.
  • Residues include E112, T116, S120, Q122, S126, K128, E139, T143, Q145, T147, S151, and K155 (charged residues in bold).
  • FIG. 1A shows a surface representation of a pore (e.g., a CytK pore)
  • FIG. 3B depicts a cartoon representation of a ⁇ -barrel region, with N-terminal strands depicted as dark gray and C-terminal strands depicted as light gray.
  • Residues include E112, T116, S
  • a mutant pore e.g., a mutant CytK pore
  • the mutant pore can be in 1 M KCl, pH 7.5 ⁇ (p(K)/p(Cl) of 4.04 ⁇ 0.07, and p(K)/p(Cl) of 1.3 at pH 3.8) ⁇ .
  • Substitutions of the CytK nanopore can comprise one or more amino acid substitutions comprising K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, K155Q, T116D, S126D, T143D, Q145D, T147D, or any combination thereof.
  • a nanopore described herein can comprise one or more CytK monomers.
  • a CytK monomer may comprise a wildtype CytK monomer.
  • a CytK monomer may comprise a mutant CytK monomer.
  • a CytK monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107.
  • a CytK monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107. In some embodiments, a CytK monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution) in an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107.
  • a CytK monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112.
  • a CytK monomer of a nanopore described here may comprise an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112. In some embodiments, a CytK monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112. [0386] In some embodiments, a pore comprises engineered pores. In some embodiments, a pore comprises an oligomeric accessory protein coupled to a transmembrane domain. In some embodiments, a pore can be derived from naturally existing nanopores.
  • a pore comprises a protein pore templated by a DNA structure.
  • a pore comprises a de novo pore based on de novo alpha-helical or beta-barrel transmembrane regions.
  • a pore can comprise a de novo pore based on de novo beta- barrel pore forming protein and/or peptide and/or alpha-helical pore forming protein and/or peptide.
  • a pore can be a portion of an existing pore or mutations thereof.
  • a pore comprises a combination of elements of distinct pores or mutations thereof.
  • a pore comprises an assembly of genetically engineered pleurotolysin (Ply) A and B subunits.
  • a pore comprises a protein of the Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin (MACPF/CDC) protein superfamily.
  • MACPF/CDC Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin
  • a pore comprises perforin, complement C9, pneumolysin, or lysteriolysin.
  • a pore comprises a recognition or a binding portion configured to recognize or bind to an analyte.
  • a pore comprises a transmembrane portion comprising a channel for an analyte to pass through.
  • the pore comprises a molecular or proteinaceous adapter.
  • the molecular or proteinaceous adapter acts as a recognition or binding site for analytes.
  • the molecular or proteinaceous adapter can be a cyclic adapter.
  • the adapter can be bound covalently or non-covalently.
  • a pore comprises a proteasome.
  • the proteasome can be a S20 proteasome, a 26S proteasome, a 11S proteasome, a ClpXP proteasome, open reading frame number 854 in the M. mazei genome, or combinations thereof.
  • the proteasome comprises a subunit or components of a proteasome.
  • a proteasome can be a fused proteasome.
  • a C-terminus of a subunit of a ring-forming (multimeric) protein comprising a flanked transmembrane sequence can be genetically fused to a N-terminus of a proteasome subunit.
  • a ring-forming protein subunit can be fused to an N-terminally truncated proteasome subunit such that the proteasome gate can be left open towards.
  • a pore comprises a proteasome fused to the pore.
  • a pore comprises a proteasome fused to the pore such that the proteasome can be located on the first side (e.g., cis side) of the pore when the pore can be disposed in a membrane.
  • a proteasome can be coupled to a pore.
  • a proteasome can be coupled non-covalently to a pore.
  • a proteasome may be fused and/or coupled to a pore by a linker described herein.
  • the linker may be a helical linker, a non-helical linker, a flexible linker, a cleavable linker, or any combination thereof.
  • a proteasome can be coupled to a pore on a first side (e.g., cis side) of a membrane. In some embodiments, a proteasome can be coupled to a pore on a second side (e.g., trans side) of a membrane. In some embodiments, a pore comprises one or more proteasome subunits. In some embodiments, a pore comprises a proteasome ⁇ -subunit. In some embodiments, a pore comprises a proteasome ⁇ -subunit. In some embodiments, a pore comprises a ring of proteasome ⁇ -subunits. In some embodiments, a pore comprises a ring of proteasome ⁇ -subunits.
  • a pore comprises a catalytically active subunit. In some embodiments, a pore comprises a protease. In some embodiments, a pore comprises a protease having a trypsin-type or chymotrypsin-type of activity. [0389] In some embodiments, a pore comprises prokaryotic AAA+ unfoldase ClpX. ClpX can unfold analytes by NTP-driven translocation of the polypeptide chain through the central pore of its hexameric assembly. In complex with the ClpP peptidase, ClpX can carry out protein degradation by translocating unfolded analytes directly into the ClpP proteolytic chamber.
  • a pore comprises a multi-protein pore sensor complex comprising an artificial ClpP pore, e.g. by fusion to PA, which sensor complex further comprises ClpX or a homologous protein unfoldase.
  • a pore comprises an oligomeric Fragaceatoxin C (FraC) pore.
  • FraC can be a type II pore.
  • a type II FraC pore comprises an apparent heptameric stoichiometry, and/or a conductance of about 1.22-1.26 nS when assayed at pH 7.5 in a 1M NaCl solution or about 0.99-1.08 nS when assayed at pH 4.5 in a 1 M KC solution.
  • Conductance values can be determined by collecting single channels under -50 mV applied potential using 1 M NaCl, 15 mM Tris pH 7.5, or 1 M KCl, 0.1 M citric acid, 180 mM Tris base pH 4.5.
  • a type II FraC pore can comprise a pore size (at the narrowest constriction) of about 1.1 nm, which can be determined from homology modeling.
  • FraC can be a type III pore.
  • a type III FraC pore comprises an apparent hexameric stoichiometry, and/or a conductance of about 0.37-0.43 nS when assayed at pH 4.5 in a 1M KC solution.
  • a type III FraC pore comprises a pore size (at the narrowest constriction) of about 0.8 nm, which can be determined from homology modeling.
  • a nanopore described herein can comprise one or more FraC monomers.
  • a FraC monomer may comprise a wildtype FraC monomer.
  • a FraC monomer may comprise a mutant FraC monomer.
  • a FraC monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52.
  • a FraC monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52. In some embodiments, a FraC monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52.
  • a nanopore described herein can comprise one or more alpha-hemolysin ( ⁇ - hemolysin) monomers. An alpha-hemolysin ( ⁇ -hemolysin) monomer may comprise a wildtype alpha- hemolysin ( ⁇ -hemolysin) monomer.
  • An alpha-hemolysin ( ⁇ -hemolysin) monomer may comprise a mutant alpha-hemolysin ( ⁇ -hemolysin) monomer.
  • An alpha-hemolysin ( ⁇ -hemolysin) monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 106.
  • an alpha-hemolysin ( ⁇ -hemolysin) monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 106.
  • an alpha-hemolysin ( ⁇ - hemolysin) monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 106.
  • a nanopore described herein can comprise one or more CsgG monomers.
  • a CsgG monomer may comprise a wildtype CsgG monomer.
  • a CsgG monomer may comprise a mutant CsgG monomer.
  • a CsgG monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 128.
  • a CsgG monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 128.
  • WSGR Docket Number: 64828-710.601 a CsgG monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 128.
  • a MspA monomer may comprise a wildtype MspA monomer.
  • a wildtype MspA monomer may comprise an amino acid sequence as set forth in MGLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEGTLELG YQIGFPWSLGVGINFSYTTPNILIDDGDITAPPFGLNSVITPNLFPGVSISADLGNGPGIQEVATFSVDVS GAEGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDSVTTYGEPWNMNGSAGSAWSHPQFEK (SEQ ID NO.: 171).
  • a nanopore described herein can comprise one or more MspA monomers.
  • a MspA monomer may comprise a mutant MspA monomer.
  • a MspA monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 171.
  • a MspA monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 171.
  • a MspA monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 171.
  • an MspA nanopore may comprise a mutation in a lumen-facing region, or constriction region, or any combination thereof.
  • a mutation of an MspA nanopore can comprise a in a lumen-facing region and/or constriction region from one or more amino acid residues to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively- charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), one or more sulfur-containing amino acid residues, or any combination thereof.
  • aromatic residues e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof
  • W tryptophan
  • F pheny
  • a monomer of a MspA nanopore may comprise a mutation at position D90, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171.
  • a monomer of a MspA nanopore may comprise a mutation at position D91, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171.
  • the mutation at position D90 and/or D91 of the MspA monomer can comprise one or more mutations (e.g., a substitution mutations) to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively- WSGR Docket Number: 64828-710.601 charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171.
  • a substitution mutations to one or more aromatic residues
  • aromatic residues e.g., tryp
  • the MspA nanopore can comprise a mutation (e.g., a substitution mutation) at position D90 to an asparagine residue, wherein the mutation comprises D90N and wherein the residue numbering corresponds to SEQ ID NO: 171.
  • the MspA nanopore can comprise a mutation (e.g., a substitution mutation) at position D91 to an asparagine residue, wherein the mutation comprises D91N and wherein the residue numbering corresponds to SEQ ID NO: 171.
  • a monomer of a nanopore described herein may comprise a mutation at a position 83, 88, 103, 105, or 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 171.
  • a nanopore e.g., a MspA nanopore
  • the mutations of one or more monomers may comprise one or more mutations at position S103, I105, N108, T83, or L88, or any combination thereof, to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof.
  • aromatic residues e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof
  • W tryptophan
  • F
  • nanopore (e.g., MspA nanopore) comprises at least one mutation (e.g., a substitution mutation) in one or more monomers, wherein the mutation comprises S103E, I105E, N108E, L88E, or T83E, or any combination thereof, and wherein the residue numbering corresponds to SEQ ID NO: 171.
  • a monomer of a nanopore (e.g., MspA nanopore) described herein may comprise one or more mutations at positions D90, D91, D93, A96, T83, L88, S103, I105, or N108, or any combination thereof.
  • a nanopore described herein may comprise any combination of monomers comprising an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128.
  • a nanopore described herein may comprise one or more monomers comprising an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 1
  • a nanopore described herein may comprise one or more WSGR Docket Number: 64828-710.601 monomers comprising an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128.
  • a nanopore described herein may comprise one or more monomers comprising at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128.
  • a CsgG monomer may comprise a wildtype CsgG monomer.
  • a wildtype CsgG monomer may comprise an amino acid sequence as set forth in CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVPQSATAML VTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSIIGYESNVKSG GVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVG YTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES (SEQ ID NO.: 211).
  • a nanopore described herein can comprise one or more CsgG monomers.
  • a CsgG monomer may comprise a mutant CsgG monomer.
  • a CsgG monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 211.
  • a CsgG monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 211.
  • a CsgG monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 211.
  • a nanopore described herein can comprise an engineered CsgG nanopore. [0402]
  • the CsgG pore may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more monomers.
  • the CsgG pore may be a CsgG/F pore.
  • the CsgG/F pore may comprise a CsgG nanopore with a channel region and constriction region.
  • the pore may further comprise a CsgF protein (e.g., a CsgF peptide adapter).
  • a monomer of a CsgG nanopore may comprise a mutation at position Y51, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211.
  • a monomer of a CsgG nanopore may comprise a mutation at position N55, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211.
  • a monomer of a CsgG nanopore may comprise a mutation at position F56, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211.
  • a monomer of a CsgG nanopore may comprise a mutation at position F48, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211.
  • a monomer of a CsgG nanopore may comprise a mutation at position F58, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211.
  • the WSGR Docket Number: 64828-710.601 mutation at position Y51, N55, F56, F48, F58, or any combination thereof of the CsgG monomer can comprise one or more mutations (e.g., insertion mutations, deletion mutations, substitution mutations) to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211.
  • mutations
  • a pore can be coupled or fused to one or more accessory partner proteins that aids the binding and/or functioning of a protein translocase.
  • a translocase comprises an unfoldase, a helicase, an exonuclease, a protease translocase, or a topoisomerase.
  • a pore can be coupled to an inactive ClpP.
  • a pore can be coupled to an active ClpP.
  • nucleic acid molecules encoding any one of the pore proteins disclosed herein.
  • expression vectors comprising nucleic acid molecules disclosed herein.
  • a nanopore may comprises a steric or electrostatic obstruction.
  • the steric or electrostatic obstruction may be added to a natural or a mutant variant of a biological nanopore.
  • An adaptor can provide a steric or electrostatic obstruction.
  • An adaptor can be a separate entity from the nanopore.
  • An adaptor can provide an additional constriction zone, or an additional recognition element, or combinations thereof.
  • An adaptor can be proteinaceous or chemical.
  • An adaptor can comprise at least a portion of a molecule that couples to a nanopore and modifies a steric or electrostatic arrangement of a nanopore channel.
  • An adaptor can be coupled to a nanopore.
  • the coupling can be covalent or non-covalent.
  • the covalent coupling can be a covalent bond.
  • the covalent bound can be a polar covalent bond or a nonpolar covalent bond.
  • the noncovalent coupling can be a noncovalent bond.
  • Non- limiting examples of noncovalent bond include hydrogen bonds, electrostatic interactions, van der Waals interactions, hydrophobic interactions, and cysteine bonds.
  • the adaptor can be coupled to the channel of the nanopore via a cysteine bond, a hydrogen bond, or an electrostatic interaction, or combinations thereof.
  • the adaptor can be coupled to the nanopore via a linker.
  • linkers include (GGGGS)3, (SG)n, (GGGGS)n, (Gly)8, (Gly)6, (EAAAK)3, (EAAAK)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRR, GFLG, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, disulfide bond, or cysteine linkages, and any combination thereof.
  • a linker can comprise any combination of amino WSGR Docket Number: 64828-710.601 acids.
  • the amino acids can be canonical amino acids.
  • the amino acids can be non- natural amino acids.
  • the linker can comprise any combination of canonical amino acids and non- natural amino acids.
  • the linker can be ethylene glycol.
  • the linker can be polyethylene glycol..
  • the linker can be a cysteine linkage.
  • the adaptor can be a separate entity from the nanopore channel.
  • the adaptor can be a protein coupled to the channel of the nanopore.
  • the adaptor may not be a portion of the amino acid sequence of the nanopore channel. In some cases, the adaptor may not modify the sequence of the nanopore channel. In some examples, the adaptor may not modify the amino acid residue sequence of the nanopore channel.
  • An adaptor can be coupled to a subunit, a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer, or a nonamer of a nanopore. [0409] In some embodiments, at least one adaptor can be coupled to a nanopore.
  • between about one adaptor to about 10 adaptors can be coupled to a nanopore.
  • at least about one adaptor, at least two about adaptors, at least about three adaptors, at least about four adaptors, at least about five adaptors, at least about six adaptors, at least about seven adaptors, at least about eight adaptors, at least about nine adaptors, or more can be coupled to a nanopore.
  • At most about nine adaptors, at most about eight adaptor, at most about seven adaptors, at most about six adaptors, at most about five adaptors, at most about four adaptors, at most about three adaptors, at most about two adaptors, at most about one adaptor, or less can be coupled to a nanopore. In some cases, about one adaptor, about two adaptors, about three adaptors, about four adaptors, about five adaptors, about six adaptors, about seven adaptors, about eight adaptors, or about nine adaptors can be coupled to a nanopore.
  • An adaptor can comprise one or multiple proteinaceous subunits. In some cases, the adaptor protein can be a protein complex.
  • the protein complex can comprise one or more proteins (e.g., proteinaceous subunits).
  • the adaptor can comprise between about one to about 50 proteinaceous subunits.
  • the adaptor can comprise between about one to about five proteinaceous subunits, between about five to about 10 proteinaceous subunits, between about 10 to about 15 proteinaceous subunits, between about 15 to about 20 proteinaceous subunits, between about 20 to about 25 proteinaceous subunits, between about 25 to about 30 proteinaceous subunits, between about 30 to about 35 proteinaceous subunits, between about 35 to about 40 proteinaceous subunits, between about 40 to about 45 proteinaceous subunits, or between about 45 to about 50 proteinaceous subunits.
  • the adaptor can comprise at least about one proteinaceous subunit, at least about five proteinaceous subunits, at least about ten proteinaceous subunits, at least about 15 proteinaceous subunits, at least about 20 proteinaceous subunits, at least about 25 proteinaceous subunits, at least about 30 proteinaceous subunits, at least about 35 proteinaceous WSGR Docket Number: 64828-710.601 subunits, at least about 40 proteinaceous subunits, at least about 45 proteinaceous subunits, at least about 50 proteinaceous subunits, or more.
  • the adaptor can comprise at most about 50 proteinaceous subunits, at most about 45 proteinaceous subunits, at most about 40 proteinaceous subunits, at most about 35 proteinaceous subunits, at most about 30 proteinaceous subunits, at most about 25 proteinaceous subunits, at most about 20 proteinaceous subunits, at most about 15 proteinaceous subunits, at most about 10 proteinaceous subunits, at most about 5 proteinaceous subunits, at most about one proteinaceous subunit.
  • An adaptor can provide a steric obstruction, or an electrostatic change, or combinations thereof.
  • An adaptor can obstruct flow of an analyte through a channel of the nanopore.
  • An adaptor can selectively obstruct flow through a nanopore.
  • an adaptor can obstruct flow through the nanopore by modifying a charge of a nanopore or a geometry of a nanopore.
  • the adaptor can obstruct flow of the analyte through the channel nanopore by decreasing the circumference of the channel.
  • the adaptor can obstruct flow of the analyte through the channel nanopore by causing the nanopore channel to have a net negative charge.
  • An adaptor can narrow a portion of the channel of the nanopore so as to selectively obstruct larger molecules from passing through the nanopore channel by providing a steric barrier. The selectivity can be based at least in part on a charge characteristic of the adaptor.
  • the adaptor can prevent translocation of a nucleic acid analyte by causing the nanopore channel to have a net negative charge.
  • an adaptor can increase a positive net charge so as to decrease a flow of positively charged molecules, such as cations, by providing an electrostatic barrier.
  • an adaptor can increase a positive net charge so as to increase a flow of negatively charged molecules, such as anions, by creating attractive electrostatic interactions.
  • an adaptor can decrease a positive net charge as to increase a flow of positively charged molecule by providing an electrostatic barrier.
  • an adaptor can increase a negative net charge so as to decrease a flow of negatively charged molecules, such as anions or nucleic acid molecules, by providing an electrostatic barrier.
  • the adaptor can modify a net charge of the channel of the nanopore to be a negative net charge.
  • the adaptor can comprise surface exposed negatively charge amino acid residues (e.g., aspartic acid, glutamic acid).
  • WSGR Docket Number: 64828-710.601 [0413]
  • the adaptor can extend at least a portion of a channel of a nanopore. In some embodiments, the adaptor can extend at least about one subunit of the nanopore.
  • the adaptor can extend at least about one subunit of the nanopore, at least about two subunits of the nanopore, at least about three subunits of the nanopore, at least about four subunits of the nanopore, at least about five subunits of the nanopore, at least about six subunits of the nanopore, at least about seven subunits of the nanopore, at least about eight subunits of the nanopore, or at least about nine subunits of the nanopore.
  • the adaptor can extend at most about nine subunits of the nanopore, at most about eight subunits of the nanopore, at most about seven subunits of the nanopore, at most about six subunits of the nanopore, at most about five subunits of the nanopore, at most about four subunits of the nanopore, at most about three subunits of the nanopore, at most about two subunits of the nanopore, at most about one subunits of the nanopore, or less.
  • the adaptor can extend about one subunit of the nanopore, about two subunits of the nanopore, about three subunits of the nanopore, about four subunits of the nanopore, about five subunits of the nanopore, about six subunits of the nanopore, about seven subunits of the nanopore, about eight subunits of the nanopore, or about nine subunits of the nanopore.
  • An adaptor can constrict at least a portion of a channel of a nanopore. In some embodiments, the adaptor can decrease the circumference of the channel of the nanopore. In some embodiments, the adaptor can constrict at least about one subunit of the nanopore.
  • the adaptor can constrict at least about one subunit of the nanopore, at least about two subunits of the nanopore, at least about three subunits of the nanopore, at least about four subunits of the nanopore, at least about five subunits of the nanopore, at least about six subunits of the nanopore, at least about seven subunits of the nanopore, at least about eight subunits of the nanopore, or at least about nine subunits of the nanopore.
  • the adaptor can constrict at most about nine subunits of the nanopore, at most about eight subunits of the nanopore, at most about seven subunits of the nanopore, at most about six subunits of the nanopore, at most about five subunits of the nanopore, at most about four subunits of the nanopore, at most about three subunits of the nanopore, at most about two subunits of the nanopore, at most about one subunits of the nanopore, or less.
  • the adaptor can constrict about one subunit of the nanopore, about two subunits of the nanopore, about three subunits of the nanopore, about four subunits of the nanopore, about five subunits of the nanopore, about six subunits of the nanopore, about seven subunits of the nanopore, about eight subunits of the nanopore, or about nine subunits of the nanopore.
  • the adaptor can block a portion of the nanopore channel. In some cases, blocking a portion of the nanopore channel can reduce the ability of an analyte to move through the nanopore channel. In some cases, blocking a portion of the nanopore channel can reduce the ability of one or more salts to move through the nanopore channel.
  • the adaptor can be a proteinaceous adaptor.
  • the proteinaceous adaptor can be a protein.
  • the proteinaceous adaptor can be a monomer.
  • the proteinaceous adaptor can comprise more than one protein subunits.
  • the proteinaceous adaptor can comprise at least about 1 protein subunit, at least about 2 protein subunits, at least about 3 protein subunits, at least about 4 protein subunits, at least about 5 protein subunits, at least about 6 protein subunits, at least about 7 protein subunits, at least about 8 protein subunits, at least about 9 protein subunits, at least about 10 protein subunits, at least about 12 protein subunits, at least about 15 protein subunits, at least about 18 protein subunits, at least about 20 protein subunits, at least about 25 protein subunits, at least about 30 protein subunits, at least about 35 protein subunits, at least about 40 protein subunits, at least about 45 protein subunits, at least about 50 protein subunits, or greater than about 50 protein subunits.
  • the proteinaceous adaptor can comprise at most about 50 protein subunits, at most about 45 protein subunits, at most about 40 protein subunits, at most about 35 protein subunits, at most about 30 protein subunits, at most about 25 protein subunits, at most about 20 protein subunits, at most about 18 protein subunits, at most about 15 protein subunits, at most about 12 protein subunits, at most about 10 protein subunits, at most about 9 protein subunits, at most about 8 protein subunits, at most about 7 protein subunits, at most about 6 protein subunits, at most about 5 protein subunits, at most about 4 protein subunits, at most about 3 protein subunits, at most about 2 protein subunits, at most about 1 protein subunit, or less than about 1 protein subunit.
  • the adaptor can be at least about 0.2 kDa in size. In some cases, the adaptor can be between about 0.2 kDa to about 100 kDa in size. In some cases, the adaptor can be between about 0.2 kDa to about 1 kDa, between about 1 kDa to about 5 kDa, between about 5 kDa to about 10 kDa, between about 10 kDa to about 15 kDa, between about 15 kDa to about 20 kDa, between about 20 kDa to about 25 kDa, between about 25 kDa to about 30 kDa, between about 30 kDa to about 35 kDa, between about 35 kDa to about 40 kDa, between about 40 kDa to about 45 kDa, between about 45 kDa to about 50 kDa, between about 50 kDa to about 55 kDa, between about 55 kDa to about 60 k
  • the proteinaceous adaptor can comprise one or more protein subunits. In some cases, the one or more protein subunits can be the same. In some cases, the one or more protein subunits can be different. In some cases, the one or more different subunits can be from different proteins. WSGR Docket Number: 64828-710.601 [0419]
  • a proteinaceous adaptor can comprise a CsgF subunit, a CsgF subunit truncation, or CsgF subunit homologs, or any combination thereof. In some cases, the proteinaceous adaptor can be a monomeric adaptor. In some cases, the proteinaceous adaptor can be an oligomeric adaptor.
  • the proteinaceous oligomeric adaptor can comprise one or more protein subunits. In some cases, the one or more protein subunits can be identical. In some cases, the one or more protein subunits can be different. In some cases, the one or more different protein subunits can be derived from different proteins. [0420] In some embodiments, the adaptor can be a chemical adaptor. In some cases, the chemical adaptor can comprise a non-protein based molecule. In some cases, the chemical adaptor can comprise a non-peptide based molecule. In some cases, a non-peptide based molecule can comprise cyclodextrin, cucurbituril, or a cyclic peptide, or any combination thereof.
  • the chemical adaptor can comprise a macrocyclic molecule.
  • the macrocyclic molecule can comprise crown ethers, calixarenes, porphyrins, cyclosporines, cyclems, or cyclams, or any combination thereof.
  • a chemical adaptor can comprise a cyclodextrin, a cyclic peptide, a cucurbituril, crown ethers, calixarenes, porphyrins, cyclosporines, cyclems, or cyclams, or any combination thereof.
  • a pore comprises a molecular motor.
  • a molecular motor comprises a translocase.
  • a molecular motor comprises a protein translocase.
  • a pore comprises a protein translocase.
  • a protein translocase can be active or inactive.
  • a pore comprises a translocase-analyte complex on a first side (e.g., cis side). In some embodiments, a pore comprises a translocase- analyte complex on a second side (e.g., trans side). In some embodiments, a pore can be coupled to a translocase. In some embodiments, a pore may not be coupled to a translocase. In some embodiments, an electro-osmotic force pulls a translocase to a pore. In some embodiments, an electro-osmotic force pulls a translocase:analyte complex to a pore.
  • an electro-osmotic force pulls a translocase to a pore in a direction counter to an electrophoretic force.
  • a translocase comprises a protease domain.
  • a proteolytic activity of the protease components can be disabled.
  • a translocase does not comprise a protease domain.
  • a proteolytic activity of the protease components can be active or inactive.
  • a translocase moves an analyte through a pore.
  • a molecular motor can be ATP or NTP driven.
  • a molecular motor’s rate of translocation can be ATP-dependent or NTP-dependent.
  • a translocase increases a rate of translocation through a pore relative to translocation by an electrophoretic force alone.
  • a translocase increases a rate of translocation through a pore relative to translocation by an electro-osmotic force alone.
  • a translocase decreases a rate of translocation through a pore relative to translocation by an electrophoretic force alone.
  • a translocase decreases a rate of translocation through a pore relative to translocation by an electro-osmotic force alone.
  • a translocase increases a rate of translocation through a pore relative to translocation by an electro-osmotic force in combination with an electrophoretic force. In some embodiments, a translocase decreases a rate of translocation through a pore relative to translocation by an electro-osmotic force in combination with an electrophoretic force. [0424] Provided herein are some methods of forming a complex between a translocase and an analyte. The ratio of translocase to an analyte may be about 1 to 1 (translocase to analyte).
  • the ratio of translocase to an analyte may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to 1 (translocase to analyte).
  • the ratio of translocase to an analyte may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 to 1 (translocase to analyte).
  • the ratio of translocase to an analyte may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to 1 (analyte to translocase).
  • the ratio of translocase to an analyte may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to 1 (analyte to translocase).
  • a translocase can assist in translocating an analyte (e.g., a mixture of analytes, for example mixed amino-acid composition proteins) through a pore.
  • analyte e.g., a mixture of analytes, for example mixed amino-acid composition proteins
  • a polymer analyte in a system comprising a nanopore in a membrane, can be translocated through the nanopore from the cis compartment to the trans compartment with the aid of a translocase motor that progresses along the polymer analyte in the direction of the subset arrow (moving away from termini P A towards termini P B of polymer analyte).
  • a pore comprises a protein translocase on a first side (e.g., cis side). In some embodiments, a pore comprises a protein translocase on a second side (e.g., trans side).
  • a pore comprises a protein translocase on a first side (e.g., cis side) and/or a second side (e.g., trans side). In some embodiments, a pore comprises a protein translocase and its accessory protease on a first side (e.g., cis side) and/or a second side (e.g., trans side).
  • an analyte can be added to a first side (e.g., cis side) to be fed through a pore via a translocase on the first side (e.g., cis side), and a translocase on the WSGR Docket Number: 64828-710.601 second side (e.g., trans side) aids the progression of the analyte through the pore and/or prevents the folding of protein.
  • a first side e.g., cis side
  • a translocase on the WSGR Docket Number: 64828-710.601 second side e.g., trans side
  • a translocase binds an analyte added to a first side (e.g., cis side) after it has been captured and partially translocated through a pore cis-to-trans, then pulls the analyte through the pore from cis-to-trans.
  • a translocase added to a second side binds an analyte added to a second side (e.g., trans side) after it has been captured and partially translocated through a pore trans-to-cis, then pulls the analyte through the pore from trans-to-cis.
  • one or more unfoldases, translocases, unfoldase domains, or translocase domains, or any combination thereof can be positioned proximal to the nanopore. In some embodiments, one or more unfoldases, translocases, unfoldase domains, or translocase domains, or any combination thereof can be positioned proximal to the nanopore upon a binding with an analyte (e.g., a polypeptide, peptide, or protein, or fragments thereof, or any combination thereof). In some embodiments, a motor protein described herein may comprise at least a portion of a translocase (e.g., a translocase domain).
  • a translocase domain can be a portion of a larger protein complex.
  • a translocase binds an analyte and unwinds the analyte prior to translocation through a pore. In some embodiments, more than one translocase can bind to an analyte. In some embodiments, a translocase binds an analyte and an electro-osmotic force pulls on the analyte as the analyte translocates through the pore. In some embodiments, the analyte transmits the electro-osmotic force up to the bound translocase.
  • the transmitted force keeps the translocase adjacent to the pore during translocation of the analyte.
  • the translocase unfolds a structure it encounters. In some embodiments, unfolding a structure can increase capture of the analyte by the pore.
  • the translocase modulates a movement of the analyte into the pore such that analyte-dependent changes in ionic current, conductivity, or impedance can be measured and/or characterized.
  • a translocase pulls an analyte in the cis to trans direction. In some embodiments, a translocase pulls an analyte in a trans to cis direction.
  • a translocase can be coupled to an opening of a pore.
  • a plurality of translocases can be coupled to an opening of a pore.
  • a central cavity of a translocase can be aligned with an entrance to a pore.
  • a translocase can be genetically fused to a protomer of a pore.
  • a translocase can be fused to a protomer of a pore via chemical attachment (e.g. via cysteines), enzymatic attachment, or via hybridization of complementary DNA tags that may be attached to the top of the pure and/or the translocase.
  • a translocase can be bound to an analyte prior to contacting with a pore, which may be referred to as “preloading” herein in appropriate contexts.
  • preloading one or more translocases are bound and/or loaded onto the target analyte (e.g., target protein) prior to addition to the nanopore system of WSGR Docket Number: 64828-710.601 the invention.
  • the preloading can be performed under conditions that favor high efficiency of binding and/or loading, and/or optimal translocase movement along the target protein (whether modified or unmodified).
  • the translocase and one or more target proteins can be incubated at relatively higher concentration, and then diluted when added to the nanopore system of the invention.
  • the preloading may be performed in conditions closer to the optimal binding conditions than employed in the nanopore system of the invention.
  • preloading can be performed in solutions that are closer to the optimal salt concentration and salt types, the optimal pH, the optimal temperature, and in the presence of optimal co-factors (e.g. NTP, or M2+ ions, or any combination thereof).
  • Preloading may also be performed in combination with accessory cofactors that aid in binding.
  • the preloading can be performed under conditions that enable multiple translocases to bind to a single target protein.
  • preloading to load multiple translocases can be performed under relatively high ratio of translocase to target protein. This may be performed in combination with target proteins that are modified to optimally bind multiple translocases, for example through attachment of sufficiently long leaders to the terminus(i) of the target proteins.
  • leaders can be designed to have sufficiently long binding and/or stall motifs to accommodate the footprint of the multiple translocases and/or stall the multiple translocases respectively.
  • the translocases can be topologically closed around the leader and/or target protein to reduce or prevent unbinding, for example especially when the translocase:target-protein can be added to nanopore systems that employ conditions relatively unfavorable to binding (e.g. high salt concentration).
  • oligomers can be connected by covalent coupling, e.g. by cross reaction between suitably placed cysteines between subunits.
  • genetic fusion and chemical coupling can be used in combination.
  • the preloading solution can be dilution prior to adding to one or more analytes.
  • the preloading solution can be diluted in solvent.
  • the preloading solution:solvent dilution can comprise from about 1:1 to about 1:100.
  • the preloading solution:solvent dilution can be at least about 1:1, at least about 1:2, at least about 1:5, at least about 1:10, at least about 1:20, at least about 1:25, at least about 1:30, at least about 1:40, at least about 1:50, at least about 1:60, at least about 1:70, at least about 1:75, at least about 1:80, at least about 1:90, at least about 1:100, or more than 1:100.
  • the preloading solution:solvent dilution can be at most about 1:100, at most about 1:90, at most about 1:80, at most about 1:75, at most about 1:70, at most about 1:60, at most about 1:50, at most about 1:40, at most about WSGR Docket Number: 64828-710.601 1:30, at most about 1:25, at most about 1:20, at most about 1:10, at most about 1:5, at most about 1:2, at most about 1:1, or less than 1:1.
  • the preloading solution:solvent dilution can comprise about 1:1, about 1:2, about 1:5, about 1:10, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:75, about 1:80, about 1:90, or about 1:100.
  • the preloading solution can comprise one or more translocases.
  • an analyte can be contacted with the preloading solution to form an analyte-translocase complex.
  • the analyte-translocase complex can be added to the nanopore system.
  • the analyte-translocase complex can be added to the first side (e.g., cis side) of the nanopore system. In some cases, the analyte- translocase complex can be added to the second side (e.g., trans side) of the nanopore system.
  • an EOF of a nanopore system described herein may maintain a translocase at a nanopore. A first side to second side electro-osmotic force can maintain the translocase of a complex at a first side entrance of a channel of a nanopore.
  • an EOF may maintain the translocase at the first entrance of the nanopore with feeding one or more analytes with an EOF (e.g., translocating one or more analytes in a same direction as the EOF). In some embodiments, an EOF may maintain the translocase at the first entrance of the nanopore with feeding one or more analytes against an EOF (e.g., translocating one or more analytes in an opposite direction as the EOF). [0434] In some embodiments, the preloading solution can further comprise one or more factors (e.g., cofactors).
  • the one or more cofactors can comprise NTP, M2+, NblA/B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, Ficoll, iron manganese, cobalt, copper, penicillamine, trientine, sodium calcium edetate, ethylenediaminetetraacetic acid, and any combinations thereof.
  • the preloading solution can have between about 1 to about 100 cofactors.
  • the preloading solution can have at least about 1 cofactor, at least about 10 cofactors, at least about 20 cofactors, at least about 30 cofactors, at least about 40 cofactors, at least about 50 cofactors, at least about 60 cofactors, at least about 70 cofactors, at least about 80 cofactors, at least about 90 cofactors, at least about 100 cofactors, or more.
  • the preloading solution can have at most about 100 cofactors, at most about 90 cofactors, at most about 80 cofactors, at most about 70 cofactors, at most about 60 cofactors, at most about 50 cofactors, at most about 40 cofactors, at most about 30 cofactors, at most about 20 cofactors, at most about 10 cofactors, at most about one cofactor, or less.
  • the preloading solution can have about one cofactor, about 10 cofactors, about 20 cofactors, about 30 cofactors, about 40 cofactors, about 50 cofactors, about 60 cofactors, about 70 cofactors, about 80 cofactors, about 90 cofactors, or about 100 cofactors.
  • the one or more cofactors or the one or more chemicals enhance binding of the analyte to the one or more translocases.
  • the one or more chemicals can comprise chaperone molecules.
  • the chaperone molecules can comprise heat shock proteins.
  • the heat WSGR Docket Number: 64828-710.601 shock proteins can comprise Hsp10, Hsp 60, Hsp70, Hsp40, Hsp90, or Hsp100, or any combination thereof.
  • the chaperone molecules can comprise phage growth defect, overcome by mutation in page gene E, large subunit protein (GroEL).
  • the chaperone molecules can comprise Hsp10, Hsp60, Hsp70, Hsp40, Hsp90, Hsp100, GroEL, GRP78/BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase, peptidyl prolyl cis-trans isomerase, prolyl isomerase, or ERp57, or any combination thereof.
  • the chaperone molecule can keep the analyte in a folded structure.
  • the one or more chemicals can comprise crowding agents.
  • the crowding agents can reduce the volume of solvent available for other components of the preloading solution.
  • the crowding agents can stabilize analytes in the preloading solution.
  • the crowding agents can comprise polyethylene glycol, dextran, or Ficoll, or any combination thereof.
  • the one or more chemicals can comprise one or more metal cofactors.
  • the one or more metal cofactors can comprise iron, magnesium, manganese, cobalt, copper, zinc, or molybdenum, or any combination thereof.
  • the one or more chemicals can comprise one or more chelating agents.
  • the one or more chelating agents can comprise deferoxamine, deferiprone, deferasirox, dimercapto succinic acid (DMSA), penicillamine, trientine, sodium calcium edetate, or ethylenediaminetetraacetic acid, or any combination thereof.
  • DMSA dimercapto succinic acid
  • penicillamine trientine
  • sodium calcium edetate sodium calcium edetate
  • ethylenediaminetetraacetic acid or any combination thereof.
  • the one or more chemicals can comprise Hsp10, Hsp60, Hsp70, Hsp40, Hsp90, Hsp100, GroEL, GRP78/BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase, peptidyl prolyl cis-trans isomerase, prolyl isomerase, ERp57, nucleotide triphosphates, glycine betaine, glycerol, dithiothreitol (DTT), iron, magnesium, manganese, cobalt, copper, zinc, molybdenum, Tris(2-carboxyethyl)phosphine (TCEP), glutathione, polyethylene glycol, dextran, Ficoll, deferoxamine, deferiprone, deferasirox, dimercapto succinic acid (DMSA), penicillamine, trientine, sodium
  • the one or more cofactors or the one or more chemicals increases binding of the analyte to the one or more translocases by between about 0.1% to about 500% compared to the binding of the analyte to the one or more translocases without the one or more cofactors or the one or more chemicals. In some embodiments, the one or more cofactors or the one or more chemicals increases binding of the analyte to the one or more translocases by between about 0.1% to about 1%, between about 10% to about 100%, or between about 100% to about 500%.
  • the one or more cofactors or the one or more chemicals increases binding of the analyte to the one or more translocases by at least about 0.1%, at least about 1%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more compared to the binding of the analyte to the one or more translocases without the one or more cofactors or the one or more chemicals.
  • the one or more chemicals increases binding of the analyte to the one or more translocases by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 1%, at most about 0.1%, or less compared to the binding of the analyte to the one or more translocases without the one or more cofactors or the one or more chemicals.
  • the preloading solution may further comprise a solvent.
  • the solvent can comprise water, phosphate buffer solution (PBS), Tris buffer, Tris-HCL, Tricine buffer, Bicarbonate buffer, MOPS buffer, Bis-tris methan buffer, Bicine buffer, HEPES buffer, MES buffer, or CAPS, or any combination thereof.
  • PBS phosphate buffer solution
  • Tris buffer Tris-HCL
  • Tricine buffer Tricine buffer
  • Bicarbonate buffer MOPS buffer
  • Bis-tris methan buffer Bicine buffer
  • HEPES buffer HEPES buffer
  • MES buffer MES buffer
  • CAPS phosphate buffer solution
  • the container can be a microcentrifuge tube, a centrifuge tube, a test tube, a beaker, a flask, a pipette, or a culture tube, or any combination thereof.
  • the preloading solution and the analyte can be combined together to form a translocase-analyte complex.
  • the translocase-analyte complex can be formed in a container separate from the nanopore system. In some cases, once the translocase-analyte complex can be formed, the translocase-analyte complex can be added to the nanopore system.
  • the translocase-analyte complex can be added to the first side (e.g., cis side) of the nanopore system. In some embodiments, the translocase-analyte complex can be added to the second side (e.g., trans side) of the nanopore system. In some embodiments, the translocase-analyte complex can be added to the first side (e.g., cis side) and the second side (e.g., trans side) of the nanopore system. In some cases, the translocase-analyte complex may not be added to the first side (e.g., cis side) of the nanopore system.
  • the translocase-analyte complex may not be added to the second side (e.g., trans side) of the nanopore system.
  • the translocase-analyte complex can be added to the first side (e.g., cis side) of the nanopore system and may not be added to the second side (e.g., trans side) of the nanopore system.
  • the translocase-analyte complex can be added to the second side (e.g., trans side) of the nanopore system and may not be added to the first side (e.g., cis side) of the nanopore system.
  • the preloading solution can comprise one or more translocases.
  • the preloading solution can comprise one or more leader constructs. In some embodiments, the preloading solution can comprise one or more analytes. In some cases, the preloading solution can comprise one or more translocases and one or more leader constructs. In some cases, the preloading solution can comprise one or more translocases and one or more analytes. In some cases, the preloading solution can comprise one or WSGR Docket Number: 64828-710.601 more leader constructs and one or more analytes.
  • the preloading solution can comprise one or more translocases, one or more leader constructs, and one or more analytes [0442]
  • preloading can be performed under conditions that favor high efficiency of binding and/or loading, and/or optimal translocase movement along an analyte.
  • a translocase and an analyte may be incubated at a higher concentration, and then diluted to a lower concentration when being contacted with a pore.
  • preloading can be performed in solutions that may be closer to the optimal salt concentration and salt types, the optimal pH, the optimal temperature, and/or in the presence of optimal co-factors (e.g.
  • preloading can be performed in combination with an accessory cofactor that aids in binding.
  • an accessory cofactor comprises analyte adaptors, e.g., NblA/B, ClpS, ClpF.
  • an accessory cofactor comprises engineered binding cofactors such as ones derived from antibodies, nanobodies, or affimers.
  • preloading can be performed under conditions sufficient for multiple translocases to bind to a single analyte.
  • preloading with multiple translocases can be performed under a higher ratio of translocase to analyte.
  • preloading with multiple translocases can be performed in combination with an analyte that can be configured to bind multiple translocases, for example, through attachment of one or more leaders to a terminus of the analyte.
  • one or more translocases can be topologically closed around a leader and/or an analyte to reduce or prevent unbinding.
  • one or more translocases and an analyte can be added to a solution, to contact with a pore, that comprises conditions unfavorable to binding.
  • a translocase comprises PAN of Methanococcus jannaschii, which can be a complex of relative molecular mass 650,000 that can be homologous to the ATPases in the eukaryotic 26S proteasome.
  • a translocase comprises AMA, an AAA protein from Archaeoglobus and methanogenic archaea.
  • a translocase comprises the open reading frame number 854 in the M. mazei genome.
  • a translocase comprises MBA (membrane-bound AAA) or SAMPs (small archaeal modifier proteins).
  • a translocase comprises ClpA, a member of the two-domain AAA ATPases, from Escherichia coli.
  • a translocase comprises ClpC, an AAA protein from Staphylococcus Aureus.
  • a translocase comprises ClpE, a member of the two-domain AAA ATPases from Bacillus subtilis.
  • a translocase comprises HslU/ClpY, an AAA protein from Escherichia coli.
  • a translocase comprises Lon, a protease from Escherichia coli containing an AAA ATPase domain.
  • a translocase comprises FtsH, a membrane bound protease from Escherichia coli containing an AAA ATPase domain.
  • a translocase comprises ARC/Mpa, an AAA ATPase from Mycobacterium tuberculosis.
  • a translocase comprises Msp1, a membrane associated AAA ATPase from Saccharomyces cerevisiae.
  • a translocase comprises CDC48, an AAA ATPase from Saccharomyces cerevisiae.
  • a translocase comprises Cpa, a CDC48 homologue from actinobacteria.
  • a translocase comprises SecA, a protein translocase from Escherichia coli.
  • a pore comprises a translocase.
  • a translocase comprises Thermoplasma VCP-like ATPase.
  • a translocase comprises a proteasome-activating nucleotidase (PAN).
  • PAN proteasome-activating nucleotidase
  • a translocase comprises AMA, an AAA protein from Archaeoglobus and methanogenic archaea.
  • a translocase comprises an open reading frame number 854 in the M. mazei genome.
  • a translocase comprises MBA (membrane-bound AAA) or SAMPs (small WSGR Docket Number: 64828-710.601 archaeal modifier proteins).
  • a translocase comprises helicases (e.g. gp4), exonucleases (lambda exonuclease), proteases translocases (e.g. Ftsk), or topoisomerases (e.g. topoisomerase II).
  • a translocase can translocate a polypeptide in the N-to-C direction, the C-to-N direction, or both.
  • a translocase may translate a polypeptide in the C-to-N or N-to-C direction. In some embodiments, a translocase binds to the N-terminus or the C-terminus of a polypeptide. [0450] In some embodiments, a translocase may translocate a biopolymer with a step size between about 0.5 amino acids per step to about 50 amino acids per step.
  • a translocase may translocate a biopolymer with a step size of at least about 0.5 nanometers per step (nm/step), 1 nm/step, 2 nm/step, 3 nm/step, 4 nm/step, 5 nm/step, 10 nm/step, 20 nm/step, 30 nm/step, or greater than about 30 nm/step.
  • a translocase may translocate a biopolymer with a step size of at most about 30 nm/step, 20 nm/step, 10 nm/step, 5 nm/step, 4 nm/step, 3 nm/step, 2 nm/step, 1 nm/step, 0.5 nm/step, or less than about 0.5 nm/step.
  • a translocase may translocate a biopolymer with a step size between about 0.5 nanometers per step (nm/step) to about 50 nm/step.
  • a translocase may translocate a biopolymer with a step size between about 0.5 nm/step to about 1 nanometer per step, about 0.5 nm/step to about 2 nm/step, about 0.5 nm/step to about 3 nm/step, about 0.5 nm/step to about 4 nm/step, about 0.5 nm/step to about 5 nm/step, about 0.5 nm/step to about 10 nm/step, about 0.5 nm/step to about 15 nm/step, about 0.5 nm/step to about 20 nm/step, about 0.5 nm/step to about 30 nm/step, about 0.5 nm/step to about 40 nm/step, about 0.5 nm/step to about 50 nm/step, about 1 nanometer per step to about 2 nm/step, about 1 nanometer per step to about 3 nm/step, about 1 nanometer per step to about 4 nm
  • an analyte e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof
  • a rate of translocation may be of a free analyte (e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof).
  • a free analyte may refer to an analyte (e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof) translocating without the use of a motor protein.
  • the rate of translocation may be of an analyte (e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof) translocating with a motor protein (e.g., a translocase).
  • an average rate of translocation with a motor protein can be at least about 1 amino acid/sec, at least about 2 amino acids/sec, at least about 3 amino acids/sec, at least about 4 amino acids/sec, at least about 5 amino acids/sec, at least about 10 amino acids/sec, at least about 20 amino acids/sec, at least about 30 amino acids/sec, at least about 40 amino acids/sec, at least about 50 amino acids/sec, at least about 75 amino acids/sec, at least about 100 amino acids/sec, or greater than about 100 amino acids/sec.
  • an average rate of translocation with a motor protein can be between about 1 amino acid/sec to about 2 amino acids/sec, about 1 amino acid/sec to about 3 amino acids/sec, about 1 amino acid/sec to about 4 amino acids/sec, about 1 amino acid/sec to about 5 WSGR Docket Number: 64828-710.601 amino acids/sec, about 1 amino acid/sec to about 10 amino acids/sec, about 1 amino acid/sec to about 20 amino acids/sec, about 1 amino acid/sec to about 30 amino acids/sec, about 1 amino acid/sec to about 40 amino acids/sec, about 1 amino acid/sec to about 50 amino acids/sec, about 1 amino acid/sec to about 75 amino acids/sec, about 1 amino acid/sec to about 100 amino acids/sec, about 2 amino acids/sec to about 3 amino acids/sec, about 2 amino acids/sec to about 4 amino acids/sec, about 2 amino acids/sec to about 5 amino acids/sec, about 2 amino acids/sec to about 10 amino acids/sec, about 2 amino acids/sec to about 20 amino acids
  • a translocase can function at a temperature of at least about 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, about 90 °C, 100 °C, or greater than about 100°C.
  • a translocase can function at a temperature of at most about 100 °C, 90 °C, 80 °C, 70 °C, 60 °C, 50 °C, 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 4 °C, 3 °C, 2 °C, 1 °C, or less than about 1 °C.
  • nucleic acid molecules encoding any one of the translocase proteins disclosed herein.
  • expression vectors comprising nucleic acid molecules disclosed herein.
  • host cells comprising expression vectors disclosed herein.
  • the translocase can be capable to moving an analyte. In some embodiments, the translocase may not be capable of separating the strands of double-stranded nucleic acids. In some cases, the translocase may not be a helicase. In some embodiments, the translocase may not be capable of replicating nucleic acids. In some cases, the translocase may not be a nucleic acid polymerase. In some cases, the translocase may not be an DNA polymerase or an RNA polymerase. In some embodiments, the translocase may not be involved in nucleic acid replication. In some embodiments, the translocase may not be capable of cleaving the analyte.
  • the translocases may not be topoisomerase.
  • the translocase may be coupled to a nanopore.
  • the translocase may be coupled covalently (e.g., genetically fused) or non-covalently (e.g., by a recognition element).
  • the translocase may not be coupled to the nanopore.
  • the translocase may not be coupled to the opening of the nanopore.
  • the translocase may not be coupled to the membrane adjacent to the nanopore.
  • the translocase may not be bound to the nanopore.
  • the translocase may not be bound to the opening of the nanopore.
  • a portion of the translocase can be captured in the nanopore due to the electro-osmotic force and the electrophoretic force.
  • the portion of the translocase captured in the nanopore can be a charged linker or a peptide extension of the translocase.
  • This orientation may be provided by the analyte being drawn into the nanopore by the electro-osmotic force, which can bring the point of connection of the analyte to the translocase (e.g., the translocase channel) adjacent to the nanopore channel and the translocase channel are aligned.
  • the translocase can control the rate of analyte translocation.
  • the rate of translocation can be the result of the translocase acting on the analyte as a molecular motor.
  • the rate of translocation can be from about 0.1 amino acids per second (aa/s) to about 1,000 aa/s.
  • the rate of translocation can at least most about 1,000 aa/s, at most about 500 aa/s, at most about 100 aa/s, at most about 50 aa/s, at most about 10 aa/s, at most about 5 aa/s, at most about 1 aa/s, at most about 0.5 aa/s, at most about 0.1 aa/s, or less than 0.1 aa/s.
  • the rate of translocation can be about 0.1 aa/s, about 0.5 aa/s, about 1 aa/s, about 5 aa/s, about 10 aa/s, about 50 aa/s, about 100 aa/s, about 500 aa/s, or about 1,000 aa/s.
  • the translocation orientation can be such that a feed direction of the translocase aligns with the channel of the nanopore.
  • the feed direction can be oriented cis to trans or trans to cis. There may or may not be a gap between the lumen of the translocase channel and the lumen of the nanopore channel.
  • the translocase can be held such that the translocase can feed the analyte through the nanopore in the direction of the electro-osmotic force, or it can be held such that the translocase pulls the analyte through the nanopore against the electro- osmotic force.
  • WSGR Docket Number: 64828-710.601 [0463]
  • the translocase can feed the analyte through the nanopore in the direction of the electroosmotic force such that it translocates at a rate faster or slower than the analyte that translocates with the electro-osmotic force alone.
  • the rate of translocation of the analyte through the nanopore with the translocase can be faster than a rate of translocation of the analyte through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 500% faster than a rate of translocation of the analytes through the nanopore without the translocase.
  • the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about
  • the rate of translocation of the analyte through the nanopore with the translocase can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%
  • the rate of translocation of the analyte through the nanopore with the translocase can be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 47
  • the rate of translocation of the analyte through the nanopore with the translocase can be slower than a rate of translocation of the analyte through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 500% slower than a rate of translocation of the analytes through the nanopore without the translocase.
  • the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about
  • the main force in the translocation of the analyte through the nanopore can be the EOF.
  • the translocation of the analyte through the nanopore can occur using the EOF.
  • the translocation of the analyte through the nanopore can occur in the absence of a translocase.
  • the translocation of the analyte through the nanopore can occur using the EOF and in the absence of a translocase.
  • the translocation of the analyte through the nanopore may not occur in the absence of the EOF.
  • the translocation of the analyte through the nanopore may not occur in the WSGR Docket Number: 64828-710.601 presence of a translocase. In some cases, the translocation of the analyte through the nanopore may not occur in the absence of the EOF and in the presence of a translocase.
  • the translocase can be held at the cis or second side (e.g., trans side) of the nanopore without needing to be coupled to the nanopore. The electro-osmotic force can hold the translocase adjacent to the nanopore without additional coupling to the nanopore channel.
  • the electro-osmotic force can hold the translocase adjacent to the nanopore so that the nanopore can couple with the translocase.
  • the translocase can be held adjacent to the nanopore channel while the analyte translocates through the nanopore. After the analyte has fully translocate through the nanopore, the translocase may continue to be held adjacent to the nanopore, or the translocase may be released from the position adjacent to the nanopore. A released translocase may then form a translocase-analyte complex with another analyte.
  • the translocase may not be coupled to the nanopore. In some cases, the translocase may not be coupled adjacent to the nanopore.
  • the non-covalent bonds can comprise electrostatic interactions, hydrogens bonds, van der Waals interactions, or hydrophobic interactions, or any combination thereof.
  • the translocase can be coupled to the nanopore via a linker.
  • the linkers can comprise (GGGGS) 3 , (GGGGS) n , (SG) n , (Gly) 8 , (Gly) 6 , (EAAAK) 3 , (EAAAK) n , VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRR, GFLG, A(EAAAK) 4 ALEA(EAAAK) 4 A, PAPAP, AEAAAKEAAAKA, (Ala-Pro) n , disulfide bond, or cysteine linkages, or any combination thereof.
  • a method comprises using an electro-osmotic force (EOF) to feed an analyte from a first side (e.g., cis side) of a pore to a second side (e.g., trans side) of the pore.
  • EEF electro-osmotic force
  • An EOF can pull on a polymer as it translocates through a pore, and can allow electrical signals, which depends on the structure and the composition of the polymer, to be measured and/or characterized.
  • a sensor system comprising a proteinaceous nanopore embedded in an amphipathic or hydrophobic membrane separating a fluid filled chamber into at least two sides (e.g., chambers).
  • one side (e.g., a first side) of a fluid filled chamber can be a first side (e.g., cis side) and another side (e.g., a second side) of a fluid filled chamber can be a second side (e.g., trans side).
  • the nanopore can be a conical shaped proteinaceous nanopore.
  • the nanopore can be a cylindrical shaped proteinaceous nanopore.
  • the nanopore can be conical shaped.
  • the nanopore can be cylindrical shaped.
  • the nanopore can be vestibule shaped.
  • the term "membrane" used herein in its conventional sense can refer to a thin, film-like structure that separates the chamber of the system into a first side (e.g., a cis side or cis compartment) and a second side (e.g., a trans side or trans compartment).
  • the membrane separating the first and second sides can comprise at least one pore (e.g., a biological nanopore).
  • the pore may be a nanopore.
  • the nanopore may be an engineered biological nanopore as described herein.
  • the nanopore may have enhanced cation-selectivity.
  • Membranes can be generally classified into synthetic membranes and biological membranes.
  • a membrane of a nanopore system described herein may comprise at least about, at most about, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000 nanopores, or any number of nanopores between two of these values.
  • the membrane can be an amphiphilic layer.
  • An amphiphilic layer can refer to a layer formed from amphiphilic molecules, such as phospholipids, which have both at least one hydrophilic portion and at least one lipophilic or hydrophobic portion.
  • Membranes comprising block- copolymers may comprise mixtures of block copolymers by length, chemistry of the units, number of units, polydispersity, etc.
  • Membranes formed of block copolymers may include di-block or tri-block copolymers for example, or mixtures thereof.
  • Block copolymers for use in the invention may comprise units of Polyethylene Oxide (PEO), Polypropylene Oxide (PPO), Poly(N-isopropylacrylamide) (PNIPAM), Polylactic Acid (PLA), Polycaprolactone (PCL), PMOXA, Polystyrene (PS), Poly(methyl methacrylate) (PMMA), Polyvinyl Alcohol (PVA), or Polyvinyl Pyrrolidone (PVP), or variants thereof, or any combination thereof.
  • PEO Polyethylene Oxide
  • PPO Polypropylene Oxide
  • PPO Poly(N-isopropylacrylamide)
  • PPA Polylactic Acid
  • PCL Polycaprolactone
  • PMOXA Polystyrene
  • PS Poly(methyl methacrylate)
  • PMMA Polyvinyl Alcohol
  • PVP Polyvinyl Pyrrolidone
  • a membrane of a system described herein may comprise a thickness.
  • a membrane may be at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, or greater than about 150 nm thick.
  • the nanopore system typically comprises a first side (e.g., cis side) comprising a first conductive liquid medium in liquid communication with a second side (e.g., trans side) comprising a second conductive liquid medium.
  • a method comprises translocating a polymer through a system comprising a pore.
  • the pore can be comprised in a membrane.
  • the membrane separates a fluidic chamber into a first side (e.g., cis side) and a second side (e.g., trans side).
  • the membrane insulates a first side (e.g., cis side) and a second side (e.g., trans side).
  • a sample can be added to a first side (e.g., cis side). In some embodiments, a sample can be added to a first side (e.g., cis side) in a solution of one or more electrolytes.
  • a system can comprise a cis to trans EOF.
  • An EOF can be generated from a net ionic current flow from a first side (e.g., cis side) to a second side (e.g., WSGR Docket Number: 64828-710.601 trans side) of a membrane.
  • a sample can be added to a second side (e.g., trans side).
  • a system can have a cis to trans EOF resulting from a net ionic current flow cis to trans over total ionic current flow (herein also referred to as Irel) of at least about -0.5, at least about -0.4, at least about -0.3, at least about -0.2, at least about -0.1, at least about 0, at least about 0.05, at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, at least about 0.5, or greater than about 0.5.
  • Irel net ionic current flow cis to trans over total ionic current flow
  • a system can have a cis to trans EOF resulting from a net ionic current flow cis to trans over total ionic current flow of at most about 0.5, at most about 0.45, at most about 0.4, at most about 0.35, at most about 0.3, at most about 0.25, at most about 0.2, at most about 0.15, at most about 0.1, at most about 0.05, at most about 0, at most about -0.1, at most about -0.2, at most about -0.3, at most about -0.4, at most about -0.5, or less than about -0.5.
  • a system can have a cis to trans EOF resulting from a net ionic current flow trans to cis over total ionic current flow of at most about 0.5, at most about 0.45, at most about 0.4, at most about 0.35, at most about 0.3, at most about 0.25, at most about 0.2, at most about 0.15, at most about 0.1, at most about 0.05, at most about 0, at most about -0.1, at most about -0.2, at most about -0.3, at most about -0.4, at most about -0.5, or less than about -0.5.
  • the pore system described herein can have an ion selectivity P (+) /P (-) of greater than about 3.0 or less than about 0.3 under an applied voltage across the membrane.
  • a pore-membrane system for translocating an analyte through a pore comprising a pore comprised in a membrane separating a fluidic chamber of the pore system into a first WSGR Docket Number: 64828-710.601 side (e.g., cis side) and a second side (e.g., trans side) and wherein the analyte is to be added to the first side (e.g., cis side), wherein the pore system has a cis to trans electro-osmotic force (EOF) resulting from a net ionic current flow cis-to-trans, so that the analyte can be captured in the pore.
  • EEF electro-osmotic force
  • a dominant cis to trans EOF can result from a net ionic current flow cis-to- trans over total ionic current flow between about 0.01 to about 0.1, about 0.01 to about 0.15, about 0.01 to about 0.2, about 0.01 to about 0.25, about 0.01 to about 0.3, about 0.01 to about 0.35, about 0.01 to about 0.4, about 0.01 to about 0.45, about 0.01 to about 0.5, about 0.01 to about 0.75, about 0.01 to about 1, about 0.1 to about 0.15, about 0.1 to about 0.2, about 0.1 to about 0.25, about 0.1 to about 0.3, about 0.1 to about 0.35, about 0.1 to about 0.4, about 0.1 to about 0.45, about 0.1 to about 0.5, about 0.1 to about 0.75, about 0.1 to about 1, about 0.15 to about 0.2, about 0.15 to about 0.25, about 0.15 to about 0.3, about 0.15 to about 0.35, about 0.15 to about 0.4, about 0.15 to about 0.45, about
  • the one or more salts can comprise sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc chloride hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr’s salt, sodium potassium sulphate, potassium permanganate, tetra amino cupric sulphate, zinc chloride hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, potassium glutamate, sodium ferricyanide, sodium ferrocyanide, potassium ferricyanide, or potassium ferrocyanide, or any combination thereof.
  • the one or more salts on the first side (e.g., cis side) of the membrane can comprise sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc chloride hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr’s salt, sodium potassium sulphate, potassium permanganate, tetra amino cupric sulphate, zinc chloride hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, or magnesium nitrate, or any combination thereof.
  • the one or more salts on the second side (e.g., trans side) of the membrane can comprise sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc chloride hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr’s salt, sodium potassium sulphate, potassium permanganate, tetra amino cupric sulphate, zinc chloride hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, or magnesium nitrate, or any combination thereof.
  • the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 1
  • the concentration of one or more salts on the second side (e.g., trans side) of the membrane can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%,
  • a method comprises providing a cis-to-trans electro-osmotic flow, or vice versa.
  • the EOF can arise from a net flow of ions (e.g. cis to trans) that creates a strong force on the solvent itself (water) sufficient to move the fluid, which further imposes a significant force on any molecules within the flux. Electroosmosis can either compete or cooperate with an electrophoretic force (EPF).
  • EPF electrophoretic force
  • a cis-to-trans EOF dominates over EPF.
  • complex and/or charged analytes can be captured and translocated through a pore, under dominant cis-to-trans EOF acting against trans-to-cis EPF.
  • dominant EOF may capture the analyte in the pore with relatively high residence time, wherein the EOF pulls on the analyte directly through a pore.
  • a non-nucleic acid based polymer analyte can be translocated using an electro-osmotic force.
  • the EOF can be greater than the EPF. In some cases, the EOF can be between about 0.1% to about 500% greater than the EPF.
  • the EOF can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about 180% to about 190%, between about 190% to about 200%, between about
  • the translocation of the analyte through the nanopore occurs in the direction of the EPF and opposite the direction of the EOF.
  • an EPF can be provided.
  • the EPF can be acting in an opposite direction to an EOF.
  • an electro-osmotic force may translocate at least a portion of an analyte from the first side through the nanopore to the second side against an electro-phoretic force acting in a direction opposite the electro-osmotic force.
  • use of a motor protein described herein may provide for translocation of one or more analytes against a prevailing EOF.
  • the EPF can be greater than the EOF.
  • the first side e.g., cis side
  • the applied transmembrane potential can be designated as the potential on the second side (e.g., trans side).
  • a pore system e.g., a nanopore system described herein may comprise a first side (e.g., a cis side) and a second side (e.g., a second side (e.g., trans side).
  • a pore system (e.g., a nanopore system) described herein may comprise a first side (e.g., a trans side) and a second side (e.g., a cis side).
  • An analyte can also be added to the second side (e.g., trans side) to perform trans-to-cis translocation of the analyte, e.g., in a method where the EOF can be created trans-to-cis.
  • Ion selective ion flux across membranes can be described by the Goldman-Hodgkin-Katz (GHK) flux equation, which can be used to determine the ionic current (I(S)) of ion species S across the membrane as a function of the applied potential (V m ): [0496] where P(S) can be the membrane permeability of ion species S, zs the valency of the ion, F the Faraday constant, R the gas constant, T the temperature and [S] cis and [S] trans the cis and trans concentrations of ion species S, respectively.
  • GLK Goldman-Hodgkin-Katz
  • the GHK flux equation can therefore be used to determine the separate current flow contributions (e.g. I (S1) , I (S2) , I (S3) , or any combination thereof) of various ion species (e.g. S1, S2, S3, or any combination thereof) in a system, flowing either cis-to-trans or trans-to-cis.
  • the total absolute ionic current (I total ) flowing through the pore regardless of direction can be given by the sum of absolute component currents: [0502]
  • the separate ionic currents e.g. I (S1) , I (S2) , I (S3) , or any combination thereof
  • I (S1) , I (S2) , I (S3) can also be combined to determine the separate components of the net ionic current flowing cis-to-trans ( ⁇ ⁇ ) and net ionic current flowing trans-to-cis ( ⁇ ⁇ ).
  • analyte capture and translocation can be performed with a large net cis-to-trans current >> 0 arising from a large relative difference between the cis-to-trans current and the trans-to-cis current (I ⁇ t ⁇ c), or vice versa (I ⁇ t ⁇ c >> 0 from Irel ⁇ 1).
  • Irel can be greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, or greater than 0.4 or less than -0.4.
  • I rel can be greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or about 0.9.
  • Irel can be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1.0. In some embodiments, I rel can be less than about -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, or about -0.9. In some embodiments, Irel can be greater than about - 0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, or about -1.0.
  • the GHK flux equation can indicate the conditions under which to create a large net cis-to-trans current by altering one or more of its variables, including the system ion-selectivity P(+)/P(-), the mixtures of salts used, the salt concentrations and salt asymmetries, and the applied voltage.
  • the GHK indicates that at least three methods can generate a net total ion flux across the membrane: 1) an asymmetry in electrolyte concentration (e.g.1 M KCl buffer in cis and 0.1 M KCl buffer in trans), 2) an asymmetry in electrolyte compositions with different permeabilities (e.g.1 M KCl in cis and 1 M KGlutamate in trans), 3) the use of ion-selective membrane channels. These methods can be used individually or in any combination. [0513] In some embodiments, a highly ion-selective pore system provides a directional net flow of water (the EOF) across a membrane, even without salt asymmetry.
  • the EOF directional net flow of water
  • the GHK flux equation can be solved at 0 pA for both species of ions to yield the ion- selectivity ratio: [0516] wherein P(X+) and P(Y-) denote the permeability of the pore system for cation species X and anion species Y respectively.
  • [ ⁇ ⁇ ⁇ ]and [ ⁇ ⁇ ⁇ ] are the activity of ion Y and X respectively in the indicated compartment, and can be calculated by multiplying the concentration with the mean ion activity coefficient (which may be WSGR Docket Number: 64828-710.601 tabulated for most electrolytes, see, e.g., Lide, D.
  • permeability P(S) can be determined for any ion species S employed in the pore system, and then used in the GHK flux equations to determine the underlying ionic current flows for pore systems containing mixtures of two or more ion species (e.g. asymmetric salt types).
  • ion species S employed in the pore system
  • the ion-selectivity ratio can determine the relative ion flux that may flow across the membrane cis-to-trans and trans-to-cis.
  • a method uses an ion-selectivity ratio ⁇ ⁇ ⁇ ( ⁇ ⁇ ⁇ ) / ⁇ ⁇ ⁇ ( ⁇ ⁇ ⁇ ) > 2.0 or ⁇ 0.5, > 2.5 or ⁇ 0.4, > 3.0 or ⁇ 0.33, > 3.5 or ⁇ 0.29, in combination with a symmetrical salt system can be sufficient to drive capture and translocation of complex analytes against a prevailing EPF under an applied voltage across the membrane of 20 mV to 1 V, 50 mV to 300 mV, or 75 mV to 200 mV.
  • the ion-selectivity ratio can be at least about 1.1, 1.5, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or about 10.
  • the ion-selectivity ratio can be at most about 1.1, 1.5, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or about 10.
  • the ion-selectivity ratio can be at least about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or about 0.40.
  • the ion-selectivity ratio can be at most about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or about 0.40.
  • the applied voltage can be at least about 10 mV, 20 mV, 30 mV, 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, 90 mV, 100 mV, 150 mV, 200 mV, 250 mV, 300 mV, 350 mV, 400 mV, 450 mV, 500 mV, 600 mV, 700 mV, 800 mV, 900 mV, or about 1000 mV in magnitude.
  • a first applied voltage, or a second applied voltage, or any combination thereof can be applied to any side of a nanopore system described herein.
  • a first applied voltage may be applied to a first side and a second applied voltage may be applied to a second side
  • a first applied voltage may be applied to a first side and a second applied voltage may be applied to a first side
  • a first applied voltage may be applied to a second side and a second applied voltage may be applied to a second side.
  • an EOF can be modulated by genetically engineering or selecting a pore structure which provides a specific steric and/or the electrostatic conditions within its inner channel that adjusts the preference for translocating one ion over another.
  • the net charge of the channel, or the geometry of the channel, or any combination thereof, can influence a flow of molecules through the channel.
  • the flowing molecules can be analytes, ions, water, or other molecules, or any combination thereof on a first side (e.g., cis side) or a second side (e.g., trans side) of a nanopore.
  • the flowing molecules can generate an ionic current from a flow of ions.
  • analyte translocates through a pore
  • other molecules such as ions
  • This obstruction in translocation of other molecules can change the ionic current by changing the rate of flow of ions.
  • This change in current can be measured, for example, by a pair of electrodes configured to measure a current from a first side (e.g., cis side) to a second side (e.g., trans side) across the nanopore.
  • a nanopore of a nanopore system described herein may employ alternative means of measuring the voltage-current properties of the nanopore system, such as those that employ fluorescence probes of ionic flux or field effect transistor systems than measure changes in voltage.
  • suitable detection methods such as tunneling, surface enhanced raman, plasmonics, and other spectroscopic methods that do not measure the ionic current and instead measure the properties of the target analyte in the nanopore directly.
  • the change in current can be measured by a pair of electrodes configured to measure a current from a first side (e.g., cis side) to a second side (e.g., trans side) across a membrane of which the nanopore may be disposed.
  • a narrow geometry of the channel can slow a progression of an analyte through a pore.
  • a change to a net charge or a geometry of a channel of a nanopore can change the flow of molecules through the pore.
  • changing (e.g., mutating) a channel to have a more negative net charge can reduce a flow of a negatively charged molecule (e.g., a chloride ion).
  • changing (e.g., mutating) a channel to have a more positive net charge can reduce a flow of a positively charged molecule (e.g., a potassium ion).
  • changing a channel to have a wider geometry can increase a flow of a larger molecule (e.g., a glucose molecule or a peptide analyte).
  • changing a channel to have a more negative net charge and a WSGR Docket Number: 64828-710.601 narrower geometry can reduce a flow of a large, negatively charged molecule (e.g., a glutamate ion).
  • the net charge of the channel can influence the flow of charged molecules through the nanopore.
  • the net charge of the channel can influence the flow of charged molecules through the nanopore.
  • a shift in the net charge can make some charged molecules translocate more easily through the pore.
  • At most about 10 amino acid residues, 5 amino acid residues, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues, 1 amino acid residue, or less than about 1 amino acid residue may be mutated in a nanopore described herein.
  • a nanopore may comprise a mutation (e.g., an insertion, deletion, substitution, or chemical modification, or any combination thereof) of one or more amino acid residues to one or more positively-charged amino acid residues, one or more negatively-charged amino acid residues, one or more aromatic amino acid residues, one or more acidic amino acid residues, one or more neutrally-charged amino acid residues, one or more basic amino acid residues, one or more amidic amino acid residues, or one or more sulfur-containing amino acid residues, or any combination thereof.
  • a mutation e.g., an insertion, deletion, substitution, or chemical modification, or any combination thereof
  • an EOF of the nanopore described herein comprising a a genetically engineered negatively charged region of the channel may comprise an EOF of at least about 1.1-fold, at least about 1.2- fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 7.0-fold, at least about 8.0-fold, at least about 9.0-fold, or at least about 10.0-fold greater than an EOF generated by a wild-type nanopore.
  • an EOF of the nanopore described herein comprising a a genetically engineered negatively charged region of the channel may comprise an EOF of at most about 10.0-fold, at most about 9.0-fold, at most about 8.0-fold, at most about 7.0-fold, at most about 6.0-fold, at most about 5.0-fold, at most about 4.5-fold, at most about 3.5-fold, at most about 3.0-fold, at most about 2.5-fold, at most about 2.0-fold, at most about 1.5-fold, at most about 1.4-fold, at most about 1.3-fold, at most about 1.2-fold, or at most about 1.1-fold greater than an EOF generated by a wild- type nanopore.
  • the net charge of the channel of a pore can be increased so as to electrostatically limit the flux of one species of ions in one direction across the pore, while retaining/enhancing the flux of an oppositely WSGR Docket Number: 64828-710.601 charged species of ions flowing in the opposite direction under an applied voltage.
  • the EOF can be enhanced by either adding more charges to the residues lining the walls of the channel, or narrowing the channel dimensions, or a combination thereof.
  • Charge and/or steric barriers to the flow of specific ions can also be created by chemical modification of the inner surface of a pore. For example, cysteine residues can be reacted with derivates of maleimide or iodoacetate.
  • a wide array of chemical modifications and/or reaction types can be used to modulate ion selectivity, including but not limited to modification of cysteines, modification of lysines, incorporation of unnatural amino acids, modification of unnatural amino acids with click chemistry groups, and the like.
  • Charge and/or steric barriers to the flow of specific ions can also be created by use of proteinaceous or chemical adapters inside the pore channel.
  • circular chemical adapters such as cyclodextrins or cucurbiturils can be incorporated into the pore.
  • protein based adapters can be employed, such as the CsgF subunit of the CsgG pore, which can separately be mutated and/or engineered to create steric and/or electrostatic barriers.
  • the protein or chemical adapters can be attached either by non-covalent docking or by covalent approaches.
  • Charge and/or steric barriers can be engineered into a pore channel by the addition of amino acids into the sequence in and around the regions that comprise the channel (e.g. into the beta-barrel transmembrane region of a beta-barrel pore such as alpha-hemolysin) to create a loop, turn, constriction or other extrusion that reduces the diameter of the pore.
  • charge and/or steric barriers can be created at either the cis or trans entrance to the pore channel, and away from the narrowest parts of the pore where analyte discrimination can be strongest, to create a locally depleted regions of charge that alter the ion-selectivity through the pore.
  • Ion-selectivity biases can also be generated by altering the system conditions or adding additives that change the properties of the water-facing residues in the channel of the pore.
  • the pH of the system can be adjusted, either on both side of the membrane or just one side of the membrane, to change the protonation state of the pore.
  • low pH can be employed (e.g.
  • high pH can be used (e.g. >8.0, or >10.5) to increase the net negative charge inside the pore to increase the bias towards cation flow.
  • additives that interact with the water- facing residues can be added to the solution to change the ionic or steric properties of the water-facing residues inside the pore.
  • EOF can be generated by providing different salt type(s), providing salt asymmetries, providing a certain pH, providing additives such as Guanidinium chloride or guanidine hydrochloride (abbreviated GdmCl and sometimes GdnHCl or GuHCl, GuCl) and osmotics.
  • GdmCl Guanidinium chloride or guanidine hydrochloride
  • EOF can be generated with a chemical gradient of an ion between two sides of a pore.
  • EOF can be generated with a strong asymmetric ion flow where there can be an asymmetric ion concentration between two sides of a pore.
  • the salt gradient between the compartments can be at least about 0.001 M, 0.01 M, 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, or greater than about 1.0 M. In some embodiments, a salt gradient between the compartments can be at most about 1.0 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.05 M, 0.01 M, 0.001 M, or less than about 0.001 M. In some embodiments, a salt gradient between the compartments can be between about 0.01 M to about 1 M.
  • a salt gradient between the compartments can be between about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.2 M, about 0.01 M to about 0.3 M, about 0.01 M to about 0.4 M, about 0.01 M to about 0.5 M, about 0.01 M to about 0.6 M, about 0.01 M to about 0.7 M, about 0.01 M to about 0.9 M, about 0.01 M to about 0.9 M, about 0.01 M to about 1 M, about 0.05 M to about 0.1 M, about 0.05 M to about 0.2 M, about 0.05 M to about 0.3 M, about 0.05 M to about 0.4 M, about 0.05 M to about 0.5 M, about 0.05 M to about 0.6 M, about 0.05 M to about 0.7 M, about 0.05 M to about 0.9 M, about 0.05 M to about 0.9 M, about 0.05 M to about 1 M, about 0.1 M to about 0.2 M, about 0.1 M to about 0.3 M, about 0.1 M to about 0.4 M, about 0.05 M to
  • a system described herein may comprise a fluidic chamber; and a membrane comprising a nanopore that separates the fluidic chamber into a first side (e.g., cis side) and a second side (e.g., trans side).
  • the first side can comprise a first solution.
  • the second side can comprise a second solution.
  • the first solution or concentration thereof and/or the second solution or concentration thereof may generate an EOF.
  • the first solution may comprise a concentration of a solute (e.g., a first solute).
  • a second solution may comprise a concentration of a solute (e.g., a second solute).
  • a solute may comprise one or more WSGR Docket Number: 64828-710.601 salts, electrolytes, osmolytes, ions, or any combination thereof.
  • the first solution comprises a first concentration of a solute and/or the second solution comprises a second concentration of a solute.
  • a difference between the first concentration of the solute and the second concentration of the solute may generate an electro-osmotic force (EOF).
  • EEF electro-osmotic force
  • the electro- osmotic force comprises a net ionic current flow from the first side (e.g., cis side) to the second side (e.g., trans side).
  • the electro-osmotic force comprises a net ionic current flow from the second side (e.g., trans side) to the first side (e.g., cis side).
  • a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be at least about 0.01 M, 0.05 M, 0.10 M, 0.20 M, 0.30 M, 0.40 M, 0.50 M, 0.60 M, 0.70 M, 0.80 M, 0.90 M, 1.00 M, 1.10 M, 1.25 M, 1.50 M, 1.75 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, 4.5 M, 5 M, or greater than about 5 M.
  • a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be at most about 5 M, 4.5 M, 4 M, 3.5 M, 3 M, 2.5 M, 2 M, 1.75 M, 1.5 M, 1.25 M, 1.10 M, 1.0 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.05 M, 0.01 M, or less than about 0.01 M.
  • a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be between about 0.01 M to about 5 M.
  • a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be between about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.25 M, about 0.01 M to about 0.5 M, about 0.01 M to about 0.75 M, about 0.01 M to about 1 M, about 0.01 M to about 2 M, about 0.01 M to about 3 M, about 0.01 M to about 4 M, about 0.01 M to about 5 M, about 0.05 M to about 0.1 M, about 0.05 M to about 0.25 M, about 0.05 M to about 0.5 M, about 0.05 M to about 0.75 M, about 0.05 M to about 1 M, about 0.01 M to about 2 M,
  • a salt or electrolyte comprises sodium chloride, potassium chloride, guanidinium chloride, guanidine hydrochloride, potassium glutamate, an alkali metal salt, a halide salt, an ionic liquid, or an WSGR Docket Number: 64828-710.601 organic salt.
  • salt applied electro-osmosis can be highly advantageous for creating or enhancing EOF under lower voltages where repulsive EPF effects on the polymer analyte may be reduced.
  • High asymmetry salt conditions may be used in combination with pores that may be engineered with enhanced ion-selectivity.
  • a difference in a concentration of a molecule between two sides of a membrane can modify an electro- osmotic flux by providing a competing or assisting osmotic flux.
  • a difference in concentration across a membrane can create an osmotic gradient, wherein a solvent (e.g., water) may diffuse across a membrane in the direction of a higher concentration of the molecule so as to minimize the difference in concentration between the sides of the membrane.
  • the osmotic gradient can be oriented so as to drive a water flow in the same direction as the electro-osmotic force, or in a different direction.
  • a high ion concentration on a first side (e.g., cis side) relative to a second side (e.g., trans side) can create an osmotic gradient that competes with a cis to trans electro-osmotic force, as the osmotic gradient can drive water flow in a trans to cis direction.
  • the ion concentrations may support a cis to trans electro-osmotic flow even if they also provide an osmotic gradient.
  • the EOF can be generated by an asymmetric salt distribution between the first side (e.g., cis side) of the membrane and the second side (e.g., trans side) of the membrane.
  • the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be different from the concentration of the one or more salts on the second side (e.g., trans side) of the membrane.
  • the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be higher than the concentration of the one or more salts on the second side (e.g., trans side) of the membrane.
  • the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be lower than the concentration of one or more salts on the second side (e.g., trans side) of the membrane.
  • the concentration of one or more salts on the second side (e.g., trans side) of the membrane can be higher than the concentration of the one or more salts on the first side (e.g., cis side) of the membrane. In some cases, the concentration of one or more salts on the second side (e.g., trans side) of the membrane can be lower than the concentration of the one or more salts on the first side (e.g., cis side) of the membrane. [0531] In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be between about 1 nanomolar (nM) to about 1,000 nM.
  • the concentration of one or more salts on the first side (e.g., cis side) of the membrane can at most about 1,000 nM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 95 nM, at most about 90 nM, at most about 85 nM, at most about 80 nM, at most about 75 nM, at most about 70 nM, at most about 65 nM, at most about 60 nM, at
  • a salt, ion, osmolyte, or electrolyte concentration on the first side can be at most about 5 M, at most about 4.5 M, at most about 4 M, at most about 3.5 M, at most about 3 M, at most about 2.5 M, at most about 2 M, at most about 1.75 M, at most about 1.50 M, at most about 1.25 M, at most about 1 M, at most about 0.90 M, at most about 0.80 M, at most about 0.70 M, at most about 0.60 M, at most about 0.50 M, at most about 0.40 M, at most about 0.30 M, at most about 0.20 M, at most about 0.10 M, at most about 0.05 M, at most about 0.01 M, or less than about 0.01 M.
  • a salt, ion, osmolyte, or electrolyte concentration on the first side can be from about 0.01 M to about 5 M.
  • the concentration of one or more ions on the first side (e.g., cis side) of the membrane can be between about 1 nanomolar (nM) to about 1,000 nM. In some instances, the concentration of one or more ions on the first side (e.g., cis side) of the membrane can be between about 1 nM to about 10 nM, between about 10 nM to about 100 nM, or between about 100 nM to about 1,000 nM.
  • the concentration of one or more ions on the first side (e.g., cis side) of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about WSGR Docket Number: 64828-
  • the one or more ions can comprise chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, or oxide, or any combinations thereof.
  • the one or more ions on the first side (e.g., cis side) of the membrane can comprise chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, or oxide, or any combinations thereof.
  • the one or more ions on the second side (e.g., trans side) of the membrane can comprise chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, or oxide, or any combinations thereof.
  • the one or more ions on the first side (e.g., cis side) of the membrane can be the same types of ions as the one or more ions on the second side (e.g., trans side) of the membrane.
  • one or more ions on the first side (e.g., cis side) of the membrane can be different types of ions from the one or more ions on the second side (e.g., trans side) of the membrane.
  • Salt imbalances on two sides of a pore can create strong osmotic gradients, which can either enhance or compete with EOF depending on the relative direction of the fluid flow.
  • the osmotic gradient can compete with the EOF.
  • the low salt compartment can contain an osmolyte to fully or partially balance the osmotic imbalance created by salt concentration asymmetry.
  • an osmolyte can comprise a non-ionic or a zwitterionic solute, e.g., glycine betaine, glucose, sucrose, glycerol, PEGs, or dextrans, or any combination thereof.
  • a salt imbalance of 0.5 M KCl can be balanced with about 1 M Glycine betaine.
  • Specific osmolytes can be selected and balanced based on their osmolarity and their concentrations.
  • osmolytes can be added either to symmetrical salt concentration or asymmetric salt concentration systems to create an osmotic gradient that acts in the same direction as the EOF to enhance the capture and/or translocation of an analyte.
  • osmolyte e.g.1 M glycine betaine
  • an ion-selective pore system e.g.
  • osmolytes can be added either to symmetrical salt concentration or asymmetric salt concentration systems to create an osmotic gradient that acts in a different direction as the EOF to enhance the capture and/or translocation of an analyte.
  • high mobility ions can be used on one side of a membrane and low mobility and/or sterically inhibited counterions on the other side of a membrane.
  • a salt with a high mobility ion can be used on the first side (e.g., cis side) of a membrane, and a salt with a low mobility (counter) ion used on the second side (e.g., trans side) of a membrane to create a stronger cis-to-trans ion-selectivity under an appropriate applied voltage.
  • low mobility ions comprises all or a part of a total ionic content in the system.
  • low mobility ions can comprise greater than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the low mobility ions flow across the pore.
  • low mobility ions can comprise less than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the low mobility ions flow across the pore.
  • high mobility ions can comprise greater than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the high mobility ions flow across the pore.
  • high mobility ions can comprise less than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the high mobility ions flow across the pore.
  • an analyte can be added to the first side (e.g., cis side) and translocated cis-to-trans via a strong cis-to-trans EOF –
  • the system in this instance can be a pore system set up with a highly mobile cation salt on the cis (e.g. K+, Na+, NH 4 +) and a low mobility anion salt on the trans (e.g. glutamate, or acetate, or any combination thereof), wherein a negative voltage can be applied to the trans.
  • a highly mobile cation salt on the cis e.g. K+, Na+, NH 4 +
  • a low mobility anion salt on the trans e.g. glutamate, or acetate, or any combination thereof
  • the system can be set up with 1M KCl in the cis compartment and 1M KGlu in trans compartment, so that a greater EOF can be achieved cis-to-trans under negative applied voltage to trans than the EOF generated trans-to-cis WSGR Docket Number: 64828-710.601 when a positive voltage can be applied to the trans, due to relative lower mobility of glutamate anions versus chloride anions.
  • immobile and/or sterically hindered ions on one side of the membrane may be combined with ion selective pores to limit the flux of one or more of the ions.
  • the ion permeability of large and/or immobile salts in ion-selective pore systems e.g.
  • the flux of immobile or sterically hindered ions can be effectively zero under an applied voltage, either for an open state and/or the analyte-filled state of a pore, so that all ionic flux can be in one direction (e.g. cis to trans).
  • small and/or highly mobile ions on one side of the membrane can be combined with ion selective pores to further increase flux of given ions. For example, small and/or highly mobile cations (e.g.
  • K+, Na+, or NH4+, or any combination thereof can be combined with ion-selective pores with high internal net negative charge.
  • the net negative charge inside a pore can interact favorably with cations, which can increase the absolute flux of the cations relative to the same pore with less negative charge.
  • the increased cation flux can increase the relative proportion of net electro-osmotic flux in one direction (e.g. cis-to-trans), and it can increase the absolute net electro-osmotic flux at a given voltage, which can create a stronger EOF versus EPF.
  • the EOF can be generated by a cation biased flux through a pore.
  • a cation- biased EOF (P (+) >> P (-) ) can be created or enhanced by the choice of salts in either the cis or trans compartments.
  • High cation biased EOF can be further enhanced by exploiting salts with large anions that may be relatively immobile or otherwise restricted from translocating through a pore.
  • an anion can comprise high molecular mass inorganic anions (e.g., Br, Phosphate, sulphate, or FeCN6, and or any combination thereof), organic anions (e.g., Acetate, Glutamate, succinate, maleate, or butyrate dextrans, or any combination thereof), or ionic liquid anions (e.g., tetrafluoroborate (BF4), hexafluorophosphate (PF6), bis-trifluoromethanesulfonimide (NTf2), trifluoromethanesulfonate (OTf), dicyanamide (N(CN)2), hydrogen sulphate (HSO4), and ethyl sulphate (EtOSO3)).
  • high molecular mass inorganic anions e.g., Br, Phosphate, sulphate, or FeCN6, and or any combination thereof
  • organic anions e.g., Acetate, Glutamate, succinate, maleate, or but
  • a nanopore system described herein can be set up with an ionic salt on the first side (e.g., cis side) and an ionic salt on the second side (e.g., trans side).
  • a nanopore system described herein can be set up with a cation salt on the first side (e.g., cis side) and an anion salt on the second side (e.g., trans side).
  • a nanopore system described herein can be set up with a anion salt on the first side (e.g., cis side) and an cation salt on the second side (e.g., trans side).
  • the cation may comprise any positively charged ion (e.g., K+, Ca2+, Na+, H+, H 3 O+, or NH 4 +, or any combination thereof).
  • the anion may comprise any WSGR Docket Number: 64828-710.601 negatively charged ion (e.g., Cl-, I-, OH-, O2-, SO 2- 4 , or NO - 3, or any combination thereof).
  • a nanopore system described herein can be set up with a highly mobile cation salt (e.g. K+, Na+, or NH + 4 , or any combination thereof) on the first side and a low mobility anion salt (e.g. glutamate, acetate, or FeCN 6 , or any combination thereof) on the trans.
  • a negative voltage may be applied to the first side (e.g., cis side), or the second side (e.g., trans side), or any combination thereof.
  • the cis and trans compartments can contain >0.3 M, >0.5 M, >1.0 M, >2.0 M, >3.0 M, or >4.0 M K Glu under an applied voltage of -40 mV to - 200 mV.
  • IrelV can be greater than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or about 1.0 pA/mV. In some embodiments, I relV can be less than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or about 1.0 pA/mV. In some embodiments, a pore can have greater than about 2, 4, 6, 8, 16, 24, 32, or about 64 pA of net electro- WSGR Docket Number: 64828-710.601 osmotic flow in one direction.
  • a chamber comprising a membrane comprising a pore can be subjected to an electrical potential such that an analyte can be electrophoretically and/or electroosmotically translocated through the pore.
  • the optimal value (absolute and direction) of the potential may depend on parameters such as the ion-selectivity of the pore, characteristics like size, shape and pI of the analyte, and whether analytes may be captured from the second side (e.g., trans side) or first side (e.g., cis side) of the pore.
  • a membrane comprises a solid state material (e.g. SiN).
  • a pore may comprise a solid state hole.
  • a pore can comprise a biological pore inserted in a solid state material.
  • a membrane may be a solid- WSGR Docket Number: 64828-710.601 state membrane.
  • a membrane may comprise silicon dioxide, silicon nitride, graphene, anodic alumina, polycarbonate, poly(ethylene terephthalate), polytetrafluoroethylene, polyurethane, thermoplastic polyurethane, boron nitride, molybdenum disulfide, transition metal carbide, gold, or platinum, or combinations thereof.
  • a head group comprises a neutral head group, such as diacylglycerides (DG) and ceramides (CM); a zwitterionic head group, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM); a negatively charged head group, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); or a positively charged headgroup, such as trimethylammonium-Propane (TAP).
  • DG diacylglycerides
  • CM ceramides
  • a zwitterionic head group such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM)
  • a negatively charged head group such as phosphatidylglycerol (PG); phosphati
  • a lipid can also be chemically-modified.
  • the bead group or the tail group of a lipid may be chemically- modified.
  • a membrane may be at less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or about 150 nm thick.
  • the system may further comprise methods for measuring a signal based on ionic current flowing through the nanopore during a period of time of translocation. These measuring mechanisms are set up to detect changes in the signal that reflect characteristics of the analyte as it can be translocated.
  • the analyte measured can be a protein.
  • the characteristic of the nucleic acid molecule measured can comprise a nucleotide sequence of the nucleic acid molecule, nucleotide mutations in the sequence of the nucleic acid molecule, methylation of the nucleic acid molecule, acetylation of the nucleic acid molecule, length of the nucleic acid molecule, net charge of the nucleic acid molecule, or conformation of the nucleic acid molecule.
  • the analyte measured can be an oligosaccharide.
  • the characteristic of the oligosaccharide measured can comprise a sequence of the oligosaccharide, the length of the oligosaccharide, the net charge of the oligosaccharide, presence or absence of coupled lipids, presence or absence of coupled peptides, or structure of the oligosaccharide.
  • the analyte measured can be a lipid molecule.
  • the characteristic of the lipid measured can comprise length of the lipid, the net charge of the lipid, or a structure of a lipid.
  • a change in ionic current can be measured while the analyte translocates through the nanopore.
  • the change in ionic current can be measured by a voltage based chip.
  • the voltage based chip can measure the voltage and/or change in current across the nanopore.
  • the voltage based chip can be a trans electrode (e.g. electrodes adjacent to the membrane/nanopore to measure the voltage across the nanopore).
  • the characterization methods may involve measuring the ion current flow through the pore, typically by measurement of a current. Alternatively, the ion flow through the pore may be measured optically. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore.
  • an analyte comprises a polymer analyte.
  • the analyte can comprise a nucleic acid based polymer analyte or a non-nucleic acid based polymer analyte.
  • the analyte can be of synthetic, semi- synthetic, or biological origin.
  • a synthetic analyte may comprise an analyte constructed by a non- biological chemical process, such as polyethylene glycol (PEG), or a synthetically constructed DNA molecule.
  • a synthetic analyte may comprise an analyte constructed by a non-biological chemical process, such as polyethylene glycol (PEG), synthetically constructed peptides of proteins, or a synthetically constructed DNA molecule.
  • a biological analyte can comprise an analyte produced by a biological process, such as a protein produced by a cell or by systems employing cellular (or cellular derived) components (e.g. enzymatic in vitro translation systems).
  • a biological analyte can comprise an analyte produced by a biological process, such as a protein produced by a cell.
  • the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore.
  • the characterization methods may be carried out using a patch clamp or a voltage clamp.
  • the characterization methods preferably involve the use of a voltage clamp.
  • the characterization methods may be carried out on an array of nanopores or wells where each array comprises 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more nanopores or wells.
  • the characterization methods may involve the measuring of a current flowing through the pore.
  • the method can be typically carried out with a voltage applied across the membrane and pore.
  • the voltage used can be typically from +2 V to -2 V, typically -400 mV to +400mV.
  • the voltage used can be in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from +10 mV, 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV.
  • the voltage used can be in the range 20 mV to 240mV, or the the voltage can be in the range of 120 mV to 220 mV.
  • an analyte comprises a polymer analyte.
  • the analyte can comprise a nucleic acid based polymer analyte or a non-nucleic acid based polymer analyte.
  • the analyte can be of synthetic, semi- WSGR Docket Number: 64828-710.601 synthetic, or biological origin.
  • a synthetic analyte may comprise an analyte constructed by a non- biological chemical process, such as polyethylene glycol (PEG), or a synthetically constructed DNA molecule.
  • a biological analyte can comprise an analyte produced by a biological process, such as a protein produced by a cell.
  • a semi-synthetic analyte can comprise portions created by biological and non-biological origins, for example, a biologically-produced protein conjugated to a PEG molecule.
  • an analyte comprises a protein (e.g., a polypeptide) or peptide.
  • a protein or peptide can comprise a folded state, an unfolded state, or intermediate states thereof (e.g., a partially unfolded state).
  • a folded state comprises a state of a protein or peptide in which the polymer can be at a low-energy state such that the protein or peptide maintains a two or three dimensional structure.
  • This low-energy state can be based on the interactions of the amino acids of the peptide or protein with each other.
  • An unfolded state can comprise a state of a protein or peptide in which the polymer can be at a high-energy state such that the protein or peptide does not maintain a two or three dimensional structure.
  • An intermediate state between a folded and unfolded state can be an energy state at which a portion or portions of the peptide or protein may maintain a two or three dimensional structure, and other portions of the peptide or protein may not maintain a two or three dimensional structure.
  • a protein e.g., polypeptide
  • a peptide can comprise a folded protein structure.
  • a peptide can comprise a linear structure.
  • a peptide can comprise a portion of a protein.
  • methods described herein may comprise translocating a plurality of analytes. The plurality of analytes may be translocated through (i) a nanopore disposed in a membrane, (ii) a plurality of nanopores disposed in a membrane, or (iii) a plurality of nanopores disposed in a plurality of membranes, or (iv) any combination thereof.
  • the plurality of analytes can comprise a plurality of non-nucleic acid based polymer analytes (e.g., a plurality of proteins, a plurality of polypeptides, a plurality of peptides, or any combination thereof).
  • a plurality of signals or changes thereof e.g., plurality of currents or changes thereof
  • the plurality of signals or changes thereof may be used to generate a plurality of characteristics associated with the plurality of analytes.
  • a characteristic of the plurality of characteristics can comprise a characteristic of an analyte described herein.
  • the WSGR Docket Number: 64828-710.601 plurality of characteristics comprises differences between at least a subset of analytes of a plurality of analytes.
  • the plurality of characteristics can comprises one or more differences in sequence.
  • the differences in sequence can be of at least about 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 10 amino acids, or greater than about 10 amino acids.
  • the differences in sequence can be of at most about 10 amino acids, at most about 5 amino acids, at most about 4 amino acids, at most about 3 amino acids, at most about 2 amino acids, at most about 1 amino acid, or less than about 1 amino acid.
  • the difference in sequence may be of at most 10 units between least the subset of analytes.
  • a relative concentration and/or an absolute concentration of one or more analytes may be determined.
  • the relative concentration and/or absolute concentration of one or more analytes may be a concentration in a plurality of analytes.
  • a plurality of analytes may comprise a subset (e.g., percentage) of analytes that can be modified.
  • a percentage of modified analytes may be determined from a plurality of analytes.
  • one or more analytes may be captured by a nanopore with an EOF.
  • the one or more analytes may be captured by a nanopore with an EOF and/or a motor protein (e.g., a translocase).
  • the translocase may comprise a translocase as described herein.
  • the translocase may unfold the one or more analytes and/or assist in translocating the analyte through the nanopore.
  • analyte e.g., a peptide, polypeptide, or protein, or fragment thereof, or combination thereof
  • the analyte may be captured from a second side (e.g., a trans side).
  • an analyte may be captured on one side of a nanopore system and translocated by a motor protein (e.g., a translocase).
  • the analyte may be translocated with a motor protein in a same direction as a net EOF.
  • the analyte may be translocated with a motor protein in a different direction as a net EOF.
  • a target protein may be translocated as shown in FIGs. 138A-138E by operations of (i) adding the analyte (7) to a second side (e.g., a trans side) and one or more motor proteins (8) to a first side (e.g., a cis side) (FIG. 138A).
  • a voltage may be applied and/or set up in the nanopore system to provide an EOF (e.g., a strong net trans-to-cis EOF).
  • An analyte may be captured from a second side (e.g., a trans side) via EOF and/or electrophoresis acting with the analyte and/or a leader construct (1).
  • the analyte may be able to translocate under the strong net trans-to-cis EOF until encountering a stopper domain (2).
  • a stopper domain may be coupled to a C-terminus of an analyte.
  • a stopper domain may be coupled to a N- terminus of an analyte.
  • the stopper domain may prevent further translocation of the analyte through the nanopore (FIG.138B).
  • the translocase in the first side (e.g., cis side) of the system may bind to a leader construct and/or at least a portion of the analyte (FIG. 138C). An applied voltage may be reversed.
  • the reversal of the applied voltage may switch the direction of a net EOF of the system.
  • WSGR Docket Number: 64828-710.601 reversing the voltage may switch a net EOF from a trans-to-cis direction to a cis-to-trans direction.
  • An analyte may translocate back through to a side of the system (e.g., a trans side) after reversal of the voltage.
  • a bound motor protein e.g., translocase
  • the translocase on the top of the nanopore may continue to translocate along an analyte.
  • the analyte may be translocated in a direction opposite to a net EOF.
  • the translocase may translocate an analyte in a trans-to-cis direction against the net cis-to-trans direction of the EOF.
  • the net cis-to-trans direction of the EOF may maintain a stretch of the analyte and/or maintain the translocase against the nanopore (FIG. 138D).
  • Leader constructs can comprise one or more highly charged polymeric capture motifs as described herein.
  • a capture motif may comprise high polyanion (if negative voltage may be applied at a second side (e.g., trans side)) or polycation (if positive voltage may be applied at a second side (e.g., trans side)) content.
  • the motif may comprise (R)n, (K)n, or (H)n, or variations thereof, or any combination thereof.
  • the motif may comprise (D)n, or (E)n, or variations thereof, or any combination thereof.
  • Amino acid sequences of leader constructs are shown in Table 45. Table 45. Amino acid sequences of leader constructs. Leader SEQ ID number Leader description Leader sequence NO.
  • a stopper domain may comprise one or more steric stopper motifs comprising GFP, titin, or maltose binding protein (MBP), or any combination thereof.
  • a stopper domain may comprise a plurality of folded proteins that may fulfill two criteria: (i) the proteins may be too large to pass through a nanopore without being unfolded, and/or (ii) the proteins may be able to be unfolded by the force of a translocase pulling the analyte or at least a portion of an analyte into the nanopore after it binds on the opposite side of the membrane.
  • a stopper domain may comprise one or more electrostatic stopper motifs comprising one or more regions of opposite charge to the capture motif. For example, if a capture motif may be polyanionic, then a stopper domain may be is polycationic. These “dipole” combination of one or more charges can prevent full translocation as the one or more charges between a capture motif and a stopper motif can balance under electrophoresis at the mid-point when both may be in the nanopore. At this point, an analyte may be left to reside in the port.
  • a stopper domain can comprise one or more steric motifs, or one or more electrostatic motifs, or variants thereof, or any combination thereof.
  • FIG.139 shows a schematic of the electrophysiology signal obtained from implementing a nanopore system described herein and as depicted in FIGs.138A-138E.
  • State (i) can correspond to an unoccupied open- pore state; state (ii) can correspond to a capture of an analyte and/or at least a portion of an analyte.
  • the analyte may be captured by a leader construct under electrophoretic forces (and against the EOF) into the second side (e.g., trans side) of the nanopore. This may lead to an block (e.g., an instantaneous block) measured in the current (e.g., state (iii)).
  • the nanopore system may detect state (ii) and may hold a system static for the duration of state (iii) to enable one or more translocases to bind to at least a portion of a leader construct in a first side (e.g., cis side).
  • a system may reverse a voltage and an EOF may be reversed.
  • the analyte may then translocates in an opposite direction (e.g., a cis-to-trans direction) quickly until a bound translocase contacts the top of the nanopore.
  • the translocase may pull the analyte out of the nanopore in a direction (e.g., WSGR Docket Number: 64828-710.601 a trans-to-cis direction), which may lead to sequence of changing current levels representative of the composition of the analyte (state v) until the analyte is pulled out of the nanopore and exits (state vi).
  • a direction e.g., WSGR Docket Number: 64828-710.601 a trans-to-cis direction
  • the analyte may be captured from a side (e.g., a trans side).
  • an analyte may be captured on one side of a nanopore system and translocated by one or more motor proteins (e.g., one or more translocases).
  • the analyte may be translocated with a motor protein in a same direction as a net EOF (e.g., a net cis-to-trans EOF).
  • a net EOF e.g., a net cis-to-trans EOF
  • FIG.140A an analyte may be added to a trans side of a system and a motor protein (e.g., a translocase) may be added to a cis side.
  • a voltage may be applied to the system to establish a net trans-to-cis EOF.
  • the analyte may be captured from a second side (e.g., a trans side) via EOF and/or an electrophoretic force (EPF) acting on the analyte and/or a leader construct.
  • the analyte may be able to translocate under the strong net trans-to-cis EOF until encountering a stopper domain (2) (FIG. 140B).
  • one or more motor proteins e.g., one or more translocases
  • the one or more translocases may bind along at least one portion of an analyte (FIG.140C). An applied voltage may be reversed.
  • the reversal of the applied voltage may switch the direction of a net EOF of the system. For example, reversing the voltage may switch a net EOF from a trans-to-cis direction to a cis-to-trans direction.
  • An analyte may translocate back through to a side of the system (e.g., a trans side) after reversal of the voltage.
  • a bound motor protein e.g., translocase
  • FIG.141 shows a schematic of the electrophysiology signal obtained from implementing the system described herein and as depicted in FIGs. 140A-140E. State (i) can correspond to the unoccupied open-pore state, state (ii) can correspond to a capture of an analyte via its leader construct under electrophoretic forces and/or against an EOF into the nanopore.
  • the system can detect state (ii) and may hold the system static for a duration of state (iii). Holding the system static may enable translocases to bind to the portion of the leader in a first side (e.g., a cis side).
  • a system may then reverse a voltage and the EOF can be reversed.
  • the reversal of EOF may translocate the analyte in an opposite direction (e.g., cis-to-trans) very quickly until one or more bound translocases contact the top of a nanopore.
  • FIGs. 142A-142E illustrate a system for translocating an analyte (e.g., a peptide, polypeptide, or protein, or fragment thereof, or combination thereof) through a nanopore.
  • the analyte (7) may be captured from a side (e.g., a trans side).
  • an analyte may be captured on one side of a nanopore system WSGR Docket Number: 64828-710.601 and translocated by one or more motor proteins (e.g., one or more translocases).
  • the analyte may be translocated with a motor protein (8) in a same direction as a net EOF (e.g., a net cis-to-trans EOF).
  • the analyte may be translocated with a motor protein in a different direction as a net EOF.
  • An analyte may be added to a second side (e.g., a trans side) and a motor protein to a first side (e.g., a cis side) (FIG. 142A).
  • a voltage may be applied or set up in the system so that a strong net cis-to-trans EOF can be applied to the system.
  • An analyte can be captured from the second side (e.g., trans side) via electrophoresis acting on a charged leader construct (1) comprising a stopper domain (2).
  • the stopper domain may allow at least a portion of an analyte to translocate across the nanopore so that a portion of the substrate may be exposed to the cis solution (FIG. 142B).
  • the motor protein in a first side (e.g., cis side) may bind to at least a portion of a leader.
  • the motor protein may translocate along the analyte until it contacts an opening of a nanopore (FIG. 142C).
  • the translocase may overcome a stopper domain and pull an analyte through the nanopore in a direction that can be opposite to the EOF.
  • a translocase may overcome a stopper domain and pull an analyte through the nanopore in a trans-to-cis direction against a net cis-to-trans EOF (FIG. 142D).
  • FIG.143 shows a schematic of the electrophysiology signal obtained from implementing the system described herein and in FIGs. 142A-142E.
  • State (i) corresponds to the unoccupied open-pore state
  • state (ii) corresponds to the capture of an analyte via its leader under electrophoretic forces (and against the EOF) into the nanopore. This capture may lead to an instantaneous block measured in the current (state iii).
  • one or more translocases can bind to at least a portion of a leader construct in the first side (e.g., cis side) compartment and begin to translocate along the polypeptide (in the cis-to-trans direction).
  • a leader construct in the first side e.g., cis side
  • the translocase contacts the top of the nanopore it begin to start pulling the polypeptide through the nanopore trans-to-cis (state iv) leading to sequence of changing current levels representative of the composition of the polypeptide (state v) until the polypeptide is pulled out of the nanopore and exits to the cis side (state vi).
  • analyte e.g., a peptide, polypeptide, or protein, or fragment thereof, or combination thereof
  • the analyte (7) may be captured from a side (e.g., a cis side).
  • an analyte may be captured on one side of a nanopore system and translocated by one or more motor proteins (e.g., one or more translocases).
  • the analyte may be translocated with a motor protein (8) in a same direction as a net EOF (e.g., a net cis-to-trans EOF).
  • a net EOF e.g., a net cis-to-trans EOF
  • an analyte may be added to a first side (e.g., a cis side) and one or more motor proteins may be added to the first side (e.g., a cis side).
  • One or more motor proteins e.g., one or more translocases
  • the bound one or more translocases may translocate along the analyte (e.g., forming a “train of enzymes” along the analyte) (FIG.144A).
  • a voltage may be applied and/or a system may be set up to provide a net EOF (e.g., a net cis-to-trans EOF) to the system.
  • An analyte and/or at least a portion of an analyte may be WSGR Docket Number: 64828-710.601 captured with one or more bound translocases.
  • the analyte:translocase complex may be capture from a first side (e.g., a cis side) via an EOF and/or EPF which may act on the analyte.
  • the EOF and/or EPF can act on an analyte comprising a leader construct.
  • the analyte may be translocated near a nanopore.
  • One or more translocases may contact a nanopore (FIG.144B).
  • the one or more translocases may continue to control translocation of the analyte and/or at least a portion of the analyte.
  • the at least a portion of the analyte may translocate into the nanopore in a same direction as a net EOF (e.g., both in a cis-to-trans direction) (FIG. 144C).
  • the analyte may be released on a first side (e.g., a cis side) (FIG. 144D) and/or an analyte may be released on a second side (e.g., a trans side).
  • FIG.145 shows a schematic of the electrophysiology signal obtained from implementing the system described herein and in FIGs.144A-144D.
  • State (i) corresponds to the unoccupied open-pore state
  • state (ii) corresponds to the capture of the analyte with one or more translocases bound, which may continue to control the translocation of the analyte cis-to-trans (state iii) until the analyte exits the nanopore on the trans side (state iv).
  • DEVICES [0574]
  • the present disclosure provides systems, apparatus, instruments, devices, components, and/or elements comprising a pore, a membrane, an analyte, a sample, a processor, and/or a computer disclosed herein.
  • a device can provide an electric potential (e.g., from an electrical energy source such as a battery) across a membrane.
  • an electric potential e.g., from an electrical energy source such as a battery
  • a pore can be embedded in a membrane, and the membrane can be provided in a device, wherein the membrane provides a partition between two solutions.
  • FIGS. 71A- 71B illustrate a device, in accordance with some embodiments.
  • One or both of the solutions can comprise an electrolyte, and an electric potential can be applied across the membrane such that a net ionic current can be generated from one side of the membrane to the other.
  • the net ionic current can be a result of ions in the electrolyte passing through the pore, in response to the applied electric potential.
  • the ionic current can be faradaic current.
  • the net ionic current can be expected to maintain a constant value when the electric potential and other environmental variables are held constant. However, when an analyte can be captured by a pore, the analyte can translocate through the pore from one side of the membrane to another. During the translocation, the analyte can create a perturbation in the net ionic current.
  • the perturbation can arise from multiple mechanisms, including at least one of: (1) steric repulsion of ions from the channel – the analyte can physically obstruct a pore channel, thereby inhibiting or prohibiting ions from passing through the channel; (2) electrostatic attraction/repulsion of ions from/to the channel – the analyte can carry electrostatic charges which repel like- WSGR Docket Number: 64828-710.601 charged ions, and can attract ions to the channel – an ion may “piggy-back” with the analyte through a pore; (3) induction of conformational change of the pore – the presence of an analyte can change the conformation of a pore, which can promote or demote ion passage through the channel.
  • an analyte passing through a pore can cause net ionic current to be perturbed.
  • a device can be configured to measure a perturbation of the net ionic current.
  • the perturbation created by a translocation event can be measured in one or more ways. One method can be to measure the ionic current from one side of the membrane to the other side. Another method can be to measure electric potential from one side to the other side.
  • the impedance can be measured.
  • the conductivity can be measured.
  • current rectification can be measured.
  • an optical signal can be measured.
  • a tunneling current can be measured.
  • fluorescence probes for reporting ionic flux or field effect transistor systems can be used to measure properties of a translocation event.
  • surface enhanced Raman, plasmonics, or other spectroscopic techniques can be used to measure the property of the analyte, the pore, or both, directly.
  • perturbations can be measured without the application of an electric potential, e.g., a chemical gradient of ions and/or analytes can provide the driving force for translocation of analytes and create measurable signals.
  • the applied potential can be a chemical potential, electric potential, or another form of potential that can be sufficient to provide a gradient for analyte translocation and measurable perturbations.
  • a device can comprise electrodes, spectroscopy tools, or microscopes, or any combination thereof to measure the signals.
  • An applied electric potential can be maintained at a constant or fluctuating voltage for a fixed period (milliseconds, seconds, minutes, hours).
  • the voltage can be changed in discrete steps to alter the sensing conditions and/or obtain different information from the analytes.
  • the voltage can be constantly changing, such as periodic waveforms (e.g. square wave, triangular wave, or sinusoidal, or any combination thereof). Waveforms of different amplitudes, frequencies, and shapes can be used to translocate analytes, which can produce different signals from the same analytes.
  • an applied electric potential can have a voltage from +50 mV to -50 mV, or +100 mV to -100 mV. In some embodiments, an applied electric potential can have a voltage of at least about -300 mV, -300 mV, -150 mV, -100 mV, -50 mV, -20 mV and about 0 mV.
  • an applied electric potential can have a voltage of at most about +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, about +300 mV. In some embodiments, an applied electric potential can have a voltage of +/-50 mV to +/-150 mV or +/- 50 mV to +/-100 mV.
  • a device can comprise one or more active electronics to provide a constant or a modulating electric potential.
  • measurements can be made using a single channel recording equipment. In some embodiments, measurements can be made using multi-channel systems.
  • measurements can be made using multi-channel systems that may be capable of simultaneously acquiring signals from multiple independent pore systems (e.g. a plurality of membranes containing inserted pores).
  • a recording equipment or multi-channel system can comprise integrated circuits, e.g., an ASIC, and/or transistor, e.g., CMOS.
  • a device can comprise a pore embedded in a membrane, and a source for applying an electrical potential to the membrane, and a recording device. The device can be used to identify any one of the analytes disclosed herein.
  • a device can comprise a sensor.
  • a sensor can comprise a membrane and a pore.
  • a device can comprise an array of sensors. In some embodiments, a device can be configured to receive a sample. In some embodiments, a sensor can be configured to contact a sample. In some embodiments, a device can be configured to process the sample. In some embodiments, a sensor can comprise a membrane and a pore. In some embodiments, a sensor can comprise a membrane and an array of pores disposed in the membrane. In some embodiments, an array of pores may comprise more than about 1, 2, 4, 8, 16, 24, or 32 pores. In some embodiments, a sensor can comprise an array of membranes. In some embodiments, a sensor can comprise an array of different membranes. In some embodiments, a sensor can comprise an array of different pores.
  • a sensor can comprise an array of identical membranes. In some embodiments, a sensor can comprise an array of identical pores. In some embodiments, a sensor can comprise two or more electrodes. In some embodiments, an electrode comprises a sensing electrode. In some embodiments, an electrode comprises an anode or a cathode. In some embodiments, a sensor comprises a plurality of electrodes, comprising at least anodes and cathodes. In some embodiments, a sensor can comprise two sensing electrodes. In some embodiments, a sensor can comprise a voltage sensor configured to measure a voltage difference between the two sensing electrodes. In some embodiments, a sensor can comprise a current sensor configured to measure a current between the two sensing electrodes.
  • a sensor can comprise a current sensor configured to measure a difference in current between the two sensing electrodes.
  • a sensor can comprise a FET sensor.
  • the sensor electrodes can be located near the nanopore or membrane to sense the local voltage.
  • a sensor may be a part of a sensor array.
  • each sensor in a sensor array comprises its own set of one or more electrodes. The one or more electrodes can be electrically coupled to a channel in the sensor array.
  • a device comprises multiple different pores to generate different characteristic signals from analytes, which signal can be compared or combined to improve their discrimination and/or characterization.
  • a device can be configured to perform single molecule analysis.
  • a membrane may have a surface area from about 5 nm2 to about 1000 mm2. In some embodiments, a membrane may have a surface area less than about 5nm2. In some embodiments, a membrane may have a surface area greater than about 1000 mm2. In some embodiments, a membrane may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2.
  • a membrane may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 ⁇ m2.
  • a membrane may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2.
  • a combined surface area of a plurality of membranes may have a surface area from about 5 nm2 to about 1000 mm2. In some embodiments, a combined surface area of a plurality of membranes may have a surface area less than about 5nm2.
  • a combined surface area of a plurality of membranes may have a surface area greater than about 1000 mm2. In some embodiments, a combined surface area of a plurality of membranes may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2.
  • a combined surface area of a plurality of membranes may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 ⁇ m2.
  • a combined surface area of a plurality of membranes may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2.
  • a pore may have a surface area on a membrane of more than about 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 36, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450 or about 500 nm2. In some embodiments, a pore may have a surface area on a membrane of less than about 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 36, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450 or about 500 nm2.
  • each sensor in the sensor array can be fluidically coupled to the same fluidic system. In some embodiments, at least two sensors in the sensor array can be fluidically coupled to different fluidic systems. In some embodiments, the device can provide the same electrolyte solution to a side of the membrane of each sensor in the sensor array. In some embodiments, the device can provide a different WSGR Docket Number: 64828-710.601 electrolyte solution to a side of the membrane of each sensor in the sensor array. In some embodiments, the fluid environment of each sensor in the sensor array can be controlled individually. In some embodiments, each sensor in the sensor array can be fluidically isolated from one another.
  • a sensor array comprises a plurality of chambers.
  • a sensor array comprises a plurality of chambers, wherein a plurality of membranes form a plurality of surfaces of the plurality of chambers.
  • a plurality of electrodes may be disposed on a second plurality of surfaces within the plurality of chambers.
  • the sensor array comprises an adhesive configured to adhere the plurality of membranes to the plurality of chambers.
  • a plurality of chambers comprise at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 ⁇ L in volume. In some embodiments, a plurality of chambers comprise less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 ⁇ L in volume.
  • a plurality of chambers comprise a thickness of less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.50, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.25, 4.50, 4.75, or about 5 mm.
  • a plurality of chambers comprise a thickness of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.50, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.25, 4.50, 4.75, or about 5 mm.
  • a chamber of the plurality of chambers comprise at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, 1000, 5000 or about 10000 nL in volume.
  • a chamber of the plurality of chambers comprise less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, 1000, 5000 or about 10000 nL in volume.
  • a plurality of surfaces may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2.
  • a plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 ⁇ m2.
  • a plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2.
  • WSGR Docket Number: 64828-710.601 In some embodiments, a plurality of surfaces may have a surface area greater than about 1000 mm2. In some embodiments, a plurality of surfaces may have a surface area less than about 5 nm2.
  • a surface of the plurality of surfaces may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2.
  • a surface of the plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 ⁇ m2.
  • a surface of the plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2.
  • the plurality of membranes comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or about 1000000 pores.
  • the plurality of membranes comprises less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or about 1000000 pores.
  • a device can comprise a circuit that can both apply the voltage and measure the current. In some embodiments, the device comprises one circuit to apply the voltage difference and another to measure the current.
  • a voltage difference can be created with an asymmetric salt across the membrane.
  • a device comprises one or more electrodes directly associated with a pore at or near the pore channel or the opening. In some embodiments, one or more electrodes may be placed within the cis and/or trans chambers. In some embodiments, electrodes may be capable of detecting differences in ionic current around two chambers or tunneling electrical current around the pore channel or opening.
  • a device can comprise or be coupled to a computer or a processor.
  • a processor can be configured to receive a signal from a sensor. In some embodiments, a processor can be configured to receive an electrical signal from a sensor.
  • a processor can be configured to receive an optical signal from a sensor. In some embodiments, a processor can be configured to receive a mechanical signal from a sensor. In some embodiments, a processor can be configured to process a signal to generate an identification of an analyte. In some embodiments, a processor can be configured to process a signal to generate a probability of an identification of an analyte. In some embodiments, a processor can be configured to denoise a signal. In some embodiments, a processor can be configured to WSGR Docket Number: 64828-710.601 process a signal to generate a sequence of an analyte. In some embodiments, a processor can be configured to generate a file comprising a signal.
  • a processor can be configured to generate a file comprising an identification of an analyte.
  • a device can analyze multiple analytes simultaneously. In some embodiments, a device can analyze multiple analytes at a high throughput. [0595] In some embodiments, the device can sense an analyte at a sensing throughput of 128 simultaneous reads to 16,384 simultaneous reads. In some embodiments, the device can sense an analyte at a sensing throughput of 128 to 256, 128 to 512, 128 to 1,024, 128 to 2,048, 128 to 4,096, 128 to 8,192, 128 to 16,384 simultaneous reads.
  • the device can sense an analyte at a sensing throughput of about 128, 256, 512, 1,024, 2,048, 4,096, 8,192, or 16,384 simultaneous reads. In some embodiments, the device can sense an analyte at a sensing throughput of at least about 128, 256, 512, 1,024, 2,048, 4,096, 8,192, or 16,384 simultaneous reads. [0596] In some embodiments, the device can sense an analyte at a sensing throughput of 1,000 reads per run to 100,000,000 reads per run.
  • an aqueous solution can be used to collect lipids from an internal surface of a device, and then form a lipid bilayer membrane.
  • an aqueous solution can comprise one or more properties that are close to physiological conditions.
  • a physiologically acceptable solution can be buffered to a pH of 3 to 9. The pH of the solution can be adapted to the lipids used, and the intended application of a lipid bilayer membrane.
  • an operating parameter comprises an electrolyte concentration on a first side (e.g., cis side), an electrolyte concentration on a second side (e.g., trans side), an electrical potential, a flow rate, a sample preparation operation, a recording device operating condition, a sample source, determining a portion of the plurality of chambers to direct sample flow towards, determining a portion of the plurality of chambers to stop directing sample flow towards, or a selection of a portion of the sensor array to analyze, or combinations thereof.
  • a first side e.g., cis side
  • an electrolyte concentration on a second side e.g., trans side
  • an electrical potential e.g., a flow rate, a sample preparation operation, a recording device operating condition, a sample source, determining a portion of the plurality of chambers to direct sample flow towards, determining a portion of the plurality of chambers to stop directing sample flow towards, or a selection of a portion of the sensor array to analyze,
  • a recording device can record an electrical signal at a sampling rate of at most about 1, 2 ,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, WSGR Docket Number: 64828-710.601 600, 7000, 8000, 9000, or about 10000 kHz.
  • In recording device can be configured to filter an electrical signal.
  • a recording device can perform Bessel filtering.
  • the Bessel filtering can comprise a filter frequency of at least about 1, 2 ,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 600, 7000, 8000, 9000, or about 10000 kHz.
  • the Bessel filtering can comprise a filter frequency of at most about 1, 2 ,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 600, 7000, 8000, 9000, or about 10000 kHz.
  • the plurality of electrical signals may be used to determine one or more characteristics of the plurality of analytes.
  • the one or more controllers may repeat detection, translocation, or determination of one or more characteristics, or any combination thereof at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, at least about 500 times, or greater than about 500 times.
  • one or more samples can comprise a plurality of analytes (e.g., a plurality of non-nucleic acid based polymer analytes). At least a subset of the plurality of analytes may undergo any of the methods and/or system disclosed herein. In some embodiments, (i) a plurality of currents or changes thereof, (ii) a plurality of voltages or changes thereof, or (iii) a plurality of resistances or changes thereof, or (iv) any combinations thereof, may be used to determine a plurality of characteristics.
  • analytes e.g., a plurality of non-nucleic acid based polymer analytes. At least a subset of the plurality of analytes may undergo any of the methods and/or system disclosed herein.
  • a plurality of currents or changes thereof e.g., a plurality of voltages or changes thereof, or (iii) a plurality of resistances or changes thereof, or (i
  • the plurality of characteristics can comprise at least about 1 characteristics, at least about 2 characteristics, at least about 3 characteristics, at least about 4 characteristics, at least about 5 characteristics, at least about 10 characteristics, at least about 50 characteristics, at least about 100 characteristics, or greater than about 100 characteristics. At least a subset of a plurality of characteristics may be used to characterize the one or more properties of a sample.
  • the first side, second side, third side, or fourth side, or any combination thereof may be different sizes.
  • the first side, second side, third side, or fourth side, or any combination thereof may be the same size.
  • One or more sides of a first nanopore system may be larger in size than one or more sides of a second nanopore system.
  • One or more sides of a first nanopore system may be smaller in size than one or more sides of a second nanopore system.
  • a first side and a third side may be larger in size than a second side and a fourth side, respectively.
  • a first side and a third side may be a same size as a second side and a fourth side, respectively.
  • the first side, second side, third side, or fourth side, or any combination thereof may comprise an enclosed compartment.
  • the first side, second side, third side, or fourth side, or any combination thereof may comprise an open compartment.
  • the first side, second side, third side, or fourth side, or any combination thereof may comprise a continuous phase (e.g., a continuous aqueous phase).
  • a device can comprise a computer, a software module, a non-transitory computer readable storage medium, a database, and/or a standalone application disclosed herein.
  • FIGs.137A-137E provide schematic illustrations of a nanopore system as described herein.
  • FIG.137A shows a small enclosed compartment (e.g., trans) connected to a larger compartment (e.g., cis).
  • the cis can be open, wherein it may not be enclosed by a supporting structure.
  • a membrane can be disposed between the small enclosed compartment (e.g., trans) connected to a larger compartment (e.g., cis).
  • the membrane comprises a nanopore.
  • the applied voltage may translocate one or more analytes and/or at least a portion an analyte through a nanopore of the system.
  • the trans can be connected (e.g., fluidically connected) to the cis.
  • the trans can be connected (e.g., fluidly connected) to one or more adjacent cis compartments.
  • FIG.137B shows a first compartment (e.g., cis) directly connected to a second compartment (e.g., trans).
  • the first and second compartment can be directly connected in a solid structure (e.g., supporting substrate).
  • the supporting structure can support a membrane between the cis and trans.
  • the solid structure may form chambers around the first compartment and second compartment.
  • the membrane disposed between the two enclosed compartment (cis and trans) can comprise a nanopore.
  • the membrane comprises one or more nanopores.
  • a circuit can connect an Electrode 1 disposed in cis and an Electrode 2 disposed in trans.
  • FIG. 137C shows a nanopore system comprising one or more droplets.
  • a droplet may be cis.
  • the cis droplet may be an aqueous cis droplet.
  • the first droplet may be connected to a second droplet.
  • a second droplet can comprise a trans.
  • the trans droplet may be an aqueous trans droplet.
  • the cis droplet and trans droplet can be disposed in a hydrophobic medium.
  • the hydrophobic medium may be a hydrophobic liquid and/or a semi-liquid.
  • the one or more droplets may be connected via a membrane.
  • the membrane may comprise a nanopore as described herein.
  • the one or more droplets (e.g., cis droplet and trans droplet) may be partially surround by the hydrophobic medium.
  • the one or more droplets (e.g., cis droplet and trans droplet) can be completely surrounded by the hydrophobic medium.
  • the one or more droplets (e.g., cis droplet and trans droplet) may comprise one or more electrodes.
  • a cis droplet can comprise Electrode 1. Electrode 1 may provide an applied voltage to cis in the system.
  • a trans droplet can comprise Electrode 2.
  • Electrode 2 may provide an applied voltage to trans in the system.
  • a circuit can connect Electrode 1 and Electrode 2 in the system.
  • the one or more electrodes may be connected to the one or more droplets.
  • FIG.137D shows a nanopore system comprising a droplet.
  • a droplet may be trans (e.g., a trans droplet).
  • the trans droplet may be an aqueous trans droplet.
  • the droplet can be connected to a phase.
  • the phase may be a continuous phase (e.g., a continuous aqueous phase).
  • the continuous phase e.g., a continuous aqueous phase
  • the continuous phase may be a liquid phase.
  • the continuous phase may be a semi-solid phase.
  • the trans may connected to the continuous phase by a membrane.
  • the membrane may comprise a nanopore as described herein.
  • the trans droplet may be partially surround by a hydrophobic medium.
  • an Electrode 1 may be disposed in the continuous phase (e.g., the continuous aqueous WSGR Docket Number: 64828-710.601 phase).
  • An Electrode 2 may be disposed in trans.
  • the Electrode 1 and Electrode 2 can be connected via a circuit.
  • the circuit can provide an applied voltage to Electrode 1 and/or Electrode 2.
  • FIG.137E shows a nanopore system comprising a droplet.
  • a droplet may be cis (e.g., a cis droplet).
  • the cis droplet may be an aqueous cis droplet.
  • the droplet can be connected to a compartment (e.g., trans).
  • the trans may be an open compartment.
  • the trans may be an enclosed compartment.
  • a membrane can be disposed between the cis (e.g., aqueous droplet) and trans (e.g., enclosed compartment).
  • the membrane comprises a nanopore.
  • the cis (e.g., aqueous droplet) and trans (e.g., enclosed compartment) may be fluidly connected.
  • the cis (e.g., aqueous droplet) can be disposed in a hydrophobic medium.
  • the hydrophobic medium may be a hydrophobic liquid and/or a semi-liquid.
  • An Electrode 1 can be disposed in trans (e.g., enclosed compartment).
  • An Electrode 2 can be disposed in cis.
  • the circuit can connect Electrode 1 and Electrode 2.
  • a first side of a system described herein may be larger in size than a second side.
  • a first side of a system described herein may be smaller in size than a second side.
  • a first side of a system described herein may be the same size as a second side.
  • a first side and/or a second side of a system may comprise an enclosed compartment. The enclosed compartment may be supported by a solid structure described herein.
  • the enclosed compartment may be connected (e.g., fluidly connected) to another side (e.g., another compartment) of the system.
  • a first side and/or a second side of a system may comprise an open compartment.
  • the open compartment may be a continuous open compartment.
  • the continuous open compartment can comprise a continuous aqueous phase.
  • the open compartment may be connected (e.g., fluidly connected) to another side (e.g., another compartment) of the system.
  • a first side can comprise an open compartment and the second side can comprise an enclosed compartment.
  • a nanopore system can comprise one or more open chambers, or one or more closed chambers, or any combination thereof.
  • a nanopore system described herein may comprise one or more micropipettes.
  • two or more of the nanopore systems as represented in FIGs.137A-137E may be connected in any combination to provide one or more arrays of nanopores.
  • at least about 2 nanopore systems, at least about 3 nanopore systems, at least about 4 nanopore systems, at least about 5 nanopore systems, at least about 10 nanopore systems, or greater than about 10 nanopore systems as represented in FIGs.137A-137E may be connected to provide one or more arrays of nanopores.
  • an array can comprise at least about 2, at least about 5, at least about 10, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000, or greater than about 100,000 nanopore systems.
  • an array can comprise at most about 100,000, at most about 10,000, at most about 1,000, WSGR Docket Number: 64828-710.601 at most about 100, at most about 50, at most about 10, at most about 5, at most about 2, or less than about 2 nanopore systems.
  • a compartment of a nanopore system described herein may be enclosed by a substrate (e.g., a solid substrate, see ”Supporting structure” in FIG.137B).
  • the solid substrate may be composed of organic and/or inorganic materials. Inorganic materials can comprise silicon, glass, metals, ceramics, or plastics, or any combination thereof.
  • the compartment of a nanopore system described herein may be enclosed by a semi-solid substrate, for example an organogel.
  • the compartment of a nanopore system described herein may be enclosed by a hydrophobic liquid (e.g., oil, silicone, or alkane, or any combination thereof).
  • a nanopore system described herein can comprise an amphipathic membrane (e.g., a lipid membrane).
  • One or more compartments of a nanopore system described herein may be connected to another compartment of the nanopore system.
  • a first compartment e.g., a cis compartment
  • a second compartment e.g., a trans compartment
  • the channel can comprise a microfluidic pathway, in which a solution may be introduced, removed, or flow through the microfluidic pathway.
  • a nanopore system described herein can comprise one or more compartments connected by one or more nanopores.
  • the one or more nanopores can be disposed in one or more membranes.
  • a nanopore system described herein can comprise a trans-cis-trans compartment arrangement and/or a cis-trans-cis arrangement.
  • the one or more compartments of the nanopore system may each comprise a volume.
  • the volume of the one or more compartments may be the same.
  • the volume of the one or more compartments may be different.
  • a first compartment e.g., a cis compartment
  • a second compartment e.g., a trans compartment
  • a first compartment can be smaller in volume than a second compartment (e.g., a trans compartment).
  • a first compartment e.g., a cis compartment
  • a nanopore system described herein can comprise a ratio of a first compartment (e.g., a cis compartment) to a second compartment (e.g., a trans compartment).
  • a system described herein can comprise a 1:1 ratio, where there may be 1 first compartment (e.g., a cis compartment) to 1 second compartment (e.g., a trans compartment).
  • a nanopore system and/or system described herein can comprise a 1:1 ratio, a 1:2 ratio, a 1:3 ratio, a 1:4 ratio, a 1:5 ratio, a 2:1 ratio, a 3:1 ratio, a 4:1 ratio, or a 5:1 ratio of a first compartment (e.g., a cis compartment) to a second compartment (e.g., a trans compartment).
  • a plurality of first compartments e.g., cis compartments
  • a membrane of a nanopore system can comprise any membrane as described herein. The membrane may be pre-formed (e.g., formed prior to insertion in the nanopore system).
  • the WSGR Docket Number: 64828-710.601 membrane may not be pre-formed.
  • each compartment and/or droplet of a nanopore system described herein may be separated by one or more membranes.
  • the one or more membranes can comprise one or more nanopores described herein.
  • the compartments and/or droplets of a nanopore system may comprise one or more electrodes.
  • a first compartment e.g., a cis compartment
  • a second compartment e.g., a trans compartment
  • a first droplet e.g., a cis droplet
  • a second droplet e.g., a trans droplet
  • one or more electrodes e.g., a first droplet
  • a second droplet e.g., a trans droplet
  • one or more compartments can comprise separate (e.g., distinct) electrodes.
  • one or more compartments are connected (e.g., fluidly connected, electrically connected, or ionically connected, or any combination thereof), they can comprise a shared electrode (e.g., an electrode common to the one or more compartments).
  • a nanopore system described herein can comprise at least about 1 electrode, at least about 2 electrodes, at least about 3 electrodes, at least about 4 electrodes, at least about 5 electrodes, at least about 10 electrodes, or greater than about 10 electrodes. In some embodiments, a nanopore system described herein can comprise at most about 10 electrodes, at most about 5 electrodes, at most about 4 electrodes, at most about 3 electrodes, at most about 2 electrodes, at most about 1 electrode, or less than about 1 electrode. One or more electrodes may enable separate application of voltage across one or more membranes of the nanopore system. One or more electrodes may enable separate readout of a voltage and/or current difference across a nanopore, or membrane, or any combination thereof.
  • an array of one or more nanopore systems described herein may be addressed by circuitry.
  • the circuitry may comprise control circuitry, or acquisition circuitry, or any combination thereof.
  • a circuit can apply a voltage. In some embodiments, the circuit can apply the voltage across a membrane and/or a nanopore disposed in a membrane.
  • a circuit can measure a current or change thereof (e.g., an ionic current or change thereof). In some embodiments, the circuit can measure the current or change thereof (e.g., an ionic current or change thereof) across a membrane and/or a nanopore disposed in a membrane.
  • the nanopore and/or membrane of the nanopore system (or each nanopore and/or membrane in an array comprising two or more nanopore systems) can be addressed by an individual circuitry.
  • the nanopore and/or membrane in a nanopore system and/or array of connected nanopore systems can be individually addressed by control and/or acquisition circuitry to enable each nanopore and/or membrane to be controlled and/or measured separately.
  • a measured current or change thereof and/or an applied voltage can be amplified, or digitized, or any combination thereof.
  • current or voltage signals across one or more membranes and/or one or more nanopores disposed in one or more membranes may be amplified and/or digitized in circuitry.
  • a device described herein can comprise one or more electrochemical mediators.
  • An electrochemical mediator can enable a flow (e.g., a continuous flow) of current through a system.
  • the electrochemical mediator can comprise a solid mediator.
  • the solid mediator can comprise a metal mediator.
  • the electrochemical mediator may comprise silver/silver chloride (Ag/AgCl).
  • the electrochemical mediator can comprise ferrocene methanol (FcMeOH), ferricyanide (Fe(CN) 6 ), Iodide (I), hexacyanoferrate (II), Meldola's Blue (MB), Dichlorophenolindophenol (DCPIP), p-Benzoquinone (p-BQ), or o-Phenylenediamine (o-PD), or any combination thereof.
  • Readout of a device described herein may involve light.
  • a readout of the device can comprise using one or more fluorescent reporters to provide a quantification of the ionic current flowing through one or more nanopores of the one or more compartments of the device.
  • one or more controller circuits may be the same as one or more acquisition circuits of system described herein. In some embodiments, one or more controller circuits may be different from one or more acquisition circuits of system described herein. The controller circuit and acquisition circuit may use the same one or more electrodes. In some embodiments, the controller circuit and acquisition circuit may use different electrodes. In some embodiments, one or acquisition circuits and/or one or more controller circuits may not use at least one electrode. For example, one or acquisition circuits and/or one or more controller circuits may use one or more light sensors. The light sensor may be located adjacent to a pore and/or membrane. In some embodiments, an acquisition by one or more acquisition circuits may be performed by at least one optical reporter.
  • the optical report can provide an indication of an ionic current.
  • the acquisition circuit can be a raman based readout.
  • the raman based readout can comprise a readout of a composition of an analyte.
  • the raman based readout may not provide a readout of the current.
  • COMPUTERS [0637]
  • the present disclosure describes a computer-implemented system comprising: a digital processing device comprising: at least one processor, an operating system configured to perform executable instructions, a memory, and a computer program.
  • the computer program can include instructions executable by the digital processing device to control a manufacture a membrane in a device.
  • the computer program can include instructions executable by the digital processing device to prepare a sample.
  • the computer program can include instructions executable by the digital processing device to control a device to process a sample.
  • the computer program can include instructions executable by the digital processing device to analyze a signal produced by an analyte in a sample.
  • the computer program can include instructions executable by the digital processing device to store a signal produced by an analyte into a database.
  • the present disclosure describes a computer-implemented method, implementing any one of the methods disclosed herein in a computer system.
  • FIG.70 a block diagram is shown depicting an exemplary machine that WSGR Docket Number: 64828-710.601 includes a computer system 7700 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and/or methodologies for preparing a sample for analysis, processing a sample to generate a signal from an analyte in the sample, analyzing a signal to identify an analyte, storing a signal in a database, or any combination thereof.
  • the components in FIG.70 are examples only and do not limit the scope of use or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments.
  • Computer system 7000 may include one or more processors 7001, a memory 7003, and a storage 7008 that communicate with each other, and with other components, via a bus 7040.
  • the bus 7040 may also link a display 7032, one or more input devices 7033 (which may, for example, include a keypad, a keyboard, a mouse, or a stylus, or any combination thereof), one or more output devices 7034, one or more storage devices 7035, and various tangible storage media 7036. All of these elements may interface directly or via one or more interfaces or adaptors to the bus 7040.
  • the various tangible storage media 7036 can interface with the bus 7040 via storage medium interface 7026.
  • Computer system 7000 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers.
  • Computer system 7000 includes one or more processor(s) 7001 (e.g., central processing units (CPUs), general purpose graphics processing units (GPGPUs), or quantum processing units (QPUs)) that carry out functions.
  • processors 7001 e.g., central processing units (CPUs), general purpose graphics processing units (GPGPUs), or quantum processing units (QPUs)
  • Computer system 7000 may be one of various high performance computing platforms.
  • the one or more processor(s) 7001 may form a high performance computing cluster.
  • the one or more processors 7001 may form a distributed computing system connected by wired and/or wireless networks.
  • Processor(s) 7001 optionally contains a cache memory unit 7002 for temporary local storage of instructions, data, or computer addresses. Processor(s) 7001 are configured to assist in execution of computer readable instructions.
  • Computer system 7000 may provide functionality for the components depicted in FIG. 70 as a result of the processor(s) 7001 executing non- transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 7003, storage 7008, storage devices 7035, and/or storage medium 7036.
  • the computer- readable media may store software that implements particular embodiments, and processor(s) 7001 may execute the software.
  • Memory 7003 may read the software from one or more other computer-readable media (such as mass storage device(s) 7035, 7036) or from one or more other sources through a suitable interface, such as network interface 7020.
  • the software may cause processor(s) 7001 to carry out one or more processes or one WSGR Docket Number: 64828-710.601 or more steps of one or more processes described or illustrated herein. Carrying out such processes or steps may include defining data structures stored in memory 7003 and modifying the data structures as directed by the software.
  • the memory 7003 may include various components (e.g., machine readable media) including, but not limited to, a random access memory component (e.g., RAM 7004) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), or phase-change random access memory (PRAM), or any combination thereof), a read-only memory component (e.g., ROM 7005), and any combinations thereof.
  • RAM 7004 e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), or phase-change random access memory (PRAM), or any combination thereof
  • ROM 7005 may act to communicate data and instructions unidirectionally to processor(s) 7001
  • RAM 7004 may act to communicate data and instructions bidirectionally with processor(s) 7001.
  • ROM 7005 and RAM 7004 may include any suitable tangible computer-readable media described below.
  • a basic input/output system 7006 (BIOS), including basic routines that help to transfer information between elements within computer system 7000, such as during start-up, may be stored in the memory 7003.
  • Fixed storage 7008 can be connected bidirectionally to processor(s) 7001, optionally through storage control unit 7007.
  • Fixed storage 7008 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein.
  • Storage 7008 may be used to store operating system 7009, executable(s) 7010, data 7011, applications 7012 (application programs), and the like.
  • Storage 7008 can also include an optical disk drive, a solid-state memory device (e.g., flash-based systems), or a combination of any of the above.
  • storage device(s) 7035 may be removably interfaced with computer system 7000 (e.g., via an external port connector (not shown)) via a storage device interface 7025.
  • storage device(s) 7035 and an associated machine-readable medium may provide non-volatile and/or volatile storage of machine- readable instructions, data structures, program modules, and/or other data for the computer system 7000.
  • software may reside, completely or partially, within a machine-readable medium on storage device(s) 7035.
  • software may reside, completely or partially, within processor(s) 7001.
  • Bus 7040 connects a wide variety of subsystems.
  • reference to a bus may encompass one or more digital signal lines serving a common function, where appropriate.
  • Bus 7040 may be any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, or a local bus, and any combinations thereof, using any of a variety of bus architectures.
  • Computer system 7000 may also include an input device 7033.
  • ISA Industry Standard Architecture
  • EISA Enhanced ISA
  • MCA Micro Channel Architecture
  • VLB Video Electronics Standards Association local bus
  • PCI Peripheral Component Interconnect
  • PCI-X PCI-Express
  • AGP Accelerated Graphics Port
  • Computer system 7000 may also include an input device 7033.
  • a user of computer system 7000 may enter commands and/or other information into computer system 7000 via input device(s) 7033.
  • input device(s) 7033 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, or a voice response system, or any combination thereof), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof.
  • an alpha-numeric input device e.g., a keyboard
  • a pointing device e.g., a mouse or touchpad
  • a touchpad e.g., a touch screen
  • multi-touch screen e.g., a joystick, a stylus, a gamepad
  • the input device can be a Kinect, Leap Motion, or the like.
  • Input device(s) 7033 may be interfaced to bus 7040 via any of a variety of input interfaces 7023 (e.g., input interface 7023) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above.
  • an input device 7033 may be used to prepare a sample for analysis, process a sample to generate a signal from an analyte in the sample, analyze a signal to identify an analyte, store a signal in a database, or any combination thereof.
  • the input device can be used using human inputs through the input device 7033.
  • computer system 7000 when computer system 7000 can be connected to network 7030, computer system 7000 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 7030. Communications to and from computer system 7000 may be sent through network interface 7020.
  • network interface 7020 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 7030, and computer system 7000 may store the incoming communications in memory 7003 for processing.
  • IP Internet Protocol
  • Computer system 7000 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 7003 and communicated to network 7030 from network interface 7020.
  • Processor(s) 7001 may access these communication packets stored in memory 7003 for processing.
  • Examples of the network interface 7020 include, but are not limited to, a network interface card, or a modem, and any combination thereof.
  • Examples of a network 7030 or network segment 7030 include, but are not limited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, or a peer-to-peer network, and any combinations thereof.
  • WAN wide area network
  • LAN local area network
  • a network such as WSGR Docket Number: 64828-710.601 network 7030, may employ a wired and/or a wireless mode of communication. In general, any network topology may be used.
  • Information and data can be displayed through a display 7032.
  • Examples of a display 7032 include, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, or a plasma display, and any combinations thereof.
  • CTR cathode ray tube
  • LCD liquid crystal display
  • TFT-LCD thin film transistor liquid crystal display
  • OLED organic liquid crystal display
  • PMOLED passive-matrix OLED
  • AMOLED active-matrix OLED
  • the display 7032 can interface to the processor(s) 7001, memory 7003, and fixed storage 7008, as well as other devices, such as input device(s) 7033, via the bus 7040.
  • the display 7032 can be linked to the bus 7040 via a video interface 7022, and transport of data between the display 7032 and the bus 7040 can be controlled via the graphics control 7021.
  • the display can be a video projector.
  • the display can be a head-mounted display (HMD) such as a VR headset.
  • HMD head-mounted display
  • suitable VR headsets include, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like.
  • the display can be a combination of devices such as those disclosed herein.
  • computer system 7000 may include one or more other peripheral output devices 7034 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices may be connected to the bus 7040 via an output interface 7024.
  • Examples of an output interface 7024 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof.
  • computer system 7000 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more steps of one or more processes described or illustrated herein.
  • Reference to software in this disclosure may encompass logic, and reference to logic may encompass software.
  • reference to a computer-readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate.
  • DSP digital WSGR Docket Number: 64828-710.601 signal processor
  • ASIC application specific integrated circuit
  • FPGA field programmable gate array
  • a general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine.
  • a processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration.
  • the steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two.
  • a software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium.
  • An exemplary storage medium can be coupled to the processor such the processor can read information from, and write information to, the storage medium.
  • the storage medium may be integral to the processor.
  • the processor and the storage medium may reside in an ASIC.
  • the ASIC may reside in a user terminal.
  • the processor and the storage medium may reside as discrete components in a user terminal.
  • suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, and tablet computers.
  • the computing device includes an operating system configured to perform executable instructions.
  • the operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications.
  • Suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®.
  • Suitable personal computer operating systems include, by way of non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®.
  • the operating system can be provided by cloud computing.
  • Suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia® Symbian® OS, Apple® Ios®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®.
  • a computer system 7000 may be accessible through a user terminal to receive user commands.
  • the user commands may include line commands, scripts, programs, or any combination thereof, and various instructions executable by the computer system 7000.
  • a computer system 7000 may receive WSGR Docket Number: 64828-710.601 instructions to prepare a sample for analysis, process a sample to generate a signal from an analyte in the sample, analyze a signal to identify an analyte, store a signal in a database, schedule a computing job for the computer system 7000 to carry out any instructions, or any combination thereof.
  • ANALYTES Provided herein are various analytes that can be detected and/or identified using a pore, membrane, device, or a method disclosed herein. The terms “analyte” and “substrate” may be used interchangeably.
  • an analyte can be an antibiotic.
  • an analyte can be a drug. In some embodiments, an analyte can be a metabolite. In some embodiments, an analyte can be a lipid. In some embodiments, an analyte can be a small biological molecule. In some embodiments, an analyte can be a steroid, a carbohydrate, an amino acid, a nucleotide, a hormone, a fatty acid, a vitamin, a flavin, a protein-cofactor, a lipid, or a phenolic compound.
  • analytes can generate different signals based on volume, shape, charge, structure, cross-linking, post-translational modifications (e.g., phosphorylation, glycosylation, or rhamnosylation, or any combination thereof), damage, oxidation, reduction, (D/L-) chirality, and/or sequence of the analyte.
  • an analyte comprises a polymer.
  • an analyte can be a protein-protein complex.
  • an analyte can be a DNA-protein complex.
  • an analyte can be an RNA-protein complex.
  • the analyte can be a non-nucleic acid based polymer analyte.
  • an analyte comprises a polymer.
  • a polymer comprises a protein.
  • a polymer comprises a polypeptide.
  • the polypeptide (e.g., one or more polypeptide) may comprise one or more expressible polypeptides.
  • a polymer comprises a peptide.
  • a polymer comprises a polyamino acid.
  • a polymer comprises a polysaccharide.
  • a polymer comprises a glycoprotein.
  • a polymer comprises a nucleic acid. In some embodiments, a polymer comprises DNA. In some embodiments, a polymer comprises RNA. In some embodiments, a polymer comprises mRNA. In some embodiments, a polymer comprises a sequence. In some embodiments, a polymer can be a label-free analyte. In some embodiments, a polymer can be labeled. In some embodiments, the termini of a polymer may be unstructured, e.g., when the polymer can be denatured or partially denatured. In some embodiments, a contour length of a polymer can be longer than a length of a channel of a pore.
  • a contour length of a polymer can be shorter than a length of a channel of a pore.
  • a polymer comprises heterogeneous charge.
  • a heterogeneously charged polymer comprises positively and negatively charged repeating units.
  • charged repeating units may be evenly distributed along the sequence of a polymer.
  • charged repeating units may be unevenly distributed along the sequence of a polymer.
  • a polymer may be labeled.
  • a polymer may be synthetic, semi-synthetic or biological in origin.
  • a polymer comprises a protein.
  • a protein comprises two or more amino acids. In some embodiments, a protein comprises two or more peptides. In some embodiments, a protein comprises a polypeptide. In some embodiments, a protein comprises a negative charge. In some embodiments, a protein comprises a positive charge. In some embodiments, a protein can be negatively charged. In some embodiments, a protein can be positively charged. In some embodiments, a protein can be zwitterionic. [0662] In some embodiments, an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or any combination thereof can comprise one or more characteristics.
  • the one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or any combination thereof can comprise a shape of the analyte, a volume of the analyte, a mass of the analyte, a structure of the analyte (e.g., a secondary structure, a tertiary structure, or a quaternary structure, or any combination thereof), a length of an analyte, a truncation of an analyte, an orientation of an analyte, one or more mutations of the analyte, a sequence of an analyte (e.g., a non-nucleic acid polymer analyte), a hydrophobicity of the analyte, a polarity of the analyte, an isoform of the analyte,
  • the sequence information of the analyte may be captured in layers.
  • the sequence information may be one or more layers of data (e.g., metadata) that may each comprise the sequence information and/or at least a portion of sequence information.
  • the one or more layers of sequence information may be annotated to a reference sequence (e.g., a reference sequence of a database) with a probabilistic metric.
  • the sequence information may comprise properties of the analyte (e.g., charge of the analyte, hydrophobicity of the analyte, aromaticity of the analytes, acidity of the analyte, chirality of the analyte, polarity of the analyte, or side chain composition of the analyte, or any combination thereof).
  • the sequence information may comprise a partial set of natural amino acids, or a partial set of unnatural amino acids, or any combination thereof.
  • the sequence information may comprise an amino acid composition of the analyte.
  • the amino acid composition can comprise the natural amino acid residues of the analyte, the unnatural amino acid residues of the analyte, or any combination thereof.
  • the sequence information can comprise one or more natural post-translational modifications, one or more unnatural post-translational modifications, one or more conjugations (e.g., drugs and/or small molecules), or any combination thereof.
  • WSGR Docket Number: 64828-710.601 one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more translation errors.
  • the translation errors may be associated with a portion of the analyte (e.g., an N- terminal portion, a C-terminal portion, or any combination thereof).
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more degradations.
  • the degradations may comprise degradations to at least a portion of the analyte, or a molecular entity coupled to the analyte, or any combination thereof.
  • an analyte may comprise an antibody.
  • the one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more variable regions and/or one or more constant regions.
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise buried and/or exposed amino acid residues. At least a portion of an analyte may be associated with buried and/or exposed amino acid residues.
  • the buried and/or exposed amino acid residues can refer to the folded and/or unfolded state of the analyte (e.g., the peptide, polypeptide, or protein, or fragments thereof, or any combination thereof).
  • the exposed and/or buried state of the one or more amino acid residues of the analyte may be present in the sample analyzed with the nanopore and/or nanopore system described herein.
  • the exposed and/or buried state of the one or more amino acid residues of the analyte may be present in the original sample (e.g., the original biological sample).
  • identification and/or determination of buried and/or exposed amino acid residues may occur by secondary analysis.
  • identification and/or determination of a modified amino acid residue may indicate an exposed and/or buried state of the amino acid residue.
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise an average speed of translocation (e.g., a rate of translocation) through a nanopore.
  • the average speed of translocation is the representative time taken for a population of polypeptides to move by translocation across a nanopore from a cis to trans, or trans to cis compartment, typically expressed in units such as nm/second.
  • the average speed of translocation is the representative time taken under specified conditions for an individual amino acid or other feature of a given polypeptide, once engaged with a nanopore, to translocate through the nanopore from an entry aperture of the nanopore to an exit aperture of the nanopore (e.g., from a first side to a second side, or a second side to a first side), typically expressed in units such as nm/second.
  • a first side can comprise a cis side or a trans side
  • a second side may comprise a cis side or a WSGR Docket Number: 64828-710.601 trans side.
  • translocation of at least a portion of an analyte through a nanopore comprises translocation from a a cis side to a trans side.
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more translocation kinetics.
  • the translocation kinetics can be expressed as a function along a sequence of the analyte.
  • the kinetics may comprise a function of speed and/or position along an analyte as the analyte (e.g., the at least a portion of the analyte) translocates through a pore.
  • the kinetics of the analyte translocation can refer to different speeds of translocation that may be seen as an analyte (e.g., a polypeptide, peptide, or protein, or fragments thereof, or any combination thereof) translocates across a nanopore.
  • the different speeds may be due to different amino acid and/or peptide sub-sequences.
  • the different speeds may be due to modifications and/or moieties (e.g., PTMs, or conjugations, or any combination thereof) along structure of the analyte (e.g., along a polypeptide chain and/or peptide chain).
  • modifications and/or moieties may interact with a nanopore (e.g., a channel, or constriction region, or any combination thereof) differently and may influence a translocation speed accordingly.
  • the modifications and/or moieties interacting with the nanopore may speed up or slow down a translocation speed of an analyte through a nanopore.
  • kinetics of translocation speed may refer to how a secondary structure, a tertiary structure, or a quaternary structure, or any combination thereof of an analyte influence translocation.
  • kinetics of translocation speed may refer to how a secondary structure, a tertiary structure, or a quaternary structure, or any combination thereof slows an analyte’s translocation through a constriction region of a nanopore. This relates to the kinetics of translocation within/along a particular polypeptide.
  • Changes in the kinetics of translocation can provide information on the secondary structure, tertiary structure, and/or quaternary structure of the domains of the polypeptide, amino acid composition of the polypeptide, molecular entities bound to the polypeptide (e.g., small molecules, drugs, co-factors, peptides, proteins, or nanoparticles, or any combination thereof), internal cross- links (e.g., cysteine-cysteine disulphide), conjugations (e.g., cysteine-cysteine disulphide crosslinks to other molecules or proteins, conjugate drugs, large PTMs such as ubiquitin and/or sumo), or any combination thereof.
  • molecular entities bound to the polypeptide e.g., small molecules, drugs, co-factors, peptides, proteins, or nanoparticles, or any combination thereof
  • internal cross- links e.g., cysteine-cysteine disulphide
  • conjugations e.g., cysteine-c
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise determining one or more molecular entities of the analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof.
  • Molecular entities may comprise variants and/or conjugations to an analyte as described herein.
  • a molecular entity of the one or more molecular entities may comprise a compound.
  • the compound may comprise a drug, or a small molecule, or any combination thereof.
  • a molecular entity of the one or more molecular entities may comprise a particle, nucleic WSGR Docket Number: 64828-710.601 acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof.
  • the one or more molecular entities may be coupled to an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof.
  • one or more molecular entities may be covalently coupled to an analyte.
  • One or more molecular entities may be coupled to an analyte by a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, a non-helical linker, or any combination thereof.
  • One or more molecular entities may be non-covalently coupled to an analyte.
  • one or more molecular entities may be coupled via electrostatic interactions to analyte.
  • a characteristic of an analyte can comprise a property of one or more molecular entities.
  • a property of one or more molecular entities can comprise a mass of the molecular entity, one or more charges of the molecular entity, one or more classes of the molecular entity, or identity of the molecular entity, or any combination thereof.
  • the one or more charges can comprise a positive charge, a neutral charge, or a negative charge, or any combination thereof.
  • an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to at least about 1 molecular entity, at least about 2 molecular entities, at least about 3 molecular entities, at least about 4 molecular entities, at least about 5 molecular entities, at least about 8 molecular entities, at least about 10 molecular entities, or greater than about 10 molecular entities.
  • an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to at most about 10 molecular entities, at most about 8 molecular entities, at most about 5 molecular entities, at most about 4 molecular entities, at most about 3 molecular entities, at most about 2 molecular entities, at most about 1 molecular entity, or less than about 1 molecular entity.
  • an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to between about 1 molecular entity to about 10 molecular entities.
  • an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to between about 1 molecular entity to about 2 molecular entities, about 1 molecular entity to about 3 molecular entities, about 1 molecular entity to about 4 molecular entities, about 1 molecular entity to about 5 molecular entities, about 1 molecular entity to about 6 molecular entities, about 1 molecular entity to about 7 molecular entities, about 1 molecular entity to about 8 molecular entities, about 1 molecular entity to about 9 molecular entities, about 1 molecular entity to about 10 molecular entities, about 2 molecular entities to about 3 molecular entities, about 2 molecular entities to about 4 molecular entities, about 2 molecular entities to about 5 molecular entities, about 2 molecular entities to about 6 molecular entities, about 2 molecular entities to about 7 mole
  • a characteristic of an analyte and/or at least a portion of an analyte may comprise a quantity of two or more molecular entities (e.g., 2 molecular entities, 3 molecular entities, 4 molecular entities, 5 molecular entities, or greater than about 5 molecular entities).
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise at least one property of one or more intra cross-linkages.
  • the intra cross-linkages may be disposed within an analyte, at least a portion of analyte, or an analyte of a plurality of analytes, or fragments thereof, or any combination thereof.
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise at least one property of one or more inter cross-linkages.
  • the one or more inter cross-linkages can comprise a linkage formed between an analyte, at least a portion of analyte, and/or an analyte of a plurality of analytes with another an analyte, a portion of an analyte, or analyte of the plurality of analytes.
  • Intra cross- linkages and/or inter cross-linkages may comprise one or more covalent or non-covalent interactions.
  • Intra cross-linkages and/or inter cross-linkages may comprise one or more linkers.
  • the one or more linkers may comprise any linker described herein.
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more couplings/linkages (e.g., one or more disulfide bonds) with a molecular entity described herein.
  • a property can comprise a positioning and/or quantity WSGR Docket Number: 64828-710.601 of the one or more intra cross-linkages, the one or more inter cross-linkages, or the one or more covalent linkages with a molecular entity, or any combination thereof.
  • a property can comprise a presence and or absence of the one or more intra cross-linkages, the one or more inter cross-linkages, the one or more covalent linkages with a molecular entity, or any combination thereof.
  • a signal or change thereof of an analyte translocating through a nanopore described herein may be used to identify a presence and or absence of the one or more intra cross-linkages, the one or more inter cross-linkages, the one or more covalent linkages with a molecular entity, or any combination thereof.
  • one or more characteristics of an analyte, at least a portion of analyte, one or more analytes (e.g., a plurality of analytes), or any combination thereof can comprise a category and/or identity associated with the analyte, at least a portion of analyte, one or more analytes (e.g., a plurality of analytes), or any combination thereof.
  • a category of an analyte, at least a portion of analyte, one or more analytes (e.g., a plurality of analytes), or any combination thereof may comprise one or more of a type, class, gene ontology, sub-domains, functional domains, secondary structure elements, tertiary structural elements, quaternary structures, protein binding cavities, or any combination thereof.
  • one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise a force, energy, or time, or any combination thereof.
  • a force, an energy, or a kinetic constant, or any combination thereof may be related to (i) structural domains, (ii) coupled molecular entities, or (iii) any combination thereof.
  • a structural domain and a coupled molecular entity may be detected by changes in the speed/kinetics during a portion of the translocation. These changes may be measured in the current or change thereof.
  • a force, energy, and/or time may comprise a folding energy, an unfolding force, denaturation dwell time kinetics, a binding energy, an unbinding force, or binding/unbinding dwell time kinetics, or any combination thereof.
  • one or more characteristics of one or more non- covalently bound entities may comprise binding force, binding energy, or unbinding dwell time, or any combination thereof. In some embodiments, one or more characteristics of one or more non-covalently bound entities may comprise unfolding force, folding energy, or denaturation dwell time, or any combination thereof. [0672] In some embodiments, the analyte may comprise a mass.
  • the analyte may comprise a mass of at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, at least about 25 kDa, at least about 30 kDa, at least about 35 kDa, at least about 40 kDa, at least about 45 kDa, at least about 50 kDa, at least about 55 kDa, at least about 60 kDa, at least about 65 kDa, at least about 70 kDa, at least about 75 kDa, at least about 80 kDa, at least about 85 kDa, at least about 90 kDa, at least about 95
  • the analyte may comprise a mass of at most about 4000 kDa, at most about 3500 kDa, at most about 3000 kDa, at most about 2500 kDa, at most about 2000 kDa, at most about 1500 kDa, at most about 1000 kDa, at most about 950 kDa, at most about 900 kDa, at most about 850 kDa, at most about 800 kDa, at most about 750 kDa, at most about 700 kDa, at most about 650 kDa, at most about 600 kDa, at most about 550 kDa, at most about 500 kDa, at most about 450 kDa, at most about 400 kDa, at most about 350 kDa, at most about 300 kDa, at most about 250 kDa, at most about 200 kDa, at most about 175 kDa, at most about 150 kDa, at most about 400
  • the analyte may comprise a mass from about 1 kDa to about 100 kDa. In some embodiments, the analyte may comprise a mass from about 1 kDa to about 5 kDa, about 1 kDa to about 10 kDa, about 1 kDa to about 20 kDa, about 1 kDa to about 30 kDa, about 1 kDa to about 40 kDa, about 1 kDa to about 50 kDa, about 1 kDa to about 60 kDa, about 1 kDa to about 70 kDa, about 1 kDa to about 80 kDa, about 1 kDa to about 90 kDa, about 1 kDa to about 100 kDa, about 5 kDa to about 10 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 30 kDa, about 5 kDa to
  • the analyte may comprise a mass from about 100 kDa to about 4,000 kDa.
  • the analyte can be from about 100 kDa to about 250 kDa, about 100 kDa to about 500 kDa, about 100 kDa to about 1,000 kDa, about 100 kDa to about 1,500 kDa, about 100 kDa to about 2,000 kDa, about 100 kDa to about 2,500 kDa, about 100 kDa to about 3,000 kDa, about 100 kDa to about 3,500 kDa, about 100 kDa to about 4,000 kDa, about 250 kDa to about 500 kDa, about 250 kDa to about 1,000 kDa, about 250 kDa to about 1,500 kDa, about 250 kDa to about 2,000 kDa, about 250 kDa to about 2,500 kDa, about 250 kDa to about 2,500
  • the analyte can be about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 125 kDa, about 150 kDa, about 175 kDa, about 200 kDa, about 250 kDa, about 300 kD
  • a protein comprises a pI of at least about 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or about 13.
  • a protein comprises a pI of at WSGR Docket Number: 64828-710.601 most about 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or about 13.
  • a protein can be denatured prior to translocating through a pore. In some embodiments, a protein may not be denatured prior to translocating through a pore.
  • a protein can be reduced prior to translocating through a pore. In some embodiments, a protein may not be reduced prior to translocating through a pore. In some embodiments, a protein can be alkylated prior to translocating through a pore. In some embodiments, a protein may not be alkylated prior to translocating through a pore. In some embodiments, a protein can be isotope labeled prior to translocating through a pore. In some embodiments, a protein may not be isotope labeled prior to translocating through a pore. In some embodiments, a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophobic amino acids.
  • a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophilic amino acids. In some embodiments, a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of charged amino acids. In some embodiments, a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of neutral amino acids.
  • a non-nucleic acid based polymer analyte (e.g., polymer) comprises a peptide. In some embodiments, a non-nucleic acid based polymer analyte comprises two or more amino acids.
  • a non-nucleic acid based polymer analyte comprises a negative charge. In some embodiments, a non-nucleic acid based polymer analyte comprises a positive charge. In some embodiments, a non-nucleic acid based polymer analyte can be negatively charged. In some embodiments, a non-nucleic acid based polymer analyte can be positively charged. In some embodiments, a non-nucleic acid based polymer analyte can be zwitterionic. In some embodiments, a non-nucleic acid based polymer analyte comprises at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 amino acids.
  • a non-nucleic acid based polymer analyte comprises at most about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises between 5 and 20 amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises proteolytically cleaved fragments of a protein. In some embodiments, a non-nucleic acid based polymer analyte comprises trypsinized fragments of a protein. A protease can be used to cleave a protein into peptides.
  • a protease comprises trypsin, chymotrypsin, pepsin, elastase, or any combination thereof.
  • a non-nucleic acid based polymer analyte can be denatured prior to translocating through a pore.
  • a non- nucleic acid based polymer analyte may not be denatured prior to translocating through a pore.
  • a non-nucleic acid based polymer analyte can be reduced prior to translocating through a pore.
  • a non-nucleic acid based polymer analyte may not be reduced prior to translocating through a pore.
  • a non-nucleic acid based polymer analyte can be alkylated prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte may not be WSGR Docket Number: 64828-710.601 alkylated prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte can be isotope labeled prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte may not be isotope labeled prior to translocating through a pore.
  • a non- nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophobic amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophilic amino acids. In some embodiments, a non- nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of charged amino acids.
  • a non-nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of neutral amino acids.
  • a polymer comprises a polypeptide.
  • a polypeptide comprises two or more peptides.
  • a polypeptide may be a protein that can be unfolded during or before translocation through a pore.
  • a polypeptide comprises a molecular weight above 20 kDa.
  • a polypeptide comprises a molecular weight greater than about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or about 5000 kDa. In some embodiments, a polypeptide comprises a molecular weight less than about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or about 5000 kDa.
  • a polypeptide can be reduced prior to translocating through a pore. In some embodiments, a polypeptide may not be reduced prior to translocating through a pore. In some embodiments, a polypeptide can be alkylated prior to translocating through a pore. In some embodiments, a polypeptide may not be alkylated prior to translocating through a pore. In some embodiments, a polypeptide can be isotope labeled prior to translocating through a pore. In some embodiments, a polypeptide may not be isotope labeled prior to translocating through a pore.
  • a polysaccharide comprises two or more monosaccharides. In some embodiments, a polysaccharide comprises a positive charge. In some embodiments, a polysaccharide can be negatively charged. In some embodiments, a polysaccharide can be positively charged. In some embodiments, a polysaccharide can be zwitterionic. In some embodiments, a polysaccharide can be linear. In some embodiments, a polysaccharide can be non-linear. In some embodiments, a polysaccharide can be branched.
  • a monosaccharide can be glucose, fructose, galactose, ribose, deoxyribose, or any other monosaccharide, or any stereoisomer thereof.
  • a polysaccharide comprises glycosidic linkages between monosaccharides.
  • a polysaccharide comprises starch, glycogen, galactogen, structural polysaccharides such as cellulose or chitin, or any combination thereof.
  • a polysaccharide can be a homopolysaccharide (or homoglycan) comprising monosaccharides of the same type.
  • a polysaccharide can be a heteropolysaccharide (or heteroglycan) comprising more than one type of monosaccharide repeating unit.
  • a polymer analyte can be a polypeptide of at least 30 peptide units and comprising positively and negatively charged residues, preferably wherein the polypeptide can be in a denatured/unfolded state, more preferably wherein the polypeptide can be added in a pre-denatured state.
  • the polymer analyte can be an unmodified protein or a portion thereof, or a naturally occurring polypeptide or a portion thereof.
  • the polymer analyte can be a full length protein or naturally occurring polypeptide. In some embodiments polypeptides or polypeptide fragments can be conjugated to form a longer polypeptide. [0681] In some embodiments, one or more characteristics of an analyte and/or at least a portion of an analyte may comprise a fold portion of an analyte and/or at least a portion of an analyte. In some embodiments, one or more characteristics of an analyte and/or at least a portion of an analyte may comprise an unfolded portion of the analyte and/or at least a portion of an analyte.
  • one or more characteristics of an analyte and/or at least a portion of an analyte may comprise a partially folded portion of the analyte and/or at least a portion of an analyte. In some embodiments, one or more characteristics may comprise a percentage of unfolded portions associated with an analyte, at least a portion of an analyte, or any combination thereof. In some embodiments, one or more characteristics may comprise a percentage of folded portions associated with WSGR Docket Number: 64828-710.601 an analyte and/or at least a portion of an analyte.
  • an analyte and/or at least a portion of an analyte can comprise a structure comprising at least about 1% unfolded portions, at least about 5% unfolded portions, at least about 10% unfolded portions, at least about 20% unfolded portions, at least about 30% unfolded portions, at least about 40% unfolded portions, at least about 50% unfolded portions, or greater than about 50% unfolded portions.
  • an analyte and/or at least a portion of an analyte can comprise a structure comprising at most about 50% unfolded portions, at most about 40% unfolded portions, at most about 30% unfolded portions, at most about 20% unfolded portions, at most about 10% unfolded portions, at most about 5% unfolded portions, at most about 1% unfolded portions, or less than about 1% unfolded portions.
  • an analyte and/or at least a portion of an analyte can comprise a structure comprising between about 1% unfolded portions to about 50% unfolded portions.
  • an analyte and/or at least a portion of an analyte can comprise a structure comprising between about 1% unfolded portions to about 5% unfolded portions, about 1% unfolded portions to about 10% unfolded portions, about 1% unfolded portions to about 20% unfolded portions, about 1% unfolded portions to about 30% unfolded portions, about 1% unfolded portions to about 40% unfolded portions, about 1% unfolded portions to about 50% unfolded portions, about 5% unfolded portions to about 10% unfolded portions, about 5% unfolded portions to about 20% unfolded portions, about 5% unfolded portions to about 30% unfolded portions, about 5% unfolded portions to about 40% unfolded portions, about 5% unfolded portions to about 50% unfolded portions, about 10% unfolded portions to about 20% unfolded portions, about 10% unfolded portions to about 30% unfolded portions, about 10% unfolded portions to about 40% unfolded portions, about 10% unfolded portions to about 50% unfolded portions, about 10% unfold
  • a protein, a polypeptide, or a peptide can comprise a post-translational modification.
  • a post-translational modification can be non-natural, for instance, chemical modifications WSGR Docket Number: 64828-710.601 introduced in a laboratory for biotechnological or biomedical purposes.
  • a post- translational modification can be natural, where a living organism or a cell performed the post-translational modification if an analyte.
  • a post-translation modification can comprise modification with a hydrophobic group, modification with a cofactor, addition of a chemical group, glycation (the non-enzymatic attachment of a sugar), biotinylation, PEGylation, oxidation, or reduction, damage, or any combination thereof.
  • FMN flavin mononucleotide
  • FAD flavin adenine dinucleotide
  • attachment of heme C for instance via a thioether bond with cysteine
  • phosphopantetheinylation the attachment of a 4’-phosphopantetheinyl group
  • retinylidene Schiff base formation post-translational modification by addition of a chemical group comprises acylation, e.g.
  • one or more post-translational modifications can comprise phosphorylation, acetylation, amidation, deamidation, glycosylation, oxidation, ubiquitination, sumolation, lipidation, carbonylation, or any combination thereof.
  • One or more analytes described herein can be associated with one or more characteristics. The characteristics may be clustered (e.g., grouped together). Grouping of characteristics can provide one or more properties of a sample comprising one or more analytes.
  • a sample can be a sample that may be known to contain or is suspected of containing one or more predetermined analytes.
  • a biological sample from a patient may be suspected of containing a protein biomarker for cancer.
  • the sample can be assayed to detect the presence of the one or more predetermined analytes.
  • a sample e.g., a biological sample described herein may be from a subject with a disease, or condition, or any combination thereof.
  • the disease or condition may comprise cancer, heart disease, neurodegenerative disease, diabetes, autoimmune disease, or infectious disease, or any combination thereof.
  • the sample may be from an aged subject (e.g., a subject who is at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or greater than about 90 years of age). [0687]
  • the sample may comprise an analyte described herein.
  • the sample may comprise one or more analytes.
  • the sample may comprise between about 1 analyte to about 50 analytes.
  • the sample may comprise between about 1 analyte to about 2 analytes, about 1 analyte to about 3 analytes, about 1 analyte to about 4 analytes, about 1 analyte to about 5 analytes, about 1 analyte to about 10 analytes, about 1 analyte to about 15 analytes, about 1 analyte to about 20 analytes, about 1 analyte to about 25 analytes, about 1 analyte to about 30 analytes, about 1 analyte to about 40 analytes, about 1 analyte to about 50 analytes, about 2 analytes to about 3 analytes, about 2 analytes WSGR Docket Number: 64828-710.601 to about 4 analytes, about 2 analytes to about 5 analytes, about 2 analy
  • One or more analytes of a sample may of the same species.
  • the one or more analytes of the sample may be of different species.
  • the sample comprises at least about 2 analytes of the same species, at least about 3 analytes of the same species, at least about 4 analytes of the same species, at least about 5 analytes of the same species, at least about 10 analytes of the same species, or greater than about 10 analytes of the same species.
  • One or more analytes of the sample may be the same type of analyte.
  • One or more analytes of the sample may be different types of analytes.
  • a sample may comprise a first type of analyte and a second type of analyte.
  • first type of analyte characteristics of the analyte may be determined.
  • first type of analyte one or more of a number of analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or any combination thereof may be determined.
  • characteristics of the analyte may be determined.
  • a number of analytes in the sample may be determined.
  • a plurality of analytes may comprise a plurality of different type of analytes.
  • the difference in type between two or more analytes can comprise a difference in identity, sequence, structure, type of protein of proteins (e.g., antibody vs. antigen), or class (e.g., human protein vs plant protein), or a combination thereof.
  • the plurality of analytes may comprise a two or more analytes with different characteristics.
  • the characteristics may be any disclosed herein.
  • less than all units e.g., amino acids
  • a presence of one or more analytes may be determined.
  • an absence of one or more analytes may be determined.
  • a presence and/or absence of one or more analytes may be determined using a current or change thereof described herein. For example, an absence of a signal may be indicative of an absence of an analyte, an analyte type, a modification of an analyte, or any combination thereof, in a sample.
  • a sample can be a sample that may be being interrogated for its composition.
  • a biological sample from a patient can be human plasma.
  • the biological sample can be analyzed to detect the presence and/or the concentration levels of various proteins in the human plasma.
  • a sample can be a biological sample.
  • a biological sample comprises blood, serum, plasma, urine, sweat, saliva, tears, mucus, phlegm, cerebrospinal fluid, cell, cell lysate, tissue, organ, organelle, bone marrow, or semen.
  • a biological sample comprises cell- free DNA, protein, or peptides, or any combination thereof.
  • a sample can be an inorganic sample.
  • a sample can comprise a water sample comprising one or more impurities, such as fluorinated substances (e.g., perfluoroalkyls; perfluorooctanoic acid; perfluorosulfonic acid), or heavy metals.
  • a sample can be obtained, isolated or extracted from any organism or microorganism. For example, it can be obtained from a human or animal, e.g. from a bodily fluid, such as urine, lymph, saliva, mucus, seminal fluid or amniotic fluid, or from whole blood, plasma or serum. The sample may be obtained from a plant e.g.
  • a sample can be produced inside (animal) cells such that it can be extracted from cells for characterization.
  • a sample may comprise the products of cellular expression of a plasmid in a (microbial) host cell.
  • a sample can be secreted from cells.
  • a sample can comprise a protein, a polypeptide, a peptide, a polysaccharide, a DNA, a RNA, an antibiotic, a drug, a metabolite, a lipid, a steroid, a carbohydrate, an amino acid, a nucleotide, a hormone, a fatty acid, a vitamin, a flavin, a protein-cofactor, a lipid, or a phenolic compound, or any combination thereof.
  • a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, WSGR Docket Number: 64828-710.601 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique analytes.
  • a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique analytes.
  • a sample comprises proteins.
  • a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or about 6000 unique proteins.
  • a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or about 6000 unique proteins.
  • proteins or peptides in a sample can be cleaved.
  • proteins or peptides in a sample can be cleaved using an enzyme.
  • the enzyme can be a protease.
  • the protease can be trypsin, chymotrypsin, Lys-C protease, elastase, or peptidase.
  • cleaved proteins comprise peptides.
  • a peptide can be reacted with phenyl isothiocyanate.
  • the peptide can form a cyclical phenylthiocarbamoyl derivative in response to reaction with phenyl isothiocyanate.
  • the derivative can be cleaved as a thiazolinone derivative.
  • a peptide can undergo at least a portion of an Edman degradation cycle.
  • products of at least a portion of an Edman degradation cycle can be translocated through a pore.
  • a sample can undergo trypsinization followed by at least a portion of an Edman degradation cycle.
  • a sample comprises peptides.
  • a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique peptides.
  • a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique peptides.
  • a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ⁇ L in volume.
  • a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 mL in volume. In some embodiments, a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ⁇ L in volume. In some embodiments, a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 mL in volume.
  • a sample can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ng of analytes. In some embodiments, a sample can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ⁇ g of analytes.
  • a sample can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ng of analytes. In some embodiments, a sample can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ⁇ g of analytes. [0702] A sample can be provided as an impure mixture of one or more analytes and one or more impurities.
  • the impurities can be proteinaceous or non-proteinaceous (e.g., carbohydrates, or lipids, or any combination thereof).
  • an analyte can be an uncleaved polypeptide, and impurities can be cleaved products of a polypeptide.
  • Impurities may comprise truncated forms of the analyte.
  • Impurities may also comprise proteins other than the protein analyte e.g. which may be co-purified from a cell culture or obtained from a sample.
  • a protein may comprise one or any combination of any amino acids, amino acid analogs, and naturally or artificial modified amino acids (i.e. amino acid derivatives).
  • a sample can be assayed in vitro.
  • a sample can be assayed in vivo, e.g., to sense insulin levels in diabetic patients.
  • a sample can be obtained from or extracted from an organism of various kinds (e.g. archaeal, prokaryotic, or eukaryotic).
  • a sample can comprise a body fluid of a patient (e.g.
  • a sample can be from a human, a mouse, a rat, a macaque, a pig, a cow, a chicken, or a plant.
  • a sample can be a non-biological sample, e.g., surgical fluids, water such as drinking water, sea water, or river water, which can provide environmental testing results, as well as reagents for laboratory tests.
  • a sample can be preprocessed prior to being assayed.
  • a sample can be purified, e.g., to isolate certain class of analytes such as proteins or peptides.
  • purification can be performed with affinity binding, such as by antibodies.
  • purification can be performed with chromatographic methods.
  • purification can be performed to isolate and/or purify specific components of the sample and/or remove unwanted background impurities.
  • a sample can be preprocessed using a denaturing agent.
  • a sample can be preprocessed using applied heat.
  • a sample can be preprocessed using chaotropic agents or detergents.
  • a sample can be preprocessed to provide a WSGR Docket Number: 64828-710.601 predetermined pH or salt condition.
  • a sample can be preprocessed using a reducing agent.
  • a sample can be preprocessed to break cross-links such as disulphide bridges, e.g., to disrupt certain secondary structures.
  • a sample can be preprocessed to label certain moieties, e.g., labeling cysteines or lysines with tags to modulate a signal arising from translocation of a labeled analyte through a pore.
  • a moiety can comprise an amine, a carbonyl, a thiol, an alkyne, or an azide.
  • a label can target a particular amino acid.
  • a moiety can be labeled or tagged with a fluorescent molecule, a radioisotopic molecule, a dye, a quantum dot, or a molecular label, or combinations thereof.
  • a label or tag can cause a greater change in measured signal when a sample can be translocated through a pore.
  • a label or tag can decrease a change in measured signal when a sample can be translocated through a pore.
  • a label or tag can increase a sensitivity of analysis for an analyte.
  • N- and C- termini of amino acid-based molecules can be modified in a sample.
  • N- or C- termini of amino acid-based molecules can be chemically modified.
  • amino acid-based molecules can be modified by adding a molecular label or tag (e.g. adding a barcode to register a precursor sample, or to facilitate capture and/or detection in a pore system).
  • a sample label or tag can comprise a peptide, a protein, or a nucleic acid, or any combination thereof.
  • a sample label or tag can comprise an amine-reactive dye, a thiol reactive dye, a carbonyl-reactive dye, a click chemistry reactive group, or a copper-free click chemistry group.
  • a sample label or tag comprises an active ester, succinimidyl ester, tetrafluorophenyl ester, sulfodichlorophenol ester, isothiocyanate, sulfonyl chloride, hydrazine, hydroxylamine, an alkoxyamine, maleimide, an iodoacetyl group, or a pyridyl disulfide.
  • an analyte can be modified.
  • the polypeptide can be modified by a leader according to embodiments involving a translocase as disclosed herein below.
  • the disclosed methods are for characterizing modifications in an analyte.
  • one or more of the amino acids/derivatives/analogs in an analyte can be post-translationally modified.
  • an analyte can be coupled to a leader.
  • a leader or leader- coupled analyte can preload and/or stall translocases.
  • an analyte may not be coupled to a leader.
  • a leader can comprise an exogenous sequence, a recognition motif, a capture motif, a stall motif, a block motif, or a coupling motif, or any combination thereof, and any permutation thereof.
  • the motifs in a leader can be arranged in various orders.
  • the leader can comprise motifs in the order of recognition-capture-stall-block-coupling, or capture-stall-block-coupling, or recognition-stall-block- coupling.
  • the leader can comprise an exogenous sequence.
  • an exogenous sequence can be greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, or about 600 amino acids long.
  • an WSGR Docket Number: 64828-710.601 exogenous sequence can be less than about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, or about 600amino acids long.
  • an exogenous sequence can be from about 2 to about 600 amino acids long.
  • an exogenous sequence can be from about 5 to about 50 amino acids long.
  • An exogenous element and/or an exogenous sequence can be one or more leader constructs, barcodes, adaptors, recognition motifs, or any combination thereof.
  • a translocase fully or partially comprises amino acids.
  • a translocase does not comprise amino acids.
  • the amino acids can be natural, non-natural, or a combination thereof.
  • translocases can move along proteins either in the C to N direction or the N to C direction.
  • the components of the leader may be arranged in either the C to N or N to C orientation, or different combinations thereof in a single construct.
  • the components of the leader may be arranged to promote translocation in either the C to N or N to C orientation.
  • a leader can comprise stretches of synthetic polymer molecules, such as PEG, PHEMA, polyacrylamide, polynucleotide, or peptide nucleic acids, or combinations thereof.
  • the overall composition of the leader may be adapted for good water solubility.
  • the composition may be adapted for low structural propensity in regions of capture to provide more efficient pore capture.
  • a leader can be added to the N- or C- terminus of a protein, or to both ends.
  • a leader e.g.
  • both termini of an analyte can be coupled to different leader designs, creating separate “leader” and “tail” ends.
  • leaders and tails can be used to control which behaviors occur at which end, for example to control the orientation of the capture of the analyte, or the loading and relative direction of the translocase, or any combination thereof. For example, in combination with a leader that directs binding of translocase and capture in a pore (e.g.
  • leader contains translocase recognition motif/capture motif/stall motif/block motif
  • a tail sequence e.g. simple unstructured amino acid sequence
  • a recognition motif promotes binding of the leader-conjugated analyte to the translocase/unfoldase.
  • a recognition motif can include a peptide tag to enhance binding of the tagged protein.
  • a recognition motif can include ssrA (AANDENYALAA; SEQ ID NO: 38) to enhance binding of the tagged protein to ClpX, ClpA, ClpC, ClpE, PAN, FtsH, or VAT or any combination thereof.
  • the recognition motif can comprise any of the motif sequences as shown in Table 25. Table 25. Sequences of recognition motifs. WSGR Docket Number: 64828-710.601 Description Sequence SEQ ID NO.
  • a recognition motif can comprise C-terminal residues from SulA (SASSHATRQLSGLKIHSNLYH) to enhance binding to HslU, Lon, or any combination thereof.
  • a recognition motif can comprise portions of Pex15 (Pex15254-309 AKSKGKQRGVKQKIHHFHEPMLHNSSEEQVKVEDAFNQRTSTDSRLQSTGTAPRKK, Pex1543-309 SEVFQECVNLFIKRDIKDCLEKMSEVGFIDITVFKSNPMILDLFVSACDIMPSFTKLGLTLQSEILNIFTL DTPQCIETRKIILGDLSKLLVINKFFRCCIKVIQFNLTDHTEQEEKTLELESIMSDFIFVYITKMRTTIDV VGLQELIEIFIFQVKVKLHHKKPSPNMYWALCKTLPKLSPTLKGLYLSKDVSIEDAILNSIDNKIQKDK AKSKGKQRGVKQKIHHFHEPMLHNSSEEQVKVEDAFNQ
  • the recognition motif can comprise between about one repeating unit to about 30 repeating units. In some cases, the recognition motif can comprise between about on repeating unit to about WSGR Docket Number: 64828-710.601 5 repeating units, between about 5 repeating units to about 10 repeating units, between about 10 repeating units to about 15 repeating units, between about 15 repeating units to about 20 repeating units, between about 20 repeating units to about 25 repeating units, or between about 25 repeating units.
  • the recognition motif can comprise at least about one repeating unit, at least about 2 repeating units, at least about 3 repeating units, at least about 4 repeating units, at least about 5 repeating units, at least about 6 repeating units, at least about 7 repeating units, at least about 8 repeating units, at least about 9 repeating units, at least about 10 repeating units, at least about 11 repeating units, at least about 12 repeating units, at least about 13 repeating units, at least about 14 repeating units, at least about 15 repeating units, at least about 16 repeating units, at least about 17 repeating units, at least about 18 repeating units, at least about 19 repeating units, at least about 20 repeating units, at least about 21 repeating units, at least about 22 repeating units, at least about 23 repeating units, at least about 24 repeating units, at least about 25 repeating units, at least about 26 repeating units, at least about 27 repeating units, at least about 28 repeating units, at least about 29 repeating units, at least about 30 repeating units, or more than 30 repeating
  • the recognition motif can comprise at most about 30 repeating units, at most about 29 repeating units, at most about 28 repeating units, at most about 27 repeating units, at most about 26 repeating units, at most about 25 repeating units, at most about 24 repeating units, at most about 23 repeating units, at most about 22 repeating units, at most about 21 repeating units, at most about 20 repeating units, at most about 19 repeating units, at most about 18 repeating units, at most about 17 repeating units, at most about 16 repeating units, at most about 15 repeating units, at most about 14 repeating units, at most about 13 repeating units, at most about 12 repeating units, at most about 11 repeating units, at most about 10 repeating units, at most about 9 repeating units, at most about 8 repeating units, at most about 7 repeating units, at most about 6 repeating units, at most about 5 repeating units, at most about 4 repeating units, at most about 3 repeating units, at most about 2 repeating units, at most about one repeating unit, or less than one repeating

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Nanotechnology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention provides a method for translocating an analyte through a nanopore disposed within a membrane, the analyte comprising a protein, a polypeptide or a peptide, wherein a current or a voltage or a change thereof is detected while the analyte is translocating through the nanopore and one or more characteristics of the analyte are determined.

Description

METHODS FOR NANOPORE-BASED ANALYTE DETECTION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of United Kingdom Patent Application No. 2313202.0, filed August 30, 2023, which is herein incorporated by references in its entirety.
BACKGROUND
[0002] Nanopore sequencing is an approach to sequencing of nucleic acid molecules. Using nanopore sequencing, a single molecule of DNA or RNA can be sequenced without the need for PCR amplification or chemical labeling of the sample. Nanopore sequencing can offer low-cost genotyping, high mobility for testing, and rapid processing of samples with the ability to display results in real-time. It has been used in the rapid identification of viral pathogens, epidemiological monitoring, environmental monitoring, food safety monitoring, human genome sequencing, plant genome sequencing, monitoring of antibiotic resistance, haplotyping, and other applications.
SUMMARY
[0003] Molecules can be detected and characterized by nanopores and nanopore sensors based on capture and modulation of ionic current. Nanopores may identify and characterize many analytes, such as nucleic acid molecules, peptides, polypeptides, or proteins, or fragments thereof, or any combination thereof. There is a need for improved nanopore, nanopore systems, and methods thereof for the detection, capture, and analysis of analytes. Recognized herein are composition, methods, and systems for enhancing proteomic characterization. [0004] In an aspect, the present disclosure provides a method for identifying a polypeptide characteristic, comprising (a) translocating a polypeptide through a nanopore disposed within a membrane; (b) detecting a current or change thereof while the polypeptide is translocating through the nanopore; and (c) using the current or change thereof detected in (b) to identify a characteristic of the polypeptide with an accuracy of at least 90%. [0005] In some embodiments, the characteristic comprises a sequence, a length, an identity, a secondary structure, a tertiary structure, a modification to the polypeptide, or combinations thereof.
[0006] In some embodiments, the nanopore is a biological nanopore.
[0007] In some embodiments, the biological nanopore is selected from FraC, a-hemolysin, CytK, Lysenin, MspA, CsgG, Aerolysin, or FhuA. In some embodiments, the biological nanopore comprises an outer membrane protein (OMP) such as OmpG or OmpF.
[0008] In some embodiments, the biological nanopore comprises one or more point mutations. WSGR Docket Number: 64828-710.601 [0009] In some embodiments, the one or more point mutations affects a diameter of the biological nanopore. [0010] In some embodiments, the one or more mutations create smaller openings on a cis side of the biological nanopore. In some embodiments, the one or more mutations create smaller openings on a trans side of the biological nanopore. In some embodiments, the one or more mutations create smaller openings in the center of the biological nanopore. In some embodiments, the one or more mutations constrict multiple point throughout a channel of the biological nanopore. [0011] In some embodiments, the diameter of the biological nanopore is from about 0.5 nm to about 2 nm. [0012] In some embodiments, the one or more point mutations affects a charge of the biological nanopore. [0013] In some embodiments, the net charge of the biological nanopore is positive. [0014] In some embodiments, the charge of the channel of the biological nanopore comprises positively charged portions. [0015] In some embodiments, the net charge of the biological nanopore is negative. [0016] In some embodiments, the charge of the channel of the biological nanopore comprises negatively charged portions. [0017] In some embodiments, the one or more point mutations allow for conductance at a set pH. [0018] In some embodiments, the pH is from about 5 to about 10. [0019] In some embodiments, the one or more point mutations are one or more lumen facing mutations. [0020] In some embodiments, the nanopore is an artificial nanopore. [0021] In some embodiments, the polypeptide is unfolded prior to translocation. [0022] In some embodiments, the polypeptide is unfolded by one or more unfoldases selected from the group consisting of prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, and Valosin-containing protein-like ATPase. [0023] In some embodiments, the one or more of unfoldases are coupled to the nanopore. In some embodiments, the one or more of unfoldases are coupled to the nanopore by covalent or non-covalent forces. In some embodiments, the one or more of unfoldases are coupled to the nanopore by Pi bonding, Pi-Pi bonding, H-bonding, electrostatic interactions, or hydrophobic interfaces, or combinations thereof. [0024] In some embodiments, the one or more unfoldases are suspended in an electrolyte solution on one side of the membrane. [0025] In some embodiments, the one or more unfoldases are configured to position proximal to the nanopore upon a binding event with the polypeptide. [0026] In some embodiments, the nanopore comprises a proteasome and/or a translocase. [0027] In some embodiments, either the proteasome or the translocase are located on a cis-side of the nanopore. In some embodiments, either the proteasome or the translocase are located on a trans-side of the nanopore. In WSGR Docket Number: 64828-710.601 some embodiments, either the proteasome or the translocase are located on both a cis-side and/or a trans-side of the nanopore. [0028] In some embodiments, both the proteasome and translocase are located on a cis-side of the nanopore. In some embodiments, both the proteasome and the translocase are located on a trans-side of the nanopore. In some embodiments, both the proteasome and the translocase are located on both a cis-side and a trans-side of the nanopore. [0029] In some embodiments, the proteasome comprises one or more subunits. [0030] In some embodiments, the proteasome is fused with the nanopore. [0031] In some embodiments, the polypeptide is translocated from a cis to a trans side of the membrane. In some embodiments, the polypeptide is translocated from a trans to a cis side of the membrane. In some embodiments, a polypeptide bound to a translocase is partially translocated from a cis to trans side of the membrane, then the translocase pulls the partially translocated portion of the polypeptide back through the pore in a trans to cis direction. [0032] In some embodiments, the one or more of unfoldases are positioned on the cis side of the membrane. [0033] In some embodiments, the one or more of unfoldases are positioned on the trans side of the membrane. [0034] In some embodiments, the polypeptide is fragmented before translocation. [0035] In some embodiments, a polypeptide fragment comprises a portion comprising a non-natural amino acid, polyethylene glycol, PNA, DNA, or RNA, or combinations thereof. [0036] In some embodiments, the polypeptide is fragmented by one or more proteases comprising trypsin-type or chymotrypsin-type of activity. [0037] In some embodiments, the one or more proteases are coupled to the nanopore. [0038] In some embodiments, the one or more proteases are positioned on a cis side of the membrane [0039] In some embodiments, the polypeptide is translocated through the nanopore sequentially. [0040] In some embodiments, translocation is affected by a molecular motor. [0041] In some embodiments, the step size of translocation is determined by the molecular motor. [0042] In some embodiments, the molecular motor is ATP driven. In some embodiments, the molecular motor is NTP driven. [0043] In some embodiments, the step size of translocation is from about 0.2 to about 80 Angstroms. In some embodiments, the step size of translocation is from about 0.5 to about 20 Angstroms. [0044] In some embodiments, translocation occurs in the absence of a molecular motor. In some embodiments, translocation occurs with a molecular motor that is not being actively driven by ATP or NTP. [0045] In some embodiments, the rate of translocation is from about 0.1 nm/s to about 300 nm/s. WSGR Docket Number: 64828-710.601 [0046] In some embodiments, the membrane is an insulating membrane. [0047] In some embodiments, the insulating membrane is a phospholipid bilayer. [0048] In some embodiments, the insulating membrane is a solid-state membrane. [0049] In some embodiments, the membrane has a thickness from about 4 nm to about 20 nm. [0050] In some embodiments, the polypeptide is unlabeled. [0051] In some embodiments, the polypeptide comprises a tag. [0052] In some embodiments, the polypeptide comprises an exogenous sequence. [0053] In some embodiments, the exogenous sequence is about 5 to about 50 amino acids in length. [0054] In some embodiments, the polypeptide is suspended in an electrolytic solution. [0055] In some embodiments, the electrolytic solution comprises water, potassium, lithium sodium, calcium, magnesium, phosphate, sulfate, or chloride, or any combination thereof. [0056] In some embodiments, the concentration of one or more electrolytes in the electrolytic solution is from about 0.1 M to about 1.5 M. [0057] In some embodiments, the pH of the electrolytic solution is from about 5 to about 10. [0058] In some embodiments, the pH of the electrolytic solution is different between a trans-side and a cis- side of a membrane. [0059] In some embodiments, a terminus of the polypeptide is chemically modified with a leader or a tail. [0060] In some embodiments, a machine learning algorithm is used to identify the characteristic of the polypeptide using the current signal. In some embodiments, the characteristic comprises a sequence. [0061] In some embodiments, identity of the characteristics is determined with an accuracy from about 90% to about 99.99%. [0062] In some embodiments, the multi pass accuracy is from about 90% to about 99.99%. [0063] In some embodiments, the single pass accuracy is from about 90% to about 99.99%. [0064] In some embodiments, the polypeptide comprises post-translational modifications. [0065] In some embodiments, the detected change in ionic current is from about 0.1 pA to about 150 pA. [0066] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane, wherein the step size of translocation is about 0.2 to about 80 Angstroms; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to characterize a property of the polypeptide or to identify a sequence of the polypeptide. [0067] In some embodiments, the step size is dependent on the polypeptide structure. WSGR Docket Number: 64828-710.601 [0068] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane, wherein the rate of translocation is about 0.1 nm/s to about 300 nm/s; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) characterize a property of the polypeptide or to identify a sequence of the polypeptide. [0069] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to identify a sequence of the polypeptide with a sensing throughput of at least 1 molecule / minute. [0070] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide is translocating through the nanopore; and using the current or change thereof detected in (b) to identify a sequence of the polypeptide wherein the average read length is at least about 1 to about 10 amino acids. [0071] In some aspects, the present disclosure provides a method for proteome analysis, comprising: providing a cell comprising a plurality of polypeptides; translocating a polypeptide of the plurality of polypeptides through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide of the plurality of polypeptides is translocating through the nanopore; using the current or change thereof detected in (b) to identify a sequence of the polypeptide of the plurality of polypeptides; and characterizing one or more properties of a proteome using the sequence of the polypeptide identified in (d), wherein proteome coverage is at least 50%. [0072] In some aspects, the present disclosure provides a method for proteome analysis, comprising: providing a cell comprising a plurality of polypeptides; translocating a polypeptide of the plurality of polypeptides through a nanopore disposed within a membrane; detecting a current or change thereof while the polypeptide of the plurality of polypeptides is translocating through the nanopore; using the current or change thereof detected in (b) to identify a sequence of the polypeptide of the plurality of polypeptides; and characterizing one or more properties of a proteome using the sequence of the polypeptide identified in (d), wherein sequence coverage is at least 10%. [0073] In some aspects, the present disclosure provides a device for identifying a polypeptide sequence, comprising: a first chamber configured to prepare a biological sample for polypeptide sequencing; a second chamber in fluidic communication with the first chamber, the second chamber comprising a support configured to connect and disconnect with a sensor array; a sensor array connected to the support, wherein the sensor array WSGR Docket Number: 64828-710.601 comprises: a plurality of membranes comprising a plurality of pores, the plurality of pores configured to translocate the plurality of polypeptides; and a plurality of electrodes configured to measure a plurality of electrical signals from the plurality of polypeptides translocating through the plurality of pores; and a recording device configured to receive and record a plurality of electrical signals from the plurality of electrodes. [0074] In some aspects, the present disclosure provides a device for identifying a polypeptide sequence, comprising: a first chamber configured to prepare a biological sample for polypeptide sequencing by binding a plurality of polypeptides in the biological sample with a plurality of biomolecules; a second chamber in fluidic communication with the first chamber, the second chamber comprising a support configured to connect and disconnect with a sensor array; a sensor array connected to the support, wherein the sensor array comprises: a plurality of membranes comprising a plurality of pores, the plurality of pores configured to translocate the plurality of polypeptides, wherein the plurality of biomolecules are configured to facilitate translocating the plurality of polypeptides; and a plurality of electrodes configured to measure a plurality of electrical signals from the plurality of polypeptides translocating through the plurality of pores; and a recording device configured to receive and record a plurality of electrical signals from the plurality of electrodes. [0075] In some embodiments, the sensor array comprises a plurality of chambers, wherein the plurality of membranes form a plurality of surfaces of the plurality of chambers. [0076] In some embodiments, the plurality of chambers comprise a volume from about 0.1 μL to about 250 μL. [0077] In some embodiments, a chamber of the plurality of chambers comprises a volume from about 0.0001 μL to about 1.0 μL. [0078] In some embodiments, the plurality of chambers comprise a thickness of at most about 3 mm. [0079] In some embodiments, the plurality of surfaces each comprise an area of at most about 5 mm2. [0080] In some embodiments, a surface of the plurality of surfaces comprises an area of at most 100 μm2. [0081] In some embodiments, the device is configured to connect to a recording device. [0082] In some embodiments, the recording device comprises an analog-to-digital converter. [0083] In some embodiments, the recording device comprises an amplifier. [0084] In some embodiments, the plurality of electrodes are disposed on a second plurality of surfaces of the plurality of chambers. [0085] In some embodiments, the sensor array comprises an adhesive configured to adhere the plurality of membranes to the plurality of chambers. [0086] In some embodiments, the device comprises a display for displaying the electrical signal. [0087] In some embodiments, the device comprises a flow cell. WSGR Docket Number: 64828-710.601 [0088] In some aspects, the present disclosure provides a microfluidic device for polypeptide characterization, comprising: one or more microfluidic channels for flowing fluid comprising a polypeptide therethrough; and one or more nanopores disposed within a membrane in fluid communication with the one or more microfluidic channels, the one or more nanopores configured to effect a change in a current applied across the membrane upon translocation of the polypeptide therethrough, wherein the changed effected in the current corresponds to a characteristic of the polypeptide. [0089] In some embodiments, the membrane comprises from about 10 to about 100,000 pores [0090] In some embodiments, the surface area of the membrane is at most about 5 mm2. [0091] In some embodiments, the surface area of the nanopores within the membrane is from about 50 to about 500 nm2. [0092] In some embodiments, the membrane is disposed within a fluidic chamber comprising an anode and a cathode. [0093] In some embodiments, each of the anode and cathode is independently positioned on either a cis side or a trans side of the membrane. [0094] In some embodiments, the device further comprises a potential generator for applying a potential difference across the anode and cathode. [0095] In some embodiments, the potential across the anode and cathode generated by the potential generator is from about 10 mV to about 1 V. [0096] In some embodiments, the microfluidic device further comprises a pump for flowing the fluid through the one or more microfluidic channels. [0097] In some aspects, the present disclosure provides a kit for use with a device for identifying a polypeptide sequence, comprising: a chip comprising a sensor array, the sensor array comprising a plurality of chambers or wells comprising a plurality of lipids and pores; and a biomolecule configured to bind to a polypeptide to facilitate translocation of the polypeptide through a pore. [0098] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal and a database, the database comprising a plurality of reference signals for the polypeptide, proteoforms thereof, and post-translationally modified variants thereof. [0099] In some aspects, the present disclosure provides a method for characterizing and/or identifying a polypeptide sequence, comprising: translocating a polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and WSGR Docket Number: 64828-710.601 assigning an identification to the polypeptide based on the electrical signal and a machine learning algorithm, the machine learning algorithm configured to distinguish between a plurality of reference signals for the polypeptide, proteoforms thereof, and post-translationally modified variants thereof. [0100] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: translocating a polypeptide from a biological sample through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal and a database, the database comprising a plurality of reference signals for a plurality of polypeptides, wherein the plurality of polypeptides comprise expressible polypeptides, or proteoforms thereof, or post-translationally modified variants thereof, or any combination thereof, based on genomic information of the biological sample. [0101] In some aspects, the present disclosure provides a method for identifying a polypeptide sequence, comprising: contacting a composition comprising a biological sample with a nanopore, the biological sample comprising an initial volume of at most about 50 μL, wherein the composition comprises a polypeptide and a nucleic acid from the biological sample; translocating the polypeptide through a nanopore disposed within a membrane; detecting an electrical signal while the polypeptide is translocating through the nanopore; and assigning an identification to the polypeptide based on the electrical signal. [0102] In some aspects, the present disclosure provides a method for generating a sample profile, comprising: receiving an electrical signal, wherein the electrical signal is derived at least from one analyte of a sample translocating across a nanopore; and generating the sample profile based on the electrical signal and a database, wherein the database comprises a plurality of reference electrical signals for a plurality of analytes; wherein the reference electrical signals for the plurality of analytes comprise expressible polypeptides, or proteoforms thereof, or post-translationally modified variants thereof, or combinations thereof. [0103] In some embodiments, the reference electrical signals further comprise small molecules, metabolites, DNA, or RNA, or combinations thereof. [0104] In some embodiments, the analyte is derived from a patient. [0105] In some embodiments, the sample profile comprises an assessment of patient health. [0106] In some embodiments, the analyte comprises a metabolite, a small molecule, a biopolymer, or a biomolecule. [0107] In some embodiments, the sample profile comprises an identification of a phenotype. [0108] In some embodiments, the sample profile comprises an identification of a metabolic state. [0109] In some embodiments, the sample profile comprises an identification of a disease. WSGR Docket Number: 64828-710.601 [0110] In some embodiments, the electrical signal is derived from a plurality of analytes of a sample translocating across a nanopore. [0111] In some embodiments, generating a sample profile comprises counting event instances of a subset of the plurality of analytes. [0112] In some embodiments, the sample profile comprises an environmental profile. [0113] In some embodiments, assigning an identification comprises determining a degree of similarity with a previously identified phenotypic profile. [0114] In some embodiments, the degree of similarity is measured by the proteomic coverage. [0115] In some aspects, the present disclosure provides a method for generating a sample profile, comprising: receiving an electrical signal, wherein the electrical signal is derived from at least one analyte translocating across a nanopore; and generating a sample profile based on the electrical signal and a machine learning algorithm configured to distinguish between a plurality of reference signals for the analyte. [0116] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein. [0117] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein. [0118] In some aspects, the present disclosure provides a method, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. [0119] In some aspects, the present disclosure provides a method for determining a characteristic of an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, or (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is WSGR Docket Number: 64828-710.601 translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte. [0120] In some aspects, the present disclosure provides a method for characterizing an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. [0121] In some aspects, the present disclosure provides a method for sample analysis, comprising: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or first fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or second fragments thereof, or a combination thereof; (c) detecting (i) (1) a first current or change thereof or (2) a first voltage or change thereof while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (3) a second current or change thereof, or (4) a second voltage or change thereof while the at least the portion of the second analyte is translocating through the second nanopore; (d) using (i) (1) the first current or change thereof or (2) the first voltage or change thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (3) the second current or change thereof or (4) the second voltage or change thereof to determine a second characteristic of the at least the portion of the second analyte; and (e) characterizing one or more properties of the sample using the first characteristic or the second characteristic determined in (d). [0122] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without WSGR Docket Number: 64828-710.601 departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE [0123] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS [0124] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and the disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which: [0125] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which: [0126] FIGs.1A-1C illustrate electro-osmotic nanopore systems for the translocation and characterization of polymer analytes such as polypeptides through the nanopores, in accordance with some embodiments. FIG.1A illustrates a strong cis-to-trans EOF across the system for capture and translocation of a polymer analyte in the cis to trans direction, in accordance with some embodiments. Arrows through the pore indicate the magnitude of the ion flow in each direction, showing that the EOF can be generated by a large net flow of ions from cis to trans. FIG.1B illustrates a strong cis-to-trans EOF established in a system with positive voltage applied to the trans compartment across the membrane. FIG.1C illustrates a strong cis-to-trans EOF established in a system with negative voltage applied to the trans compartment across the membrane. [0127] FIG. 2 shows example nanopore-based systems for characterizing and/or translocating polymer analytes. A translocase motor can aid translocation of the analyte through the nanopore, progressing along the polymer analyte in the direction of the subset arrow (moving away from termini PA towards termini PB of polymer analyte. WSGR Docket Number: 64828-710.601 [0128] FIGs.3A-3D show CytK nanopores. FIG.3A illustrates a cross-section of a surface representation of WT-CytK nanopores in 1 M KCl, pH 7.5. The nanopore was made by homology modelling from the alpha- hemolysin nanopore. FIG. 3B shows a cartoon representation of WT-CytK β-barrel region. The N-terminal strand is in dark gray and the C-terminal strand is in light gray. The charged residues are underlined. FIG.3C shows a schematic of the residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK. FIG. 3D shows a cross-section of a surface (left) and cartoon (right) representation of a high ion selectivity mutant, CytK-2E-4D. [0129] FIG. 4. shows an amino acid sequence and corresponding schematic representation of the three designed unstructured model polypeptide analytes, in accordance with some embodiments (which can be referred to as S1, tzatziki and mujdei). Solid circles indicate negatively charged amino acids, and open circles indicate positively charged amino acids. [0130] FIGs. 5A-5E illustrate translocation of analyte S1 through wildtype (WT)-CytK nanopores, in accordance with some embodiments. FIG. 5A shows a schematic representation of the translocation of S1 through WT-CytK. The arrow denotes the analyte S1 translocating through the nanopore by an electrophoretic force (EF). FIG.5B shows a voltage dependency of translocation rates for type 1 and type 2 blockades. FIG. 5C shows a voltage dependency of the excluded current (Iex(%)) for type 1 and type 2 blockades. For FIGs.5B and 5C, type 1 blockades are shown as black squares and type 2 blockades are shown as light gray circles. FIG. 5D shows representative traces at –160 mV bias, in accordance with some embodiments. IO denotes the open pore current measurement and IB denotes the blocked pore current measurement. FIG.5E shows dwell time versus current amplitude at –160 mV bias. [0131] FIGs. 6A-6E illustrate translocation of S1 through 2E-4D-CytK nanopores, in accordance with some embodiments. FIG. 6A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments. The arrows denote the analyte S1 translocating through the nanopore by an electrophoretic force (EF; solid arrow) and electro-osmotic force (EOF; dotted arrow). The EF and EOF are in the same direction. FIG.6B shows a voltage dependency of translocation rate for analyte S1, in accordance with some embodiments. FIG.6C shows a voltage dependency of the excluded current (Iex%), in accordance with some embodiments. FIG. 6D shows representative traces at –40 mV bias, in accordance with some embodiments. FIG.6E shows a dwell time versus current amplitude at –40 mV bias, in accordance with some embodiments. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. [0132] FIGs. 7A-7E illustrate translocation of tzatziki through 2E-4D-CytK nanopores, in accordance with some embodiments. FIG.7A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments. The arrows denote the analyte tzatziki translocating through the WSGR Docket Number: 64828-710.601 nanopore by an electrophoretic force (EF; solid arrow) and electro-osmotic force (EOF; dotted arrow). The EF and EOF are in opposing directions. FIG.7B shows a voltage dependency of translocation rate, in accordance with some embodiments. FIG.7C shows a voltage dependency of the excluded current (Iex%), in accordance with some embodiments. FIG. 7D shows representative traces at –160 mV bias, in accordance with some embodiments. FIG. 7E shows dwell time versus current amplitude at –160 mV bias, in accordance with some embodiments. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. [0133] FIGs. 8A-8E illustrate translocation of mujdei through 2E-4D-CytK nanopores, in accordance with some embodiments. FIG.8A shows a schematic representation of the translocation of S1 through 2E-4D-CytK, in accordance with some embodiments. The arrows denote the analyte tzatziki translocating through the nanopore by an electrophoretic force (EF; solid arrow) and electro-osmotic force (EOF; dotted arrow). The EF and EOF are in opposing directions. FIG.8B shows a voltage dependency of translocation rate, in accordance with some embodiments. FIG.8C shows a voltage dependency of the excluded current (Iex%), in accordance with some embodiments. FIG. 8D shows representative traces at –160 mV bias, in accordance with some embodiments. FIG. 8E shows dwell time versus current amplitude at –160 mV bias, in accordance with some embodiments. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. [0134] FIGs.9A-9H illustrate translocation of model substrates through nanopores, in accordance with some embodiments. For each of FIGs. 9A-9H, panel (i) shows the cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right), panel (ii) indicates the entry or translocation of analyte S1, panel (iii), if present, indicates the entry or translocation of analyte tzatziki and panel (iv), if present, shows the entry or translocation of analyte mudjei. FIG.9A shows representative current traces of substrates through WT-CytK, in accordance with some embodiments. FIG. 9B shows representative current traces of substrates through K128D-CytK, in accordance with some embodiments. FIG. 9C shows representative current traces of substrates through K128-K155Q-CytK, in accordance with some embodiments. FIG.9D shows representative current traces of K128D-K155D-CytK, in accordance with some embodiments. FIG. 9E shows representative current traces of K128D-K155Q-Q122D-CytK, in accordance with some embodiments. FIG. 9F shows representative current traces of K128D-K155D-Q145D-CytK, in accordance with some embodiments. FIG.9G shows representative current traces of substrates through K128D-K155D- T147D-CytK, in accordance with some embodiments. FIG.9H shows representative current traces of substrates through K128D-K155D-Q145D-S151D-CytK, in accordance with some embodiments. [0135] FIGs. 10A-10G show translocation of model substrates through nanopores in accordance with some embodiments. FIG. 10A shows a cut-through of a surface representation of the WT-CytK nanopore (left) and WSGR Docket Number: 64828-710.601 a cartoon representation of its β-barrel region (right). Plots depict the voltage dependency of the excluded current (Iex%) for type 1 (dark gray circle) and type 2 blockades (light gray circle) (left) and a voltage dependency of translocation rates for type 1 blockade (dark gray square) and type 2 blockades (light gray circle) (right) for the translocation of substrates through WT-CytK. FIG. 10B shows a cut-through of a surface representation of the K128D-K155Q-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right) and voltage dependency of excluded current (Iex(%)) (left) for the translocation of substrates through K128D-K155Q-CytK. FIG.10C shows a cut- through of a surface representation of the K128D-K155D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates for the two types of events, type 1 blockade (dark gray square) and type 2 blockade (light gray circle), and voltage dependency of excluded current (Iex(%)) of substrates through K128D-K155D-CytK. FIG. 10D shows a cut-through of a surface representation of the K128D-K155Q-Q122D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex(%)) (left) of substrates through K128D-K155Q-Q122D-CytK. FIG. 10E shows a cut- through of a surface representation of the K128D-K155D-Q145D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex(%)) (left) of analyte S1 and analyte tzatziki through K128D- K155D-Q145D-CytK. FIG.10F shows a cut-through of a surface representation of the K128D-K155D-T147D- CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex%)(left) of analyte S1 and analyte tzatziki through K128D-K155D-T147D-CytK. FIG. 10G shows a cut-through of a surface representation of the K128D-K155D-Q145D-S151D-CytK nanopore (left) and a cartoon representation of its β-barrel region (right). Plots depict voltage dependency of translocation rates (right), and voltage dependency of excluded current (Iex%)(left) of analyte S1, analyte tzatziki, and analyte mujdei through K128D-K155D-Q145D-S151D- CytK. [0136] FIGs. 11A-11F illustrate translocation of unfolded MalE219a across 2E-4D-CytK nanopores. FIG. 11A shows representative traces of the translocation of MalE219a across 2E-4D-CytK in 2M urea. FIG. 11B shows dwell time versus amplitude of current blockades under –100 mV. FIG. 11C shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right). FIG. 11D shows a cartoon representation of MalE219a. FIG.11E shows a voltage dependency of the translocation speed. FIG. 11F shows a voltage dependency of the excluded current. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. WSGR Docket Number: 64828-710.601 [0137] FIGs.12A-12F show translocation of unfolded H152A-GBP across 2E-4D-CytK nanopores. FIG.12A shows representative traces of the translocation of H152A-GBP across 2E-4D-CytK in 2.4 M urea showing the two levels, Level 1 (L1) and level 2 (L2), of the translocation blockades. FIG. 12B shows dwell time versus amplitude of current blockades under –100 mV. FIG.12C shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right). FIG. 12D shows a cartoon representation of MalE219a. FIG.12E shows a voltage dependency of the translocation speed. FIG.12F shows a voltage dependency of the excluded current for L1 and L2 levels as indicated in panel A. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. [0138] FIGs.13A-13E illustrate malE219a translocation through the 2E-2D CytK mutant in the presence of 1 M and 1.8 M GuHCl. FIG. 13A shows a cut-through of a surface representation of the nanopores (left) and a cartoon representation of its β-barrel region (right), in accordance with some embodiments. FIG.13B shows a voltage dependency of the dwell time of malE219a in 1 M (squares) and 1.8 M (circles) GuHCl. FIG. 13C shows a voltage dependency of the excluded current of malE219a in 1 M (squares) and 1.8 M (circles) GuHCl. FIG. 13D shows a dwell time versus amplitude dependence at various voltages in 1M GuHCl as indicated. FIG.13E shows a dwell time versus amplitude dependence at various voltages in 1M GuHCl as indicated. [0139] FIGs.14A-14D show characterizations of the 2E-4D-CytK nanopore in the two denaturants. IV curves for 2E-4D-CytK nanopores in the urea is shown in FIG. 14A and GuHCl in FIG. 14B. Numerical values of the asymmetry using the ratio of the ionic current at -100 mV and +100 mV in different concentrations of urea (FIG.14C) and GuHCl (FIG.14D). [0140] FIGs.15A-15C show MalE219a transport across WT-CytK. FIG. 15A shows translocation events of 100 nM of malE219a-D10 unfolded by 2 M urea, in accordance with some embodiments. FIG.15B shows 100 nM of malE219a added in the cis chamber did not induce events. FIG.15C shows a sequence of malE219a- D10. [0141] FIGs.16A-16C show actinoporins common sequence alignment and wild-type Fragaceatoxin C. FIG. 16A shows common sequence alignment of some actinoporins, the dots represent the same amino acid as the common sequence, other amino acid differences between the pores are represented by their single-letter code. FIG.16B shows an artistic representation of Fragaceatoxin C (PDB: 4TSY) inserted into a lipid bilayer, across which a voltage is applied. Several non-conserved positions are enlarged. FIG.16C shows representative traces of the octameric (T1) and heptameric (T2) form of wild-type Fragaceatoxin C under an applied potential of -50 mV in 1M KC1 and 50 mM citric acid titrated with bis-tris propane to pH 3.8. Traces were collected at a sampling frequency of 50 kHz, using a 10 kHz Bessel filter and 5 kHz Gaussian filter. [0142] FIG.17 shows alignment between Fragaceatoxin C homologues. Positions in homologs corresponding to D10 and G13 in Fragaceatoxin C are outlined by black boxes. WSGR Docket Number: 64828-710.601 [0143] FIGs.18A-18D show electrophysiology recordings of (mutant) Fragaceatoxin C with trypsin digested lysozyme, in accordance with some embodiments. FIG. 18A shows representative electrical ionic current traces of (mutant) Fragaceatoxin C combined with equal units of trypsin digested lysozyme added to the first side (e.g., cis side) and under an applied potential of -50 mV. The lowest current level is the open-pore current of the pore (Io), and the step-like upwards events may be the result of captured analytes occluding a portion of the ionic current flowing through the nanopore (event blockades, IB). FIG.18B shows representative trace of octameric Fragaceatoxin C (T1). FIG. 18C shows representative trace of heptameric Fragaceatoxin C (T2). FIG.18D shows representative trace of Fragaceatoxin C mutant (G13F). [0144] FIGs. 19A-19E show event count and signal correlation of (mutant)’ Fragaceatoxin C with trypsin digested lysozyme. FIGS. 19A-19D show observed excluded current (Iex%) spectra from tryptic digest of lysozyme. FIG.19A shows octameric wild-type Fragaceatoxin C (T1). FIG.19B shows heptameric wild-type Fragaceatoxin C (T2). FIG. 19C shows Fragaceatoxin C mutant G13F. FIG. 19D shows Fragaceatoxin C mutant G13N. FIG.19E shows squared first derivative Euclidean cosine correlation of residual current spectra of (mutant) Fragaceatoxin C combined with equal units of trypsin digested lysozyme. Boxes 1-3 surrounding multiple mutants represent similar signals. [0145] FIGs.20A-20C show peptide recognition of (mutant) Fragaceatoxin C. FIG.20A shows the location of mutations in the lumen of Fragaceatoxin C (modeled on PDB: 4TSY) marked by arrows. FIG. 20B shows Gaussian fits to histograms of the excluded currents from the clustered event blockade for the capture and detection of Angiotensin IV, Angiotensin III, Angiotensin I and Angiotensinogen recorded under an applied potential of -50 mV. FIG.20C shows excluded current % (IEX%) versus dwell time scatter plots of the single- molecule peptide event blockades detected by the different pore types. [0146] FIG.21 shows peptide recognition of (mutant) Fragaceatoxin C. [0147] FIG. 22 shows an electrophysiology setup of an analytical system comprising a nanopore, in accordance with some embodiments. [0148] FIGs. 23A-23D show bottom-up nanopore-based proteomics. FIG. 23A shows an artistic representation of protease protein digestion to digest a protein into a mixture of peptide fragments. FIG. 23B shows an artistic representation of the experimental setup of a nanopore system. FIG. 23C shows an artistic representation of the resulting ionic current data for detected peptides from a nanopore-based electrophysiology experiment. FIG.23D shows an artistic representation of a resulting residual current versus standard deviation spectrum obtained from analysis of the individual single-molecule event blockades. [0149] FIGs. 24A-24B show excluded current - mass calibration using peptides and the spectrum obtained from tryptic lysozyme peptides. FIG. 24A shows the mass of the synthetic model peptides (circles) plotted against the average measured excluded current (%) for each peptide when added to the G13F-FraC-T1 nanopore WSGR Docket Number: 64828-710.601 system. FIG.24B shows excluded current spectrum (histogram of the excluded currents from event blockades) recorded from addition of a mixture of all the model peptides to a G13F-FraC-T1 pore. [0150] FIGs. 25A-25B show nanopore experiments compared to electrospray ionization mass spectrometry. FIG. 25A shows residual current spectrum as obtained by nanopore electrophysiology using G13F-FraC-T1 and a tryptic digest of Gallus-gallus lysozyme. FIG.25B shows mass spectrometry results from the same tryptic digest, but measured with a mass spectrometer (ESI-MS). The resulting peptide masses were mapped to residual current using the logistic function prediction shown in FIG.24A with a standard deviation of 0.5 Iex%. [0151] FIGs.26A-26C show the reproducibility of nanopore protein spectra, using three independent repeats of the sensing of proteolytic digestions. FIG.26A shows results from bovine serum albumin (BSA). FIG.26B shows results from dihydrofolate reductase (DHFR). FIG. 26C shows results from elongation factor P (EFP). For each figure, the left-side panel shows the excluded current histograms with a normalized area of 100%, which were obtained from the excluded current versus dwell time scatters of all event blockades shown in the respective right-side panels. [0152] FIGs. 27A-27B shows spectral matching using squared first difference correlation coefficient. FIG. 27A shows example representative baseline corrected residual current spectra of the measurement of peptide fragment mixtures from 9 tryptic digested proteins. Unique spectra were observed for each protein type. For all digested proteins of FIG. 27A, the right-side panel shows the excluded current histograms with a normalized area of 100%, which were obtained from the excluded current versus dwell time scatters of all event blockades shown in the respective left-side panel. FIG. 27B shows leave-one-out spectral matching of the baseline corrected residual current spectra using Euclidean cosine cross-correlation. [0153] FIG. 28 shows the detection of proteins kemptide (LRRASLG) and phosphorylated kemptide (LRRA{pS}LG). The graph shows that the peptides can be detected as two distinct clusters, plotting residual current (Ires = blockade current/open-pore current) versus dwell time. [0154] FIG.29 shows the detection of glycopeptides. The peptides were unmodified peptide (ANVTLNTAG), peptide with one glycan (ANVT(Glc)LNTAG and peptide with two glycans (ANVT(Glc)LNTT(Glc)G). [0155] FIGs.30A-30B show the detection of rhamnosylated proteins. FIG.30A shows unmodified Elongation Factor P (EF-P), with residual current (IRes) plotted against dwell time. FIG. 30B shows and rhamnosylated EF-P, with residual current (IRes) plotted against dwell time. [0156] FIGs.31A-31C show discrimination between single amino changes. FIG.31A shows detection of two forms of enkephalin with sequences added to the cis-chamber of G13F-FraC-T1 pores, in accordance with some embodiments: YGGFL, and YdAGFdL, wherein d represents a D-amino acid; all other amino acids are L- amino acid. FIGS. 31B-31C show differences in nanopore signal due to the presence of D-amino acids, with analytes added to the cis compartment (FIG.31B) or trans compartment (FIG.31C). WSGR Docket Number: 64828-710.601 [0157] FIGs.32A-32D show detection of trypsinated lysozyme in Aerolysin nanopores, including WT-Aer at pH 7.5 (FIG. 32A), WT-Aer at pH 3.8 (FIG.32B), Aer-K238F at pH 3.8 (FIG.32C) and Aer-K238D-S264F at pH 3.0 (FIG. 32D). The open-pore current (Io) and exemplary step-like current blockades (IB) from peptide captures are marked. [0158] FIGs. 33A-33I show detection of trypsinated lysozyme in Aerolysin nanopores. The pores included WT-Aerolysin at pH 7.5 (FIG. 33A), WT-Aerolysin at pH 3.8 (FIG. 33B), K238F aerolysin at pH 3.8 (FIG. 33C), K238D aerolysin at pH 3.0 (FIG. 33D), K238D-A260F aerolysin at pH 3.0 (FIG.33E), K238D-S264F aerolysin at pH 3.0 (FIG.33F), K238D-Q268F aerolysin at pH 3.0 (FIG.33G), K238D-S272F aerolysin at pH 3.0 (FIG.33H). FIG.33I shows measurement of 4μg trypsinated lysozyme added to the cis compartment (final concentration l0ng/μl) of nanopore system comprising Aer-K238W. [0159] FIGs.34A-34C show detection of trypsinated lysozyme in Cytolysin K (CytK) nanopores. The current traces show representative sections of ionic current data for selected pores, comprising either WT-CytK at pH 3.8 (FIG.34A), CytK-K128F at pH 3.8 (FIG.34B), or CytK-S126F-K128D at pH 3.8 (FIG.34C). The open- pore current (Io) and exemplary step-like current blockades (IB) from peptide captures are marked in each plot. [0160] FIGs.35A-35H show detection of trypsinated lysozyme in Cytolysin K (CytK) nanopores. FIG. 35A shows a homology model of CytK (left) mapped onto the structure of the alpha-hemolysin nanopore from Staphylococcus aureus, and predicted beta-strand showing inward water-facing amino acids for the beta-barrel lumen of the nanopore (right). FIGS.35B-35G shows residual current versus dwell time scatter of individual peptide blockades provoked by 4μg of trypsinated lysozyme added to the trans-chamber of a system comprising either (FIG.35B) wild type (WT-CytK) at pH 3.8, (FIG.35C) K128F CytK nanopore at pH 3.8, (FIG.35D) S126F- K128D CytK nanopore at pH 3.8, (FIG. 35E) S120F - K128D CytK nanopore at pH 3.0 (FIG. 35F) Q122F - K128D CytK nanopore at pH 3.0, (FIG.35G) G124F - K128D CytK nanopore at pH 3.0. FIG.35H shows measurement of two peptides (10 μM Lys4 and 10μM Lys7) added to the trans compartment a system comprising K128W CytK nanopore. [0161] FIGs.36A-36B show detection of Lys-C digested lysozyme in Lysenin nanopores. FIG.36A shows a nanopore system comprising wildtype lysenin (Lys-WT). FIG. 36B shows a nanopore system comprising mutant lysenin (Lys-E76F). [0162] FIGs. 37A-37F show detection of non-proteinaceous small molecules. The system comprised heptameric wild-type FraC (FIG. 37A), mutant FraC_G13F nanopores with Thioflavin (FIG. 37B and FIG. 37C), octameric wild-type FraC (FIG. 37D), or mutant FraC_G13F nanopores with Vitamin B12 (FIG. 37E and FIG.37F). [0163] FIG.38 shows the design of a transmembrane protein device for single-molecule protein analysis. WSGR Docket Number: 64828-710.601 [0164] FIGs. 39A-39D show the fabrication and electrical optimization of a nanopore. FIG.39A shows the effects of linker length on the nanopore expression in E. coli cells, insertion efficiency and nanopore stability. FIG. 39B shows the electrical properties of ^4 mutant. The left schematic of FIG. 39B shows the linker sequence of ^4 mutant. The middle current representation of FIG.39B shows electrical recordings of a single nanopore at ±35 mV. The right plot of FIG. 39B shows a histogram of the unitary conductance values of 59 nanopores at-35 mV. FIG.39C shows the electrical properties of ^2 mutant. The left schematic of FIG.39C shows the linker sequence of ^2 mutant. The middle current representation of FIG.39C shows a current trace and the current histogram corresponding the insertion of individual pore into a lipid membrane at +35 mV. The right plot of FIG. 39C shows a histogram of the unitary conductance values of 59 artificial nanopores at-35 mV. FIG. 39D shows interaction of DPhPC with the artificial transmembrane pore generated by molecular dynamics simulations. [0165] FIGs.40A-40H show the electrical properties of optimized artificial pore ( ^2) and discrimination of substrates. FIG. 40A shows the schematic of an ion-current measurement setup. FIG. 40B shows a current trace recorded through an efficient single pore after optimization at ±35 mV. FIG. 40C shows averaged current—voltage (I— V) characteristics of three different nanopores. The error bars represent a standard deviation from the mean curve of the ion selectivity of the nanopore (FIG.40D). Determination of the reversal potential shows that the pore is cation-selective, as expected from the electrostatic potentials at their constrictions. FIG.40E shows the chemical structure of beta-cyclodextrin (β-CD), scatter plots of Ires% versus dwell time, and representative trace. FIG. 40F shows the chemical structure of gamma-cyclodextrin (γ-CD), scatter plots of Ires % versus dwell time, and representative trace. FIG. 40G shows peptide sequences of angiotensin I, scatter plots of Ires % versus dwell time, and representative trace. FIG. 40H shows peptide sequences of dynorphin A, scatter plots of Ires % versus dwell time, and representative trace. [0166] FIGs.41A-41E show the design of the artificial proteasome-nanopore. FIG.41A shows the structure of T. acidophilum proteasome-PA26. The C-terminal of PA26 (S231) is near L21 of the α subunit. FIG. 41B shows the reconstitution of artificial proteasome-nanopore. To obtain subcomplex 3, two separate vectors were used to express the four proteins. FIG.41C shows SDS-PAGE (left) and native PAGE (right) analyses of the purified complex 3. SDS-PAGE revealed the presence of three unique bands of PAuA20 (top), α∆12 (middle), and β (bottom) with molecular weights of 52.7, 25.8, and 22.3 kDa, respectively. FIG.41D shows behavior of a single pore at ±35 mV in 1 M NaCl, 15 mM Tris, pH 7.5. FIG. 41E depicts a cut-through of a surface representation of artificial transmembrane proteasome. [0167] FIGs.42A-42C show SDS-PAGE analysis the hydrolyzing activity of subcomplex 3. FIG.42A shows β-casein (1 mg/mL) that was incubated with subcomplex 3 at 53°C in buffer A (50 mM Tris, pH 7.5, 150 mM NaCl). FIG. 42B shows β-casein (1 mg/mL) that was incubated with subcomplex 3 for 2 hours in buffer A. WSGR Docket Number: 64828-710.601 FIG.42C shows β-casein (1 mg/mL) that was incubated with subcomplex 3 at 53°C for 0.5 hour in buffer B (50 mM Tris, pH 7.5, 0.3-1.0 M NaCl). The β-casein/subcomplex 3 concentration ratio was 42. [0168] FIGs.43A-43F show the discrimination of substrates with the proteasomal nanopore. FIG.43A shows typical current trace provoked by the analyte substrate 1 (S1) using an inactive proteasome-nanopore. FIG.43B shows translocation of S1 (20 μM) through an inactive proteasome-nanopore mediated by VAT (20.0 μM) and ATP (2.0 mM). FIG.43C shows when an inactive proteasome is used in the presence of ATP and VAT, GFP- ssrA is unfolded and translocated intact through the proteasome chamber and nanopore. FIG.43D shows typical current traces provoked by S1 using an active proteasome-nanopore. FIG. 43E shows when an active proteasome is used, in the presence of VAT and ATP, only rare and fast events may be observed suggesting that the active proteasome-nanopore can cleave S1 efficiently, producing small fragments. As shown in FIG. 43F, when an active proteasome is used in the presence of ATP and VAT, unfolded GFP-ssrA is cleaved in the proteasomal chamber and the degraded peptides may be too short to be detected by the nanopore. [0169] FIGs. 44A-44B show discrimination of substrates with proteasomal nanopore. FIG. 44A shows sequence comparison of substrate 1 (S1) and substrate 2 (S2). FIG.44B shows scatter plots of fraction blockade versus time and representative blockades induced by cleaved S1 and S2. [0170] FIGs.45A-45D show the design and membrane insertion of PA26 artificial nanopore. FIG.45A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C). FIG.45B shows a structure of PA26 (PDB ID: 1YA7). FIG.45C shows the structure of artificial PA26-nanopore. FIG.45D shows a current trace shows insertion of individual pore. [0171] FIGs. 46A-46D show the design and insertion of ATPase artificial nanopores. FIG. 46A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C). The transmembrane region is highlighted in blue. FIG. 46B shows the structure of Aquifex aeolicus ATPase (PDB ID: 3M0E). FIG. 46C shows the structure of artificial ATPase transmembrane pore. FIG. 46D shows a current trace shows insertion and ATP hydrolysis of individual pore. [0172] FIG.47 shows the design of a ClpP-artificial nanopore for single-molecule protein analysis. [0173] FIG. 48 shows the current-voltage (I-V) characteristics of three different nanopores: opened ClpP- nanopore, closed ClpP-nanopore, and PA-nanopore. [0174] FIG.49 shows the controlled translocation through the ClpP-nanopore. [0175] FIGs. 50A-50G show the preparation and characterization of type I, type II, and type III FraC nanopores. FIG. 50A shows a cut through of a surface representation of WT-FraC oligomer (PDB: 4TSY) colored according to the vacuum electrostatic potential as calculated by PyMOLPyMOL. FIG.50B shows the percentage of the distribution of type I, type II, and type III for WT-FraC, W112S-FraC, W116S-FraC and W112S-W116S-FraC at pH 7.5 and 4.5. FIG.50C shows IV curves of type II nanopores formed by WT-FraC, WSGR Docket Number: 64828-710.601 W116S-FraC and W112S-W116S-FraC at pH 7.5 (15 mM Tris-HCl, 1 M KCl). FIG. 50D shows the single nanopore conductance of W116S-FraC in 1 M KC1 (0.1 M citric acid and 180 mM Tris base) at pH 4.5. FIG. 50E shows typical current traces for the three nanopore types of W116S-FraC in 1 M KCl at pH 4.5 under -50 mV applied potential. FIG.50F shows reversal potentials measured under asymmetric condition of KCl (1960 mM cis, 467 mM trans) at pH 4.5 for the three W116S-FraC nanopore types. FIG.50G shows molecular models of three types of FraC nanopores constructed from the FraC crystals structure using the symmetrical docking function of Rosetta. [0176] FIGs. 51A-51F show single channel conductance distributions of FraC nanopores at pH 7.5 and 4.5. FIG. 51A shows a table reporting the average conductance values obtained by fitting Gaussian functions to conductance histograms, in accordance with some embodiments. S.D. represents the standard deviation of all single channels (number given as n). In FIG. 51B-51F, each panel represents a different batch of FraC nanopores as indicated, in accordance with some embodiments. [0177] FIGs. 52A-52B show discrimination of angiotensin peptides in mixture with type II W116S-FraC nanopores. FIG.52A shows (i) sequences of angiotensin I (DRVYIHPFHL), II (DRVYIHPF), III (RVYIHPF) and IV (VYIHPF) with corresponding Ires% measured at -30 mV; (ii) blockades provoked by the four angiotensin peptides; (iii) density plot of the Ires% versus the standard deviation of the current amplitude for angiotensin I added to the cis compartment; and (iv) density plot after further addition of angiotensin II, angiotensin III, and angiotensin IV to the cis chamber. FIG. 52B shows discrimination of angiotensin II and angiotensin A (ARVYIHPF), showing (i) table showing the sequences, the molecular weights and the Ires% of the peptides; (ii) representative traces of the peptide blockades; and color density plot of the Ires% versus the standard deviation of the current amplitude for angiotensin II blockades prior (iii) and after (iv) the further addition of angiotensin A to the cis chamber. [0178] FIGs. 53A-53C show an evaluation of biological peptides having different chemical compositions. Relation between the molecular weight and Ires% of peptide using: type I WT-FraC nanopores (FIG. 53A), type II W116S-FraC nanopores (FIG. 53B), and type III W112S-W116S-FraC nanopores (FIG. 53C) at pH 4.5. The solid line represents a second order polynomial fitting. [0179] FIGs. 54A-54D show a nanopore peptide mass spectrometer at pH 3.8. FIG. 54A shows amino acid sequences of four different peptides and their overall charge at different pH. The chargeable amino acids are underlined. FIG.54B shows pH dependence of the Ires% for the four peptides (cis) shown in FIG.54A using type II W116S-FraC nanopores under -30 mV applied potential. FIG. 54C shows comparison of the Ires% versus the mass of peptides at pH 4.5 and 3.8. FIG.54D shows voltage dependence of c-Myc dwell times at different pHs. WSGR Docket Number: 64828-710.601 [0180] FIGs.55A-55C show discrimination of short peptide mixture with type III FraC nanopores comprising mutant W112S-W116S-FraC. FIG. 55A shows sequence, Ires% (-50 mV) and molecular weight (M.W) of angiotensin IV (VYIHPF), angiotensin 4-8 (YIHPF), endomorphin I (YPWF), and leucine enkephalin (Leu- enkephalin; YGGFL). FIG.55B shows blockades provoked by the different peptides. FIG.55C shows density plot showing the Ires% versus the standard deviation of the current blockade for the mixture of angiotensin IV, angiotensin 4-8, endomorphin I and leucine-enkephalin. [0181] FIGs. 56A-56B show characterization of type II FraC nanopores comprising an oxidized cysteine at position 10. Difference between the DOC/ W116S type II pore (FIG.56A) and the oxidized DIOC / W116S type II pore (FIG.56B). [0182] FIGs. 57A-57D show wild type FraC (WtFraC) and D10R-K159E FraC (ReFraC) nanopores. FIG. 57A shows a cross-section through octameric WtFraC showing coulombic surface coloring. Aspartate residue 10 (D10), located in the constriction zone of WtFraC, is indicated. FIG.57B shows a top view on WtFraC (top) and ReFraC (bottom). FIG. 57C shows single channel conductance histogram for ReFraC (left) and WtFraC (right) at +50 mV in IM NaCl, 15 mM Tris-HCl pH 7.5. FIG.57D shows raw trace of WtFraC (top) and ReFraC (bottom). [0183] FIGs.58A-58F show DNA discrimination with ReFraC. FIG.58A shows representative blockades of a homopolymeric DNA strand in complex with NA using ReFraC. The cartoon representations on the right of each current trace shows the interpretation of the current blockades. FIG. 58B shows representative distributions of residual currents obtained for A20, C20, T20 homopolymeric strands with ReFraC nanopores. FIG.58C shows current blockades of a continuous trace induced by homopolymeric C20 and A20 nucleotides to the same ReFraC pore. Traces shown were digitally filtered with 100 Hz cut-off. FIG. 58D shows a distribution of residual currents imposed by mixtures of C20 and A20 homopolymeric strands. FIG.58E shows continuous trace of an experiment to resolve mixtures of homopolymeric C20 and T20 nucleotides and FIG.58F shows the distribution of residual currents imposed by mixtures of C20 and T20 homopolymeric strands. [0184] FIGs.59A-59B show unzipping/translocation of dsDNA by ReFraC. FIG.59A shows a representative trace of ReFraC capturing a NA:A(dsDNA)C complex at +50 mV. The open pore current is denoted as “1” and for comparison indicated after capture of the complex. States 2 and 3 are indicative of the block. Upon reversal of potential (“4”) the block is immediately released indicating that the double-stranded region NA:A(dsDNA)C complex was peeled off. FIG. 59B shows at + 100 mV, in more than half of the cases (insert) a single block (“2”) is observed after the dsDNA part is pushed through (deformation, brackets) and upon application of - 30 mV the block cannot be released immediately (“3”). At higher negative potentials the block can be released, indicating a rotaxane was formed. WSGR Docket Number: 64828-710.601 [0185] FIGs. 60A-60B show unitary channel conductance distribution and voltage current dependence determined for WtFraC and ReFraC nanopores. FIG. 60A shows unitary channel conductance distribution measured for WtFraC (top) and ReFraC (bottom) pre-oligomerized pores reconstituted in planar lipid bilayers. FIG.60B shows voltage current dependence measured for WtFraC and ReFraC nanopores. [0186] FIG. 61 shows hemolytic activity of the WtFraC, D10R FraC and ReFraC. Hemolysis rate was calculated as inverse of the time elapsed till 50% decrease in turbidity (measured as optical density at 650 nm wavelength) observed in 1% of horse erythrocytes suspension in 15 mM Tris-HCl pH 7.5150 mM NaCl. [0187] FIGs.62A-62B show translocation and immobilization of A(dsDNA)C DNA substrate recorded with ReFraC nanopore. A(dsDNA)C substrate (depicted above the trace) was made by annealing of ohgo I (5’ biotinylated AAAAAAAAAAAAAAAAAAAAGTGCTACGACTCTCTGTGTGCCCCCCCCCCCCCCCCCCCC) and oligo II (CACACAGAGAGTCGTAGCAC). FIG.62A shows blockades provoked on ReFraC nanopore by 1 µM of A(dsDNA)C alone (left) and in complex with 0.25 µM of neutravidin (right), substrates were added in cis under +50 mV applied potential. FIG. 62B shows blockades provoked on ReFraC nanopore by 1 µM of A(dsDNA)C alone (left) and in complex with 0.25 µM of neutravidin (right), substrates were added in cis under at + 70 mV. [0188] FIG. 63 shows representative traces showing stepwise enhancements of the residual current within A(dsDNA)C-neutravidin blockades provoked on ReFraC nanopore. 1 µM of A(dsDNA)C and 0.25 µM of neutravidin were present in cis at +50 mV. [0189] FIG. 64 shows traces depicting rotaxane formation by A(dsDNA)C-neutravidin driven into ReFraC nanopore at +100 mV applied potential.1 µM of A(dsDNA)C and 0.250 µM of neutravidin were added in cis. Voltage stepping protocols are shown with the lines below each current trace diagram. Rotaxanes were dismantled by switching the applied potential to -40 mV. [0190] FIGs. 65A-65B show representative traces showing pseudorotaxane and rotaxane formation by oligonucleotide I - neutravidin immobilized within the ReFraC nanopore. FIG. 65A shows pseudorotaxane formation provoked by 1 µM of oligo I and 0.25 µM of neutravidin present in cis. FIG. 65B shows rotaxane formation by 1 µM of oligonucleotide I and 0.25 µM neutravidin present in cis while 1 µM of oligonucleotide II was added in trans. Rotaxanes were dismantled by switching the applied potential to -40 mV (two arrows above the trace indicate the dismantling of rotaxane). [0191] FIGs.66A-66C show capture of an oligopeptide (Endothelin 1) and a protein (Chymotrypsin) with two FraC variants at two different pH conditions, in accordance with some embodiments. FIG. 66A shows cross- sections of wild type FraC (WtFraC, PDB: 4TSY) and D10R-K159E-FraC (ReFraC). FIGs. 66B-66C show representative traces induced by 1 µM endothelin 1 (FIG. 66B) and 200 nM chymotrypsin (FIG. 66C) to WSGR Docket Number: 64828-710.601 WtFraC (left) and ReFraC (right). Chymotrypsin (PDB: 5CHA) and human endothelin 1 (PDB: 1EDN) are shown as surface representations. The coloring represents the electrostatic potential of the molecular surface as calculated by APBS(13) (pH 7.5 in 1 M KC1) with light gray and dark gray corresponding to negative and positive potentials (range -4 to +4 kbT/ec), respectively. [0192] FIGs. 67A-67B show electrostatic distribution and ion-selectivity of WtFraC and ReFraC. FIG. 67A shows the monomer averaged simulated electrostatic potentials reveal the negatively and positively charged constrictions of WtFraC and ReFrac, respectively. FIG. 67B shows determination of the reversal potential shows that WtFraC and ReFrac may be respectively cation- and anion-selective, as expected from the electrostatic potentials at their constrictions. [0193] FIGs. 68A-68E show biomarker characterization with WtFraC at pH 4.5. FIG. 68A shows, from top to bottom: (i) surface representation with molecular surface and cartoon representations (PyMOL) of chymotrypsin (25 kDa, PDB: 5CHA), (ii) a representative trace obtained under -150 mV applied potential, (iii) a heatplot depicting the dwell time distribution versus Ires% at -150 mV, and (iv) the voltage dependence of Ires%, the voltage dependence of the dwell times, and the capture frequency. FIGs. 68B-68E show the same information for β2- macroglobulin (FIG. 68B; 11.6 kD, PDB: 1LDS), human EGF (FIG.68C; 6.2 kD, PDB: 1JL9), endothelin 1 (FIG. 68D; 2.5 kD, PDB: 1EDN) and angiotensin I (FIG. 68E; 1.3 kD), respectively. Angiotensin I is depicted as a random structure drawn with PyMOL. [0194] FIGs. 69A-69E show discrimination of endothelin 1 and 2 with WtFraC at pH 4.5. FIG. 69A shows molecular surface representation of endothelin 1 and endothelin 2 using electrostatic coloring (PyMOL). FIG. 69B shows amino acid sequences of endothelin 1 and 2 (top), and Ires% and dwell time for endothelin 1 and endothelin 2 blockades at -50 mV in pH 4.5 buffer (1 M KCl, 0.1 M citric acid, 180 mM Tris-Base (bottom). Lines (6901) indicate the disulfide bridges in each oligopeptide. FIG.69C shows representative endothelin 1 and endothelin 2 blockades to the same FraC nanopore under -50 mV applied potential. FIG. 69D shows histogram (left) of residual currents provoked by 2 µM endothelin 1 and corresponding heatplot depicting the standard deviation of the current amplitude versus Ires% (right). FIG. 69E shows the same as FIG. 69D but after addition of 8 µM endothelin 2 to the same pore revealing a second population. [0195] FIG. 70 shows a computer system for implementing a method, a system, or a device of the present disclosure, in accordance with some embodiments. [0196] FIGS.71A-71B show a device comprising a nanopore as described herein. [0197] FIG. 72 shows a method using devices, nanopores, systems, and sample preparation as described herein. [0198] FIG.73 shows a kit as described herein. WSGR Docket Number: 64828-710.601 [0199] FIG.74 shows an exemplary system for characterizing and translocating polymer analytes, for example mixed amino-acid composition protein analytes, through a nanopore in a membrane. [0200] FIGs.75A-75B illustrate an example leader with potential components. FIG.75A shows components of a (peptide based) “leader construct” (6) for attaching to a target protein substrate of interest, which can assist loading/binding of protein translocase motor(s) for unfolding and controlling translocation of the target protein substrate through a nanopore. The construct comprises a number of possible elements: 1. Recognition motif, 2. Capture motif, 3. Stall motif, 4. Block motif, 5. Coupling motif. FIG.75B shows an illustrative schematic of a leader construct (6) that is attached to a target protein substrate(s) of interest (7), e.g. a folded or structured protein. [0201] FIG. 76 shows an exemplary process of a method of loading a protein translocase (8) onto a leader construct (6). A translocase first binds to a leader construct (B) at or near the recognition motif, and then proceeds to translocate along the construct (C) in the direction of the subset arrow via NTP hydrolysis until encountering the stall and/or blocking motifs that stall/pause the progression of the translocase (D). [0202] FIGs. 77A-77B show an exemplary process of loading multiple protein translocases onto a leader construct. FIG. 77A shows a schematic of a substrate designed to load and stall one protein translocase. The capture (2) and/or stall (3) motifs in combination have a footprint long enough to accommodate a single translocase. FIG.77B shows a schematic of a substrate designed to load and stall n multiple protein translocase, comprising a longer combination of capture (2) and/or stall (3) motifs that can effectively stall and accommodate the binding footprints of the n multiple translocases, such that the trailing translocase motor(s) cannot push the leading translocase(s) through the stall/block motifs. [0203] FIGs. 78A-78B show exemplary methods of loading a leader construct (6) with protein translocase(s) (8) and attaching the leader construct to a protein of interest (7). FIG.78A shows leader constructs can first be coupled to target protein analytes, then loaded with translocases. FIG.78B shows leader substrates can be pre- loaded with translocases, and then coupled to target protein analytes. [0204] FIGs.79A-79D show electrical recordings of the capture of a Maltose Binding Protein substrate (MBP- 1) in wild-type alpha-hemolysin nanopores (WT αHL). Figures show selected representative regions of recording at -80 mV (FIG.79A), -120 mV (FIG.79B), -160 mV (FIG.79C) and -180 mV (FIG. 79D) (trans electrode). [0205] FIGs. 80A-80D show the same as detailed in FIGs. 79A-79D, but using wild-type CytK nanopores (WT CytK). [0206] FIGs. 81A-81C show a representative example of electrical current vs time traces for testing of a Maltose Binding Protein substrate:ClpX translocase complexes (MBP-1:ClpX) in weak EOF wild-type alpha- WSGR Docket Number: 64828-710.601 hemolysin (FIGs. 81A-81B) or wild-type CytK (WT CytK) (FIG. 81C). Measurements were carried out in a system similar to that described in FIG.1 (except with low or zero EOF nanopores). [0207] FIGs. 82A-82B show a representative examples of electrical current vs time traces for testing of a Maltose Binding Protein substrate:ClpX translocase complexes (MBP-1:ClpX) in strong EOF CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores according to the system described in FIG.74. FIGs.82A-82B show representative sections at -80 mV from separate experiments. The characteristic ClpX controlled MBP-1 translocations are marked by numbered arrows. [0208] FIG.83 shows a representative zoomed single example event of ClpX controlled MBP-1 translocation through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores. Events start with a blockade (S1) from the open pore level (state i) to an almost 0 pA level (state ii), and terminate (S3) with a return to open pore current levels (state iv) when the ClpX reaches the end of the MBP-1. [0209] FIGs.84A-84B show representative zoomed examples of ClpX controlled MBP-1 translocation events through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores. The spectra illustrate MBP-1 polypeptide translocations through CytK 4D2E. [0210] FIGs.85A-85D show examples of ClpX controlled MBP-1 translocation events for selected high EOF nanopore systems using high ion-selectivity nanopores. Events acquired from a CytK_4D2E nanopore (CytK K128D K155D S120D Q122D) system at -80 mV (FIG. 85A), a CytK_3D1F2E nanopore (CytK K128F_S120D_Q122D_K155D) system -80 mV (FIG. 85B), a CytK_4D2E_Alt nanopore (CytK K128D K155D S120D S151D) system at -80mV (FIG.85C), a CytK_2D1F2E nanopore (CytK K128F S120D Q122D) system at -120 mV (FIG.85D), all in cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, with preloaded MBP-1:ClpX in the cis compartment (to a final concentration 0.2 µM ClpX, 0.1 µM MBP-1 and 2.5 mM ATP). [0211] FIGs. 86A-86B show representative zoomed exemplary events of ClpX controlled translocation of MBP-1 substrates (FIG.86A) compared to GFP-1 substrates (FIG.86B) through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores at -80 mV. [0212] FIG. 87 shows exemplary electrical current vs. time traces for testing of a Maltose Binding Protein:ClpX translocase complexes (MBP-1:ClpX) in strong EOF CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores with non-hydrolyzable Gamma-S-ATP. [0213] FIG. 88 shows a histogram of the translocation duration for 35 full-length ClpX controlled MBP-1 translocations through a CytK 4D2E nanopore at -80 mV (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5 containing 0.2 µM ClpX:0.1 µM MBP-1, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5). WSGR Docket Number: 64828-710.601 [0214] FIGs.89A-89B show a comparison of ClpX controlled MBP-1 translocations at -80 mV through CytK 4D2E nanopore systems (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5, 2.5 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5) without (FIG.89A) and with (FIG.89B) pre-loading incubation to form the MBP-1:ClpX complexes. [0215] FIG. 90 shows exemplary capture and ClpX controlled trans-to-cis translocation of MBP-1 through CytK 4D2E nanopores that were inserted from the cis compartment (cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2). Preloaded MBP-1:ClpX complexes were added to the trans compartment (to a final concentration 0.2 µM ClpX, 0.1 µM MBP-1 and 2.5 mM ATP) and captured into the trans entrance of the nanopores at +80 mV, and translocated under ClpX control as a result of the strong trans- to-cis EOF created at positive applied voltage. [0216] FIGs. 91A-91B show gel results of ClpX activity assays. FIG. 91A shows a gel showing the results of a ClpX/ClpP degradation assay of a GFP-ssrA substrate (GFP-0) under varying concentrations of KCl. FIG. 91B shows gel showing the results of a degradation assay of GFP-0 under varying concentration of potassium glutamate (KGlu). (65 nM ClpX, 65nM ClpP, 2800 nM GFP) [0217] FIG.92 shows representative ClpX controlled translocation of MBP-1 through a CytK 4D2E nanopore at -120 mV in a nanopore system. [0218] FIGs. 93A-93E show a schematic showing an exemplary “Out mode” method for characterizing a target protein by capturing it from the first side (e.g., cis side) into the nanopore of a system setup with high net cis-to-trans EOF, in conjunction with a protein translocase orientated on the target protein such that it then pulls the polypeptide back out through the same nanopore to the first side (e.g., cis side). [0219] FIG. 94 shows a schematic of current vs time for a translocation event resulting from translocase controlled polypeptide translocation through a nanopore as described by the scheme in FIGs.93A-93E. [0220] FIGs.95A-95D show structural models of CytK. FIG.95A shows a structural model of the wild-type CytK nanopore from homology mapping onto the structure of the alpha-hemolysin nanopore. FIG.95B shows a schematic of the residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK, marking water-facing residues of the down- and up- strands most suitable for mutagenesis. FIG.95C shows a model of the CytK 4D2E nanopore (CytK K128D K155D S120D Q122D), showing very high net negative internal charge due to mutations. FIG.95D shows a schematic location of mutations to negative residues in the barrel region of the CytK 4D2E nanopore. [0221] FIGs. 96A-96E show recordings of different substrates in CytK nanopores. FIG. 96A shows a schematic of the substrate design. FIGs.96B-96E show electrophysiology reads acquired from {GFP}-{MBP- 1}, {LIVBP}-{MBP-1}, {SpuE}-{MBP-1}, and {GBP}-{MBP-1} substrates. Each substrate shows a similar ionic current pattern in the region corresponding to MBP as marked by the underlying arrows, and a unique WSGR Docket Number: 64828-710.601 ionic current signature specific to the attached protein in the second sections as marked by the indicated arrows below. [0222] FIG.97 shows a single-molecule read of a 88 kDa MBP-MBP fusion protein. [0223] FIGs.98A-98D show stalling of ClpX at 37°C using blocking domains. FIG.98A shows unfolding of GFP-1 with (+ClpX) and without ClpX (-ClpX); FIG.98B shows mNG with an alpha-helical blocking domain; FIG. 98C shows mNG with a helix-turn-helix blocking domain, and FIG. 98D shows mNG with a hairpin blocking domain. [0224] FIGs. 99A-99B show depictions of maltose-binding protein (MBP) monomers (FIG. 99A) and maltose-binding protein dimers (FIG.99B) for analysis. [0225] FIG.100 shows an electrophysiology recording of a MBP protein lacking the ssrA recognition motif, that was tagged on the C-terminus to allow binding of ClpX. ClpX controlled MBP translocations (marked by arrows) are evident from the characteristic pattern of changing ionic current signals. [0226] FIGs.101A-101D show attachment of a single-stranded DNA to ClyA nanopore. FIG.101A depicts a side view (left) and top view (right) of ClyA structure (PDB: 6mrt). Serine at position 110 (S110) was genetically mutated to cysteine to enable site-specific chemical modification. FIG. 101B shows a schematic model showing the conjugation strategy of attaching ssDNA to ClyA nanopore. FIG. 101C shows a SDS- PAGE analysis of the conjugation efficiency. Lane 1: protein ladder, lane 2: ClyA-S110C monomer, lane 3: after reaction of ClyA-S110C with maleimide-PEG4-DBCO (ClyA-DBCO), lane 4: after reaction of purified ClyA-DBCO with f-azide (ClyA-f). FIG. 101D shows a native polyacrylamide gel analysis of the oligomerization of ClyA-f. Lane 5: ClyA-f after oligomerization, lane 6: S110C mutated ClyA after oligomerization. [0227] FIGs.102A-102F depict functionalization of ClyA nanopore with Spike nanobody Ty1 and electrical characterization of the nanopore. FIG.102A shows a schematic model showing the strategy of functionalizing ClyA nanopore with Ty1 nanobody. FIG. 102B shows I-V curves of ClyA-S110C (triangle), ClyA-f (square) and ClyA-f-Ty1 (circle) at applied potential ranging from -90 to 90 mV (three independent experiments). FIG. 102C shows a histogram showing conductance distribution of ClyA-f nanopore with (white) and without (black) Ty1 nanobody. FIG.102D depicts representative current traces of ClyA-f-Ty1 under an applied potential of - 20 mV. The terms “in” and “out” represented the nanobodies locating inside (blocked pore) and outside (open pore) of the nanopore respectively. Io is the open pore current and Ib is the blocked pore current. FIG. 102E shows an all-point histogram of the current traces shown in FIG. 102D, which demonstrated well-defined distribution of the blockade signals. FIG. 102F shows a schematic model interpreting the reversible conformation change between blocked (left) and open (right) states of ClyA-f-Ty1 at applied potential of -20 mV, which corresponded to the movement of one of the Ty1 nanobodies in and out of the vestibule of the pore. WSGR Docket Number: 64828-710.601 [0228] FIGs. 103A-103F show single channel recording traces of ClyA-f-Ty1 and the analysis of residual current Ib/Io, tin and tout under different applied potentials. FIG.103A shows representative current traces of ClyA-f-Ty1 under applied potentials ranging from -10 to -40 mV. FIG. 103B shows an all-point histogram of current traces depicted in FIG.103A, showing Ty1 nanobodies tend to reside in ClyA nanopore with increasing applied potentials. FIGs.103C-103D show a histograms of logarithmic time of Ty1 locating inside and outside of ClyA, respectively. FIGs. 103E-103F show the influence of applied potentials on the average logarithmic time of Ty1 locating inside and outside of ClyA. [0229] FIGs. 104A-104B depict nanobody attachment to ClyA through DNA oligo hybridization verified using DNase I. FIG.104A shows current traces of ClyA-f-Ty1 before and after the addition of 5 U DNase I in the presence of 2.5 mM MgCl2 at an applied potential of -20 mV. FIG. 104B shows enlarged representative current traces from FIG.104A, showing that the nanobodies attached to the ClyA nanopore were removed after the addition of DNase I after about 30 mins. All-point histograms were displayed on the top of the panel showing the current distribution before and after the addition of DNaseI. The schematic model shown above depicts how the nanobodies were removed from ClyA nanopore. [0230] FIG.105 depicts detection of Spike protein by nanobody-functionalized nanopores. The upper current trace shows ClyA-f-Ty1 before and after the sequential addition of 6 µM BSA and 2.3 nM Spike protein. Below the upper current trace are enlarged representative current traces and all-point histograms of the current distribution. From left to right are: (i) representative traces before addition of BSA and spike protein; (ii) representative traces after addition of BSA and prior to spike protein; and (iii) after addition of BSA and spike protein. showed before and after the addition BSA and Spike proteins. [0231] FIGs. 106A-106G show the effect of BSA on nanobody internalization. FIGs. 106A-106C show histograms distribution of tout before and after the addition of 3 µM BSA or 6 µM BSA to the first side (e.g., a cis side) of a ClyA-f-Ty1 nanopore system. The histograms were fitted with single exponential function. FIGs. 106D-106G show the change of blockade percentage, open percentage, average time of the coupled Ty1 staying inside the ClyA nanopore (tin), average time of Ty1 staying outside the ClyA nanopore (tout) with increasing concentration of BSA, respectively. (n=4, each experiment was conducted with independent nanopores. Error bars represent standard deviations). [0232] FIGs.107A-107E show the effect of adding Spike protein to the ClyA-f-Ty1 pore. FIG.107A depicts current traces showing the transition of the pore from a dynamic state (alternating between Ty1 in the pore and out the pore) to a fully open state (with the Ty1 trapped outside the pore through binding to Spike protein) in the period immediately following the addition of 2.3 nM Spike protein. FIG. 107B shows current traces of ClyA-f-Ty1 in the period approximately 25 minutes post addition of 2.3 nM Spike protein. FIG. 107C shows an all-point histogram of the current traces presented in FIG.107B. FIGs.107D-107E show histograms of the WSGR Docket Number: 64828-710.601 logarithm of tin (FIG. 107D) and tout (FIG.107E) after the addition of 2.3 nM Spike protein in of the period about 25 minutes post addition of Spike protein. The histograms were fitted with the Gauss distribution function. [0233] FIG.108A-108D show open probability of ClyA-f-Ty1 correlates positively with Spike trimer protein concentration. FIG. 108A shows representative current traces of ClyA-f-Ty1 before and after the addition of increasing concentration of Spike trimer protein. FIG. 108B shows an all-point histograms were displayed to show the current distribution before and after the addition of increasing concentration of Spike protein. FIG. 108C shows a curve regression of the open probability in the function of spike concentrations. The curve was fitted by using Hill-Langmuir equation (n=1.31, Kd=760.6 pM). FIG.108D shows a schematic model depicting the dynamics of the interaction between ClyA-f-Ty1 and Spike protein. Ty1 nanobodies dynamically move in and out of ClyA nanopore under applied potential. [0234] FIGs.109A-109J show the influence of Spike proteins concentration on binding kinetics to the ClyA- f-Ty1 pore. FIGs. 109A-109D show histograms of log10(tout) at Spike concentration of 115 pM, 230 pM, 345 pM, 460 pM, respectively. The data were fitted with Guass distribution. FIGs.109E-109H show histograms of log10(tin) at Spike concentration of 115 pM, 230 pM, 345 pM, 460 pM, respectively. The data were fitted with Guass distribution. FIGs.109I-109J show the concentration dependency of the logarithm of tout and tin. [0235] FIGs. 110A-110H show the behavior of ClyA-f-Ty1 in the presence of blood. FIG. 110A shows a schematic model showing electrical measurement of ClyA-f-Ty1 in the presence of blood. FIG. 110B shows current traces showing the current change before and after addition of 1 µL of blood to the ClyA-f-Ty1 nanopore presenting in 500 µL electrolyte buffer. FIGs.110C and FIG. 110E show representative current traces in the presence of 6 µM BSA (FIG. 110C) and after addition of 1 µL of blood (FIG.110E). FIGs.110D and FIG. 110F show all-point histograms of the current traces before (FIG.110D) and after (FIG.110F) addition of 1 µL of blood. FIG.110G shows a histogram of the logarithm of dwell time in level0 before and after the addition of 1 µL of blood. FIG. 110H shows a histogram of the logarithm of dwell time in level1 before and after the addition of 1 µL of blood. [0236] FIGs. 111A-111B show detection of spike trimer in the presence of blood. FIGs. 111A-111B show representative current traces before (FIG.111A) and after (FIG.111B) the addition of 2.3 nM Spike protein in the presence of 1 µL blood at a bias of -20 mV. [0237] FIGs. 112A-112B show detection of Her2 with functionalized nanopores. FIG. 112A shows representative current traces of ClyA attached by 2Rs15d nanobody (ClyA-f-15d) before and after the addition of 32.8 nM Her2 protein under an applied potential of -20 mV. FIG.112B shows representative current traces of ClyA attached by 2Rb17c nanobody (ClyA-f-17c) before and after the addition of 20.8 nM Her2 protein at the same applied potential. WSGR Docket Number: 64828-710.601 [0238] FIGs.113A-113E show functionalized ClyA nanopore for the detection of muPA. FIG. 113A shows the crystal structure of muPA in complex with nb22 nanobody (PDB: 5LHR). Reported binding affinity of nb22 to muPA56: kon = (4.6 ± 0.8) x 105 M-1 s-1, koff = (7.8 ± 2.2) x 10-5 s-1, KD = 0.2 ± 0.03 nM. FIG. 113B shows representative current traces of ClyA-f-nb22 before and after adding 3 nM muPA under -15 mV applied potential. FIG. 113C shows enlarged representative current traces after adding 3 nM muPA at -15 mV. FIG. 113D shows a heatmap of the blockade events observed after the addition of 3 nM muPA with the logarithm of the dwell time against current blockade percentage. FIG. 113E shows the schematic model showing the conformation changes of ClyA-f-nb22 in response to muPA proteins. [0239] FIGs.114A-114C shows schematic illustrations of some of the options for coupling targeting moieties R to nanopores N via the hybridization of duplexed oligonucleotide (e.g. dsDNA) linkers L (where one oligonucleotide strand of the duplex linker L is coupled to the nanopore N, and the other complementary strand is coupled to the binding moiety R). FIG. 114A-114C illustrates 3 possible options for coupling the components. FIG.114A shows the nanopore N and binding moiety R components are located at opposite ends of the duplex linker L. FIG. 114B shows the nanopore N and binding moiety R components are located at the same end of the hybridized duplex linker L. FIG.114C shows one or both the nanopore N and binding moiety R components are coupled to the oligonucleotide strands of the hybridized duplex linker L at an internal position along the strand. [0240] FIGs. 115A-115D show schematic illustrations of a nanopore N with a linker L that is initially in a protected state (FIG.115A), comprising a hybridized protecting polynucleotide strand (i) that can be removed by applying voltage to the nanopore in a membrane system to capture and strip the protecting strand from the linker L (FIG.115B). The deprotected nanopore (FIG.115C) can then be combined with a selected binding moiety R which will hybridize to the linker L to create the functional N-L-R nanopore system (FIG.115D). [0241] FIG. 116 shows a depiction of a computer system that is programmed or otherwise configured to implement the methods provided herein. [0242] FIGs. 117A-117B show example workflow methods for signal extraction and processing with representative current signals (e.g., electrophysiology traces or waveforms). FIG. 117A shows a schematic illustrating a method for extracting a signal or portion thereof or change thereof and processing the signal. FIG. 117B shows the representative signals for each step shown in the method schematic of FIG.117A, beginning with raw signal (step 1), extracted and denoised portions of signal (reads, putative reads and other event types, step 2), filtering (e.g., based on read metrics) to remove events that do not meet the criteria of good reads (step 3), segmenting the reads and performing merging and other resegmentation steps to produce a segmented read (squiggle) (steps 4 and 5), and finally performing further filtering to remove reads based on the metrics of the segmented reads. WSGR Docket Number: 64828-710.601 [0243] FIG.118 shows a representative schematic of a model architecture of a model described herein. [0244] FIG.119 shows a representative workflow for scaling a reference squiggle to read as described herein. [0245] FIG. 120 shows a representative workflow for polypeptide identification. The polypeptide identification can be for a single molecule. [0246] FIG.121 shows a representative workflow for polypeptide quantification. [0247] FIG.122 shows a representative workflow for alignment and consensus of signal reads. [0248] FIGs.123A-123D show alignment and generation of consensus sequence from data. FIG.123A shows representative current signal reads for protein translocation events of data produced by a nanopore system described herein. FIG.123B shows representative reference signals (e.g., generated by machine learning from trained models as described herein). FIG.123C shows an example of an alignment schematic mapping of one of the signal reads of the detected current signals of FIG.123A and the reference signals of FIG.123B. FIG. 123D shows an example of the output consensus of multiple signal reads of FIG.123A that all aligned to one of the reference signals of FIG. 123B all piled up into a consensus plot with shared x-axis sequence position (also called index position) and scaled and normalized current (performed during scaling as described herein). [0249] FIGs. 124A-124E show representative raw current signal (specifically portions of signal reads corresponding to protein translocations through a nanopore)showing how point mutations and other small amino acid motif 2-mer or 3-mer changes in the analytes alters the ionic current signal where indicated by the dotted box. FIG. 124A shows the current signal from an analyte with the KNK motif, with point mutations relative to KWK and ENK motifs. FIG.124B shows the current signal from an analyte with the WWW motif, with 2-mer and 3-mer changes relative to analytes with the other motifs. FIG. 124C shows the current signal from the ENE motif, with a single point mutation relative to the ENK motif. FIG. 124D shows the current signal from an analyte with the KWK motif, with a point mutation relative to the KNK motif. FIG.124E shows the current signal from an analyte with the ENK motif, with a single point mutation relative to the KNK and ENE motifs. [0250] FIGs. 125A-125B show the consensus analysis of point mutation sequencing of maltose protein containing analyte MBP-1. Circles represent wildtype MBP (MBP-MBP1), triangles represent MBP1 with ENK mutation, squares represent MBP1 with KWK mutation, and downward-facing triangles represent MBP1 with WWW mutation. FIGs. 125A-125B show the consensus squiggle along index positions of the analyte (determined as described herein by aligning many reads to a reference squiggle of MBP-1 and adjusted the reference squiggle based on the deviations in the reads) as the lines and markers. Overlaid over the consensus lines and markers are the points corresponding to the average current for the given segments aligned the sequence index position for all the reads used in the consensus analysis. The spread of points are tightly clustered around the mean of the consensus squiggle at each index position, showing that all the single molecule WSGR Docket Number: 64828-710.601 reads closely match the consensus. FIG.125B shows the zoom region of FIG.125A containing the mutation, illustrating the clear deviations in current that are observed in the reads and the consensus from the point mutations. [0251] FIGs.126A-126D show consensus squiggles (overlaid with their corresponding aligned reads, plotting the 1-standard deviation spread of the difference in the current of the corresponding segment at each index position as the width of the lines) from samples containing protein analytes with and without post-translational modifications (PTMs). FIG. 126A shows detection of phosphorylation, specifically a phosphorylated serine residue, from the sequence index. FIG. 126B shows detection of glycosylation, specifically a glycosylated cysteine residue, from the sequence index. FIG.126C shows detection of acetylation, specifically an acetylated lysine residue, from the sequence index. FIG.126D shows detection of deamidation from the sequence index. Resides Q255, N203, N175, and Q174 were deaminated. [0252] FIGs.127A-127B show a poly-PTM measurements from an analyte. FIG.127A shows the consensus squiggle plots for both treated (phosphorylated) and untreated samples, obtained from alignment of the reads for the samples as described herein. The figure plots current vs index position for the two consensus squiggles obtained (lines), overlaying points corresponding to the average current for the given segments aligned the sequence index position for all the reads used in the consensus. Also overlaid is a transparent thicker line showing the 1-standard deviation spread in the points across index positions of the analyte. FIG. 127B shows the zoom of FIG.127A in the region containing two deviations that arise from two phosphorylation sites along the proteins. Both Site 1 and Site 2 showed clear deviations in current for the modified reads and consensus squiggle vs the unmodified. [0253] FIGs. 128A-128C show detection of post-translational modifications and variants of analytes in a mixed population. FIG. 128A shows a multiple aligned reads (aligned as described herein to a reference squiggle to align the segments in sequence index space) overlaid into a pile-up of reads for the sample measured at 0 hours of mixing (e.g., treatment). FIG. 128B shows the similar multiple aligned reads pile-up from the sample measured at 16 hours after mixing (e.g., treatment). The degraded sub-population can be viewed as a new population of reads containing a clear downward deflection in current at the position indicated by the arrow. FIG. 128C shows quantification of the percentage of reads aligning to the degraded sub-population in the mixture versus the sample time points. The percent of modified analyte is shown on the y-axis over time on the x-axis. Over 0 hours to 72 hours post-treatment, the amount of modified analyte increased from 0% to 57%. [0254] FIGs.129A-129B show bi-directional current signal reads of protein analyte nanopore translocations. FIG. 129A shows reading of the MBP1 analyte in C-terminus to N-terminus direction when fed through a nanopore cis-to-trans as described herein. FIG.129B shows reading of the MBP1 analyte in N-terminus to C- terminus direction when fed through a nanopore cis-to-trans as described herein. WSGR Docket Number: 64828-710.601 [0255] FIG.130 shows a representative current waveform for a MBP-1 analyte translocated through an MspA nanopore with VAT (VAT-ΔN unfoldase). S. and E. mark the start and end of the reads respectively, and IO the open-pore current. Measurements obtained with a MspA_D90N nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV. The cis compartment contained a final concentration of 0.2 µM VAT-ΔN, 0.1 µM substrate and 2.5 mM ATP. [0256] FIG. 131 shows a representative portion of the electrophysiology current signal of a substrate with truncation of a C-terminal peptide tag. The substrate had no poly-glycine region . Measurements obtained with a MspA_D90N nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM MBP and 2.5 mM ATP. [0257] FIG.132 shows translocation read of a C-terminus to C-terminus linked protein created by chemical conjugation of a tag to the substrate. The location of recognizable and repeatable features in the reads are indicated by arrows. Measurements obtained with a MspA_D90N nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM MBP-C and 2.5 mM ATP. [0258] FIGs. 133A-133F show representative portions of current signal reads for different proteins. FIG. 133A shows the current signal for translocation of alpha-synuclein through a nanopore, displayed as current (pA) versus time (s). FIG.133B shows the current signal for translocation of p53 through a nanopore, displayed as current (pA) versus time (s). FIG. 133C shows the current signal for translocation of TauF through a nanopore, displayed as current (pA) versus time (s). FIG.133D shows the current signal for translocation of a nanobody through a nanopore, displayed as current (pA) versus time (s). FIG. 133E shows the current signal for translocation of a light chain of an antibody through a nanopore, displayed as current (pA) versus time (s). FIG.133F shows the current signal for translocation of Titin I27 through a nanopore, displayed as current (pA) versus time (s). [0259] FIGs. 134A-134C show representative current signal reads for the translocation of long peptides (peptides with a length of 900 amino acids or more). FIG. 134A shows a representative current signal for translocation of analyte DNAK-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s). FIG. 134B shows a representative current signal for translocation of analyte CH60-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s). FIG. 134C shows a representative current signal for translocation of analyte ODP2-E.coli-MBP1 through a nanopore, displayed as current (pA) versus time (s). [0260] FIG. 135 shows a schematic for isoform characterization that involves comparing multiple signals reads for protein isoform translocations against multiple predicted reference squiggles to score and then identify the isoform identify of each read. WSGR Docket Number: 64828-710.601 [0261] FIGs.136A-136D show schematic current signal reads of current versus time that illustrate differences in the kinetics or speed of translocation as a function of the position through the read/sequence. FIG. 136A illustrates longer lived segments or pauses in the middle of translocations (marked by arrows), after which the read continues to translocate at the normal average speed. FIG 136B illustrates stalls (marked by arrows) that terminate the translocation of the reads (e.g. requiring the read to be ejected). FIG.136C shows representative current signal reads for CH60 protein translocations, marking pauses and changes in kinetics by arrows. FIG. 136D shows representative current signal reads for antibody protein translocations, marking pauses and changes in kinetics by arrows. Pauses and other changes in kinetics can be used for determination of polypeptide characteristics. [0262] FIGs. 137A-137E show schematic representations of electrophysiology systems as described herein. FIG. 137A shows an enclosed trans compartment fluidically connected to an open cis compartment by a membrane. FIG. 137B shows an enclosed cis compartment directly connected to an enclosed trans compartment, via a membrane, both compartments within a solid substrate. FIG. 137C shows an aqueous cis droplet connected to an aqueous trans droplet. The droplets can have electrodes in or connected to the aqueous droplet. FIG. 137D shows an aqueous droplet connected to a continuous aqueous phase. FIG.137E shows an aqueous droplet connected to an enclosed compartment (trans compartment). [0263] FIGs. 138A-138E show a schematic of a system for translocating a protein substrate through a nanopore which is initially captured from the trans, then translocated with a motor protein trans-to-cis against a net cis-to-trans EOF (after a reversal of voltage and EOF in step D and E). [0264] FIG.139 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.138A-138E. [0265] FIGs.140A-140E show a schematic of a system for translocating a protein substrate, initially captured from the trans, then translocated with a motor protein cis-to-trans with a net cis-to-trans EOF, (after a reversal of voltage and EOF in step D and E). [0266] FIG. 141 shows shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.140A-140E. [0267] FIGs. 142A-142E show a schematic of a system for translocating a protein substrate through a nanopore, which is initially captured from the trans, then translocated with a motor protein trans-to-cis against a net cis-to-trans EOF. [0268] FIG.143 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.142A-142E. WSGR Docket Number: 64828-710.601 [0269] FIG. 144A-144D show a schematic of a system for translocating a protein substrate through a nanopore, which is initially captured from the cis after binding to translocases on the cis, then translocated with a motor protein cis-to-trans with the net cis-to-trans EOF. [0270] FIG.145 shows a schematic of the electrophysiology signal obtained from implementing the system as shown in FIGs.144A-144D. [0271] FIG. 146 shows representative electrophysiology reads of ClpX controlled MBP-1 translocations through MspA nanopores, obtained from multiple different single nanopores in multiple membranes (formed on separate trans compartments with a common cis compartment) on a array chip where the nanopores are individually electrically addressed and measured in parallel. DETAILED DESCRIPTION [0272] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed. [0273] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure. [0274] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The use of the words “a” or “an” when used in conjunction with the term “comprising” herein may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [0275] It should also be noted that the term “or” can be generally employed in its sense including “and/or” unless the content clearly dictates otherwise. [0276] The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, can be meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, “about” and “approximately” may mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values. WSGR Docket Number: 64828-710.601 [0277] The compositions and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 80%, 85%, 90%, 95% identical or higher to the sequence specified. In the context of an amino acid sequence, the term “substantially identical” can be used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are (i) identical to, or (ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and/or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 80%, 85%, 90%.91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein. In the context of nucleotide sequence, the term “substantially identical” can be used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein. [0278] The term “variant” can refer to a polypeptide and/or at least a portion of a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or can be encoded by a substantially identical nucleotide sequence. In some embodiments, the variant can be a functional variant. [0279] The term “functional variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or can be encoded by a substantially identical nucleotide sequence, and can be capable of having one or more activities of the reference amino acid sequence. [0280] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In some embodiments, the length of a reference sequence aligned for comparison purposes can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, and at least about 70%, at least about 80%, at least about 90%, or at least about 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions can then be compared. When a position in the first sequence can be occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” can be WSGR Docket Number: 64828-710.601 equivalent to amino acid or nucleic acid “homology”). A nanopore described herein may comprise one or more components. The one or more components may be of a family of binary toxin, or a mutant thereof, or a functional homolog thereof, or a functional ortholog thereof, or a functional paralog thereof. “Homologs” can refer to proteins, peptides, oligopeptides, polypeptides having amino acid substitutions, deletions, or insertions, or any combination thereof relative to an unmodified (e.g., wild-type) protein and having similar biological and/or functional activity as the unmodified protein from which they are derived. “Ortholog” can refer to a gene or protein from different organisms (e.g., different species) that are derived from a common ancestral gene. “Paralog” can refer to a gene or protein from the same organism (e.g., same species) that can be a product of gene duplication of a common ancestral gene. [0281] The percent identity between the two sequences may be a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol.48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A set of parameters (and the one that should be used unless otherwise specified) can be a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. [0282] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. WSGR Docket Number: 64828-710.601 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. [0283] The term “amino acid” can embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Amino acids can include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. As used herein the term “amino acid” can comprise both the D- or L- optical isomers and peptidomimetics. [0284] A “conservative amino acid substitution” can be one in which the amino acid residue can be replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), or aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), or any combination thereof. [0285] As used herein, the term “mutation” can refer to an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. In some embodiments, the mutation may be a large-scale mutation, such as amplifications (or gene duplications) or repetitions of a chromosomal segment, deletions of large chromosomal regions, chromosomal rearrangements (e.g., chromosomal translocations, chromosomal inversions, non-homologous chromosomal crossover, and interstitial deletions), and loss of heterozygosity. In some embodiments, the mutation may be a small-scale mutation, such as insertions, deletions, and substitution mutations. As used herein, the term “substitution mutation” can refer to the transition that exchange a single nucleotide for another. A mutation herein may comprise a chemical conjugation to a non-natural amino acid. [0286] In one approach to single-molecule analyte sequencing, analytes may be unfolded and translocated through a nanopore. In some cases, analyte domains may be recognized, a complex and inconsistent current signal obtained from measurement of an analyte-translocase complex and arising from the unfolding process that the analyte undergoes in the system may prevent the recognition of protein, polypeptides, or peptide sequences or subtle characteristics thereof. In another approach, analytes might be cleaved at specific sites and nanopore currents may be used to identify the released polypeptide or peptides. Therefore, the present disclosure may be aimed at designing and engineering new, analyte-based (e.g., protein-based) nanopores and nanopore systems that may be capable (for example, as part of a multi-protein sensor complex) of unfolding proteins, controlling processive and unidirectional transit across the nanopore, and producing highly consistent and reproducible signals that enable the analytes to be accurately characterized. Thus, the properties of a sample WSGR Docket Number: 64828-710.601 containing a plurality of proteins (including complex mixtures of different protein types) may be accurately determined. [0287] The detection of analytes and the sequencing of DNA using biological nanopores has seen major advances over recent years. There remains a long-felt need for the detection and sequencing of proteins with nanopores. However, this may be complicated by the complex physio-chemical structure of polypeptides or proteins, and/or the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores. In fact, it can be challenging to measure and/or analyze one or more analytes from a real complex sample or small sample volumes, and to do so with high speed, a high degree of accuracy or sensitivity, and/or capability for real time analysis. To achieve this, requires a combination of capable measurement and data processing methods and systems disclosed herein. As an example, the alignment methods disclosed herein can work incredibly well because the movement control is achieved to yield consistent high quality data signals. [0288] Importantly, the ability to accurately characterize peptides/polypeptides/proteins (or the properties of the samples from which they are derived) can be highly dependent on the consistency (e.g., between runs, systems, nanopores, membranes) and quality of the signal, which in turn depends on the translocation properties as the molecules move through the nanopore. The methods and systems disclosed herein allow for translocation movement of single reads of non-nucleic acid based polymer analytes (e.g., one or more peptides, polypeptides, or proteins) through nanopores with high quality movement characteristics from which the step size can be obtained from reads. [0289] One of the goals of the present invention is to improve the accuracy of characterizing individually captured peptides by a nanopore sensor. To that end, engineered proteinaceous nanopores can be developed to improve the capture of unlabeled peptides and/or labeled analytes (e.g., unlabeled or labeled non-nucleic acid based polymer analytes), to increase a residence (dwell time) of analytes in a nanopore sensor, and to improve the discrimination between analyte species. The present disclosure provides engineered nanopores to improve analyte sensing under about physiological conditions as well as at low pH conditions that may be optimized for analyte detection. [0290] It was previously not thought possible to push/feed analytes into pores from, for example, the cis side in their native form (e.g. without attaching or conjugating to DNA leaders or adding other (e.g. polyanion) tags to create electrophoretic capture motifs) due to their complex composition. The diverse charge can result in the unfolded peptides being sometimes attracted and/or repelled from a nanopore depending on charge and applied voltage. Thus, it was not possible to translocate a diverse repertoire of complex peptides or polypeptides through nanopores by electrophoretic mechanisms alone. Indeed, previous studies demonstrated translocation of either very short peptides with a contour length shorter than the length of the nanopore channel or of very carefully selected (model) analytes, whose charge, structure or added electrophoretic tags favor capture and translocation WSGR Docket Number: 64828-710.601 through nanopores by electrophoresis. However, in no way is this representative for the broad amino acid composition of proteins that are found in nature. [0291] The present disclosure provides novel approaches that may be simple and/or provide robust mechanisms of feeding non-nucleic acid based polymer analyte (e.g., polypeptides, peptides, full-length proteins, or any combination thereof) through nanopores (e.g., for the purpose of sequencing and/or characterizing them). It was found that these goals can be achieved by using a large and dominant cis-to-trans electro-osmotic flow (EOF), generated by a large cis-to-trans excess of ions flowing through the nanopore, in conjunction with a translocase on the cis side of a nanopore that can controllably feed and pass a wide range of analytes from cis to trans through the nanopore against the direction of the electrophoretic forces (EPFs). [0292] The present disclosure provides novel approaches that can yield highly reproducible signals from peptide/polypeptide/protein analytes. The present disclosure further demonstrates that the portions of the signals that result from translocation of peptide/polypeptide/protein analytes may be highly consistent between different measurements of the same type of analyte. The present disclosure further demonstrates that the high signal consistency and reproducibility between different measurements of single analyte molecules (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, etc.) can enable signals from analyte molecules to be combined and compared in analytical bioinformatics methods disclosed herein. For example, the high reproducibility may enable forming databases that are generated from a plurality of the same type of molecule (where multiple reads of the same molecule improves the precision of the database information). The high reproducibility can mean molecules can be compared with high accuracy on a single molecule basis, either to each other or to an artificial reference signal that is generated by training from a plurality of training molecules. For example, the high reproducibility enables characteristics (e.g. identification, variants, modifications, length, speed, etc.) to be determined with high accuracy by comparison to reference information. High reproducibility may mean molecules can be combined into sets and analysed in aggregate (e.g. to achieve higher accuracy by averaging or to determine properties of a sample that require a plurality of molecules). The high reproducibility can mean that cleaner (less noisy and variable) signal data may be measured on a molecule by molecule basis, which can provide more detail of the analytes. For example, very small changes in the analytes (e.g. relative to an unchanged reference signal) can be detected with higher accuracy (e.g. the ability to measure many types of single point variants and larger motifs at high accuracy). [0293] Furthermore, the present disclosure in combination with the analytical methods shown herein (including comparisons to references and databases) also can demonstrate that the EOF exerts a powerful force that stretches and/or pulls on the portions of the peptide/polypeptide/protein analyte within or near the nanopore while the analyte resides in the nanopore. An advantage of the EOF may be that this force also keeps the motor WSGR Docket Number: 64828-710.601 bound to the analyte held against the nanopore entrance during motor controlled analyte translocation. Another advantage of the EOF force acting on the portions of the peptide/polypeptide/protein in the nanopore may be improved signal quality, for example, resulting from more consistent signal molecule-to-molecule, and/or by limiting variable changes in current signal that can result from the random stochastic movements of the portion of the peptide/polypeptide/protein in the nanopore (e.g. crumpling, folding, wobbling, shifting up and down of portions of the peptide/polypeptide/protein in the nanopore). The present disclosure demonstrates that the EOF in the systems and methods of the invention can further improve the consistency and reproducibility of portions of the signal, enabling signals obtained from multiple sources (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combination thereof) to be combined and measured in aggregated analyses, and/or to be compared (e.g. to each other or to signals in databases generated from other analytes) to accurately determine characteristics of the analytes or samples of a plurality of analytes (including complex mixtures of different analytes). [0294] Further, the EOF exerts a stretching force on the peptide/polypeptide/protein that can reduce the length of the portion of the peptide/polypeptide/protein that is in the “reader” region of the nanopore (for example the one or more constrictions of the nanopore that give rise to the majority of the changes in current). The reduction in the portion within the reader can have several benefits, including simplifying the signal, increasing the magnitude of current ranges as the analyte moves, provides higher resolution of closely separated features along the analyte, and/or simplifies the analysis of the sequence-to-signal (due to reduced sequence contributing to the signal). [0295] The present disclosure provides novel approaches that yield highly reproducible signals from one or more analytes (e.g., one or more peptides, polypeptides, proteins, or any combinations thereof). The present disclosure further demonstrates that the portions of the signals that can result from translocation of non-nucleic acid based polymer analytes can be highly consistent between different measurements of the same type of analyte. The present disclosure demonstrates that the high signal consistency and reproducibility between different measurements of single analyte molecules (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combinations thereof) can enable signals from analyte molecules to be combined and/or compared in analytical bioinformatics methods disclosed herein. For example, the high reproducibility enables forming databases that can be generated from a plurality of the same type of molecule (e.g., where multiple reads of the same molecule can improve the precision of the database information). The high reproducibility can demonstrate that molecules can be compared with high accuracy on a single molecule basis, to each other and/or to an artificial reference signal that can be generated by training from a plurality of training molecules. For example, the high reproducibility can enable one or more characteristics (e.g. identification, variants, modifications, length, speed, or any combinations thereof) to be WSGR Docket Number: 64828-710.601 determined with high accuracy by comparison to reference information. High reproducibility means molecules can be combined into sets and analyzed in aggregate. For example, one or more analytes may be combined and/or analyzed in aggregate to achieve higher accuracy by averaging and/or to determine properties of a sample that require a plurality of analytes. The high reproducibility can mean that cleaner (e.g., less noisy and variable) signal data may be measured on a molecule by molecule basis, which can provide more detail of the analytes. For example, enabling very small changes in the analytes (e.g. relative to an unchanged reference signal) may be detected with higher accuracy (e.g. the ability to measure many types of single point variants and larger motifs at high accuracy). [0296] Furthermore, the present disclosure in combination with the analytical methods shown herein (for example, including comparisons to references and databases) can also demonstrate that the electro-osmotic force (EOF) can exert a powerful force that stretches and pulls on the portions of the one or more analytes (e.g., one or more non-nucleic acid based polymer analytes) within or near a nanopore while the one or more analytes reside in the nanopore. An advantage of the EOF is that this force can also keep a motor protein as described herein bound to the analyte held against the nanopore entrance during motor controlled analyte translocation. An advantage of the EOF force acting on the portions of the one or more analytes (e.g., one or more non-nucleic acid based polymer analytes) in the nanopore can be improved signal quality. The improved signal quality may result from more consistent signal molecule-to-molecule, for example by limiting variable changes in current signal that can result from random stochastic movements of the portion of the one or more non-nucleic acid based polymer analytes in the nanopore. Random stochastic movements of the one or more analytes may comprise crumpling, folding, wobbling, shifting up and down, or any combination thereof of portions of the one or more analytes in the nanopore. The present disclosure demonstrates that the EOF in the systems and methods of the invention can further improve the consistency and reproducibility of portions of the signal. This may enable signals obtained from multiple sources (e.g. from different instruments, different systems, different membranes and nanopores, different times, different samples, or any combinations thereof) to be combined and/or measured in aggregated analyses, and may be compared to each other and/or to signals in one or more databases generated from other analytes. These comparisons may accurately determine characteristics of the analytes and/or samples of a plurality of analytes (e.g. including complex mixtures of different analytes). [0297] Further, the EOF can exert a stretching force on the one or more non-nucleic acid based polymer analytes that may reduce the length of the portion of the one or more non-nucleic acid based polymer analytes that may be in the “reader” region of the nanopore. The “reader” region can comprise one or more constrictions (e.g., constriction regions) of a nanopore that may give rise to one or more changes in current. The reduction in the portion within the constriction regions can have several benefits, including, but not limited to: (i) simplifying a signal, (ii) increasing a magnitude of current ranges as one or more analytes move, (iii) providing higher WSGR Docket Number: 64828-710.601 resolution of closely separated features along the one or more analytes, (iv) simplifying an analysis of the sequence-to-signal (e.g., due to reduced sequence contributing to the signal), or (v) any combination thereof. [0298] In some aspects, the present disclosure provides a membrane comprising a pore. A pore can be inserted into a membrane such as a lipid bilayer. The pore can be a biological pore. A pore can be engineered to bind with specific analytes. In some embodiments, a pore can be engineered to bind with a class of analytes (e.g., a peptide). In some embodiments, a pore can be engineered to not to bind with a class of analytes. In some embodiments, a pore can be engineered to permit certain ionic species to traverse through the pore. In some embodiments, a pore can be engineered to prevent certain ionic species from traversing through the pore. Various design aspects of pore and systems and methods for achieving those aspects are disclosed herein. [0299] In some aspects, the present disclosure provides a sensor array. The array of sensors can comprise two or more sensors. The array of sensors can comprise at least about 1, 2, 3, 4, 8, 16, 32, 64, 96, 100, 500, 1000, 2000, or greater than about 2000 sensors. The array of sensors can comprise, at most about 2000, 1000, 500, 100, 96, 64, 32, 16, 8, 4, 3, 2, 1, or less than about 1 sensor. In some embodiments, an array of sensors can comprise between about 1 sensor to about 1,000 sensors. In some embodiments, an array of sensors can comprise between about 1 sensor to about 2 sensors, about 1 sensor to about 3 sensors, about 1 sensor to about 4 sensors, about 1 sensor to about 8 sensors, about 1 sensor to about 16 sensors, about 1 sensor to about 32 sensors, about 1 sensor to about 64 sensors, about 1 sensor to about 96 sensors, about 1 sensor to about 400 sensors, about 1 sensor to about 800 sensors, about 1 sensor to about 1,000 sensors, about 2 sensors to about 3 sensors, about 2 sensors to about 4 sensors, about 2 sensors to about 8 sensors, about 2 sensors to about 16 sensors, about 2 sensors to about 32 sensors, about 2 sensors to about 64 sensors, about 2 sensors to about 96 sensors, about 2 sensors to about 400 sensors, about 2 sensors to about 800 sensors, about 2 sensors to about 1,000 sensors, about 3 sensors to about 4 sensors, about 3 sensors to about 8 sensors, about 3 sensors to about 16 sensors, about 3 sensors to about 32 sensors, about 3 sensors to about 64 sensors, about 3 sensors to about 96 sensors, about 3 sensors to about 400 sensors, about 3 sensors to about 800 sensors, about 3 sensors to about 1,000 sensors, about 4 sensors to about 8 sensors, about 4 sensors to about 16 sensors, about 4 sensors to about 32 sensors, about 4 sensors to about 64 sensors, about 4 sensors to about 96 sensors, about 4 sensors to about 400 sensors, about 4 sensors to about 800 sensors, about 4 sensors to about 1,000 sensors, about 8 sensors to about 16 sensors, about 8 sensors to about 32 sensors, about 8 sensors to about 64 sensors, about 8 sensors to about 96 sensors, about 8 sensors to about 400 sensors, about 8 sensors to about 800 sensors, about 8 sensors to about 1,000 sensors, about 16 sensors to about 32 sensors, about 16 sensors to about 64 sensors, about 16 sensors to about 96 sensors, about 16 sensors to about 400 sensors, about 16 sensors to about 800 sensors, about 16 sensors to about 1,000 sensors, about 32 sensors to about 64 sensors, about 32 sensors to about 96 sensors, about 32 sensors to about 400 sensors, about 32 sensors to about 800 sensors, about 32 sensors to about 1,000 WSGR Docket Number: 64828-710.601 sensors, about 64 sensors to about 96 sensors, about 64 sensors to about 400 sensors, about 64 sensors to about 800 sensors, about 64 sensors to about 1,000 sensors, about 96 sensors to about 400 sensors, about 96 sensors to about 800 sensors, about 96 sensors to about 1,000 sensors, about 400 sensors to about 800 sensors, about 400 sensors to about 1,000 sensors, or about 800 sensors to about 1,000 sensors. The array of sensors can comprise the same membrane, the same pore, and the same electrolyte conditions. At least two sensors in the array of sensors can comprise a different membrane, a different pore, and/or different electrolyte conditions. The array of sensors can provide signals in parallel, which can increase the throughput of analyte detection and/or identification. Sensors in the array may each analyze the same sample. In some embodiments, one fraction of sensors in the array can analyze one sample, and another fraction of sensors in the array can analyze a different sample. Various configurations and embodiments for arrays of sensors are provided herein. [0300] In some aspects, the present disclosure provides a device. The device can comprise a sensor or an array of sensors. The device can comprise an electrical energy source and two electrodes. One of the two electrodes may be disposed on one side of the membrane of a sensor, and another electrode may be disposed on the other side. The electrical energy source can apply a potential between the two electrodes, which can cause ions in an electrolyte to conduct through the fluid, and through the pore of the sensor. The potential can also cause an analyte, if charged, to translocate through the pore. The potential can create an electrophoretic force (EPF), described further in detail elsewhere in this application, which can provide a driving force for an analyte to translocate through the pore in order to generate a change in signal. The device may further comprise two or more additional electrodes. For example, these electrodes can be configured to measure the electrical potential across the nanopore and/or membrane that changes when an analyte translocates through a pore. These electrodes can be configured to measure the current across a membrane as an analyte translocates through a pore. The device can be in electrical communication with a recording device to record measured signals. The device can be in electrical communication with a computer or a processor (e.g., a circuit, or an integrated circuit, or any combination thereof), which can receive a signal from the sensor or the array of sensors, store the signals in digital form, and/or process the signal. The device can comprise a flow cell. The flow cell can comprise or be fluidically coupled to the sensor or the sensor array. The sensor or the sensor array can be integrated with the flow cell into a single piece, or they can be separate. The device can comprise or be fluidically coupled to a fluidic control system (e.g., pumps, or controllers, or any combination thereof). In some embodiments, the fluidic control system may comprise a pump, a pressure based flow controller, a pressurized reservoir, a pressure sensor, a vacuum control system, one or more valves, a bubble trap, or fluidic channels that can generate a capillary-force, or combinations thereof. In some embodiments, a pump may be a syringe pump, a peristaltic pump, or piezoelectric pump. The device can be a handheld device or a tabletop device. The device can be configured to detect a single analytes (e.g., chemical species) (e.g., detecting the presence of a particular WSGR Docket Number: 64828-710.601 pathogen like coronavirus). The device can be configured to detect a variety of analytes (e.g., chemical species). The device can be configured to identify any analytes (e.g., chemical species) in a sample. Various forms of devices and methods of using devices are disclosed herein. [0301] In some aspects, the nanopores, methods, and system provided herein comprise detecting and/or characterizing one or more characteristics of an analyte. Characteristics of the analyte (e.g., the non-nucleic acid based polymer analyte) may comprise a shape of the non-nucleic acid based polymer analyte, a structure of the non-nucleic acid based polymer analyte, one or more mutations of the non-nucleic acid based polymer analyte, a sequence of the non-nucleic acid polymer analyte, a surface charge of the non-nucleic acid based polymer analyte, one or more post-translation modifications of the non-nucleic acid based polymer analyte, or one or more ligands coupled to the non-nucleic acid based polymer analyte, or any combination thereof. [0302] In some aspects, the present disclosure provides methods for processing signals. The methods can be implemented on a computer. The methods can be written as a set of instructions, which can be stored in a non- transitory storage medium. The methods can be executable by a computer processor. The methods and algorithms can be configured to store or process one or more signals and determine one or more identifications and/or characteristics of analytes associated with the one or more signals. A computer or processor implementing the methods can be in electrical (wireless or wired) communication with the device. Various methods for processing signals to identify analytes are disclosed herein. [0303] In some aspects, provided herein are methods for translocating an analyte (e.g., a non-nucleic acid based polymer analyte). In some embodiments, the methods may comprise translocating one or more analytes (e.g., a plurality of analytes, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more analytes). The analyte may be translocated through a pore described herein. The pore (e.g., nanopore) may be disposed within a membrane. The pore may be part of a nanopore system described herein. In some embodiments, at least a portion of an analyte (e.g., a non-nucleic acid based polymer analyte) may be translocated through a pore. The at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or any combination thereof. The terms “polypeptide” and “peptide” generally to refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid (i.e., amino acid analogue). The polymer can be linear or branched and can include modified amino acids, and/or may be interrupted by non-amino acids. Polypeptides can occur as single chains or associated chains. The polymer may include a plurality of amino acids and may have a secondary and tertiary structure (i.e., protein). In some examples, the polymer comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1000, at least about WSGR Docket Number: 64828-710.601 10,000, or more amino acids. In some embodiments, the at least a portion of the analyte translocated through a pore may comprise at least a portion of an analyte described herein. [0304] A method may comprise detecting a current or change thereof. A method may comprise detecting a voltage or change thereof. In some embodiments, a signal or change thereof can comprise a measure of an ionic current, voltage, or any combination thereof. The method may comprise detecting a signal or change thereof. In some embodiments, the method may comprise detecting a current or change thereof while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore). In some embodiments, the method may comprise detecting a current or change thereof while at least a portion of an analyte translocates through a pore. In some embodiments, the method may comprise detecting a current or change thereof while at least a portion of an analyte (e.g., one or more analytes) resides in a pore. The method may comprise using a current or change thereof. The method may comprise using a voltage or change thereof. In some embodiments, the method may comprise using a current or change thereof to identify one or more characteristics of an analyte (e.g., a non- nucleic acid based polymer analyte). In some embodiments, the method may comprise using a current or change thereof to determine one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte). Determining a characteristic can comprise measuring a characteristic of an analyte, or quantitating a characteristic of an analyte, or any combination thereof. In some embodiments, the current or change thereof and/or voltage or change thereof may be used to identify a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics). In some embodiments, the current or change thereof and/or voltage or change thereof may be used to determine a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics). The characteristics of at least a portion of the analyte may comprise characteristics of an analyte described herein (e.g., a shape of the analyte, a structure of the analyte, one or more mutations of the analyte, a sequence of the non-nucleic acid polymer analyte, a surface charge of the analyte, one or more post- translation modifications of the analyte, or one or more ligands coupled to the analyte, or any combination thereof). In some embodiments, the current or change thereof may be used to identify one or more characteristics of an analyte with an accuracy. The accuracy of identifying the one or more characteristics of the analyte may be at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. The accuracy of identifying the one or more characteristics of the analyte may be at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or less than about 20%. In some embodiments, the accuracy of identifying the one or more characteristics of the analyte may be between about 20% to about 100%. In some embodiments, the accuracy of identifying the one or more characteristics of the analyte may be between about 20% to about 30%, about 20% to about 40%, about 20% to WSGR Docket Number: 64828-710.601 about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 85% to about 90%, about 85% to about 95%, about 85% to about 100%, about 90% to about 95%, about 90% to about 100%, or about 95% to about 100%. [0305] As an example, a method provided herein can comprise: (a) translocating at least a portion of an analyte through a nanopore disposed within a mem-brane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. [0306] In some embodiments, translocating of at least a portion of an analyte can comprise translocating in a C-terminus to N-terminus (C-to-N) direction, or a N-terminus to C-terminus (N-to-C) direction, or any combination thereof. The C-to-N and/or N-to-C directions can be relative to at least a portion of the analyte sequence. Determining one or more characteristics can comprise using the current or change thereof and/or the voltage or change thereof associated with a C-to-N direction, N-to-C direction, or any combination thereof. Determining one or more characteristics can comprise using an electrical signal or change thereof associated with a C-to-N direction, or N-to-C direction, or any combination thereof. [0307] In some aspects, provided herein are methods for determining one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte). The method may comprise translocating at least a portion of an analyte. The method may comprise translocating one or more analytes (e.g., a plurality of analytes, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more analytes). The analyte may be translocated through a pore described herein (e.g., a nanopore disposed within a membrane). The pore may be part of a nanopore system described herein. In some embodiments, at least a portion of analyte (e.g., at least a portion of a protein, at least a portion WSGR Docket Number: 64828-710.601 of a polypeptide, at least a portion of a peptide, or a combination thereof) may be translocated through a pore at a rate. The rate can comprise an average rate of translocation through the pore. The average rate of translocation may comprise an average of an analyte (e.g., a non-nucleic acid based polymer analyte) translocating through a pore one or more times. The average rate of translocation may comprise an average of two or more analytes translocating through a pore one or more times. A rate of translocation of an analyte through a pore may be expressed as amino acids per second (amino acids/sec) and/or nanometers/sec (nm/s). In some embodiments, a rate of translocation (e.g., an average rate of translocation) may comprise translocation of an analyte with a motor protein (e.g., a translocase) or without a motor protein. [0308] In some embodiments, an average rate of translocation can comprise at least about 0.1 amino acids/sec, at least about 0.5 amino acids/sec, at least about 0.6 amino acids/sec, at least about 0.7 amino acids/sec, at least about 0.8 amino acids/sec, at least about 0.9 amino acids/sec, at least about 1 amino acid/sec, at least about 2 amino acids/sec, at least about 3 amino acids/sec, at least about 4 amino acids/sec, at least about 5 amino acids/sec, at least about 6 amino acids/sec, at least about 7 amino acids/sec, at least about 8 amino acids/sec, at least about 9 amino acids/sec, at least about 10 amino acids/sec, at least about 11 amino acids/sec, at least about 12 amino acids/sec, at least about 13 amino acids/sec, at least about 14 amino acids/sec, at least about 15 amino acids/sec, at least about 16 amino acids/sec, at least about 17 amino acids/sec, at least about 18 amino acids/sec, at least about 19 amino acids/sec, at least about 20 amino acids/sec, at least about 30 amino acids/sec, at least about 40 amino acids/sec, at least about 50 amino acids/sec, at least about 60 amino acids/sec, at least about 70 amino acids/sec, at least about 80 amino acids/sec, at least about 90 amino acids/sec, at least about 100 amino acids/sec, at least about 200 amino acids/sec, at least about 300 amino acids/sec, at least about 400 amino acids/sec, at least about 500 amino acids/sec, at least about 600 amino acids/sec, at least about 700 amino acids/sec, at least about 800 amino acids/sec, at least about 900 amino acids/sec, at least about 1000 amino acids/sec, at least about 5000 amino acids/sec, at least about 10000 amino acids/sec, at least about 15000 amino acids/sec, at least about 20000 amino acids/sec, at least about 25000 amino acids/sec, at least about 30000 amino acids/sec, at least about 35000 amino acids/sec, at least about 40000 amino acids/sec, at least about 45000 amino acids/sec, at least about 50000 amino acids/sec, or greater than about 50000 amino acids/sec. In some embodiments, an average rate of translocation can comprise at most about 50000 amino acids/sec, at most about 45000 amino acids/sec, at most about 40000 amino acids/sec, at most about 35000 amino acids/sec, at most about 30000 amino acids/sec, at most about 25000 amino acids/sec, at most about 20000 amino acids/sec, at most about 15000 amino acids/sec, at most about 10000 amino acids/sec, at most about 5000 amino acids/sec, at most about 1000 amino acids/sec, at most about 900 amino acids/sec, at most about 800 amino acids/sec, at most about 700 amino acids/sec, at most about 600 amino acids/sec, at most about 500 amino acids/sec, at most about 400 amino acids/sec, at most about 300 amino acids/sec, at most about 200 amino acids/sec, at most about WSGR Docket Number: 64828-710.601 100 amino acids/sec, at most about 90 amino acids/sec, at most about 80 amino acids/sec, at most about 70 amino acids/sec, at most about 60 amino acids/sec, at most about 50 amino acids/sec, at most about 40 amino acids/sec, at most about 30 amino acids/sec, at most about 20 amino acids/sec, at most about 19 amino acids/sec, at most about 18 amino acids/sec, at most about 17 amino acids/sec, at most about 16 amino acids/sec, at most about 15 amino acids/sec, at most about 14 amino acids/sec, at most about 13 amino acids/sec, at most about 12 amino acids/sec, at most about 11 amino acids/sec, at most about 10 amino acids/sec, at most about 9 amino acids/sec, at most about 8 amino acids/sec, at most about 7 amino acids/sec, at most about 6 amino acids/sec, at most about 5 amino acids/sec, at most about 4 amino acids/sec, at most about 3 amino acids/sec, at most about 2 amino acids/sec, at most about 1 amino acid/sec, at most about 0.9 amino acids/sec, at most about 0.8 amino acids/sec, at most about 0.7 amino acids/sec, at most about 0.6 amino acids/sec, at most about 0.5 amino acids/sec, at most about 0.1 amino acids/sec, or less than about 0.1 amino acids/sec. [0309] In some embodiments, an average rate of translocation can be between about 0.1 amino acids/sec to about 100 amino acids/sec. In some embodiments, an average rate of translocation can comprise between about 0.1 amino acids/sec to about 0.5 amino acids/sec, about 0.1 amino acids/sec to about 1 amino acid/sec, about 0.1 amino acids/sec to about 10 amino acids/sec, about 0.1 amino acids/sec to about 20 amino acids/sec, about 0.1 amino acids/sec to about 30 amino acids/sec, about 0.1 amino acids/sec to about 40 amino acids/sec, about 0.1 amino acids/sec to about 50 amino acids/sec, about 0.1 amino acids/sec to about 60 amino acids/sec, about 0.1 amino acids/sec to about 70 amino acids/sec, about 0.1 amino acids/sec to about 80 amino acids/sec, about 0.1 amino acids/sec to about 100 amino acids/sec, about 0.5 amino acids/sec to about 1 amino acid/sec, about 0.5 amino acids/sec to about 10 amino acids/sec, about 0.5 amino acids/sec to about 20 amino acids/sec, about 0.5 amino acids/sec to about 30 amino acids/sec, about 0.5 amino acids/sec to about 40 amino acids/sec, about 0.5 amino acids/sec to about 50 amino acids/sec, about 0.5 amino acids/sec to about 60 amino acids/sec, about 0.5 amino acids/sec to about 70 amino acids/sec, about 0.5 amino acids/sec to about 80 amino acids/sec, about 0.5 amino acids/sec to about 100 amino acids/sec, about 1 amino acid/sec to about 10 amino acids/sec, about 1 amino acid/sec to about 20 amino acids/sec, about 1 amino acid/sec to about 30 amino acids/sec, about 1 amino acid/sec to about 40 amino acids/sec, about 1 amino acid/sec to about 50 amino acids/sec, about 1 amino acid/sec to about 60 amino acids/sec, about 1 amino acid/sec to about 70 amino acids/sec, about 1 amino acid/sec to about 80 amino acids/sec, about 1 amino acid/sec to about 100 amino acids/sec, about 10 amino acids/sec to about 20 amino acids/sec, about 10 amino acids/sec to about 30 amino acids/sec, about 10 amino acids/sec to about 40 amino acids/sec, about 10 amino acids/sec to about 50 amino acids/sec, about 10 amino acids/sec to about 60 amino acids/sec, about 10 amino acids/sec to about 70 amino acids/sec, about 10 amino acids/sec to about 80 amino acids/sec, about 10 amino acids/sec to about 100 amino acids/sec, about 20 amino acids/sec to about 30 amino acids/sec, about 20 amino acids/sec to about 40 amino acids/sec, about 20 amino WSGR Docket Number: 64828-710.601 acids/sec to about 50 amino acids/sec, about 20 amino acids/sec to about 60 amino acids/sec, about 20 amino acids/sec to about 70 amino acids/sec, about 20 amino acids/sec to about 80 amino acids/sec, about 20 amino acids/sec to about 100 amino acids/sec, about 30 amino acids/sec to about 40 amino acids/sec, about 30 amino acids/sec to about 50 amino acids/sec, about 30 amino acids/sec to about 60 amino acids/sec, about 30 amino acids/sec to about 70 amino acids/sec, about 30 amino acids/sec to about 80 amino acids/sec, about 30 amino acids/sec to about 100 amino acids/sec, about 40 amino acids/sec to about 50 amino acids/sec, about 40 amino acids/sec to about 60 amino acids/sec, about 40 amino acids/sec to about 70 amino acids/sec, about 40 amino acids/sec to about 80 amino acids/sec, about 40 amino acids/sec to about 100 amino acids/sec, about 50 amino acids/sec to about 60 amino acids/sec, about 50 amino acids/sec to about 70 amino acids/sec, about 50 amino acids/sec to about 80 amino acids/sec, about 50 amino acids/sec to about 100 amino acids/sec, about 60 amino acids/sec to about 70 amino acids/sec, about 60 amino acids/sec to about 80 amino acids/sec, about 60 amino acids/sec to about 100 amino acids/sec, about 70 amino acids/sec to about 80 amino acids/sec, about 70 amino acids/sec to about 100 amino acids/sec, or about 80 amino acids/sec to about 100 amino acids/sec. [0310] In some embodiments, an average rate of translocation can be between about 100 amino acids/sec to about 50,000 amino acids/sec. In some embodiments, an average rate of translocation can comprise between about 100 amino acids/sec to about 500 amino acids/sec, about 100 amino acids/sec to about 1,000 amino acids/sec, about 100 amino acids/sec to about 5,000 amino acids/sec, about 100 amino acids/sec to about 10,000 amino acids/sec, about 100 amino acids/sec to about 15,000 amino acids/sec, about 100 amino acids/sec to about 20,000 amino acids/sec, about 100 amino acids/sec to about 25,000 amino acids/sec, about 100 amino acids/sec to about 30,000 amino acids/sec, about 100 amino acids/sec to about 40,000 amino acids/sec, about 100 amino acids/sec to about 50,000 amino acids/sec, about 500 amino acids/sec to about 1,000 amino acids/sec, about 500 amino acids/sec to about 5,000 amino acids/sec, about 500 amino acids/sec to about 10,000 amino acids/sec, about 500 amino acids/sec to about 15,000 amino acids/sec, about 500 amino acids/sec to about 20,000 amino acids/sec, about 500 amino acids/sec to about 25,000 amino acids/sec, about 500 amino acids/sec to about 30,000 amino acids/sec, about 500 amino acids/sec to about 40,000 amino acids/sec, about 500 amino acids/sec to about 50,000 amino acids/sec, about 1,000 amino acids/sec to about 5,000 amino acids/sec, about 1,000 amino acids/sec to about 10,000 amino acids/sec, about 1,000 amino acids/sec to about 15,000 amino acids/sec, about 1,000 amino acids/sec to about 20,000 amino acids/sec, about 1,000 amino acids/sec to about 25,000 amino acids/sec, about 1,000 amino acids/sec to about 30,000 amino acids/sec, about 1,000 amino acids/sec to about 40,000 amino acids/sec, about 1,000 amino acids/sec to about 50,000 amino acids/sec, about 5,000 amino acids/sec to about 10,000 amino acids/sec, about 5,000 amino acids/sec to about 15,000 amino acids/sec, about 5,000 amino acids/sec to about 20,000 amino acids/sec, about 5,000 amino acids/sec to about 25,000 amino acids/sec, about 5,000 amino acids/sec to about 30,000 amino acids/sec, about 5,000 amino WSGR Docket Number: 64828-710.601 acids/sec to about 40,000 amino acids/sec, about 5,000 amino acids/sec to about 50,000 amino acids/sec, about 10,000 amino acids/sec to about 15,000 amino acids/sec, about 10,000 amino acids/sec to about 20,000 amino acids/sec, about 10,000 amino acids/sec to about 25,000 amino acids/sec, about 10,000 amino acids/sec to about 30,000 amino acids/sec, about 10,000 amino acids/sec to about 40,000 amino acids/sec, about 10,000 amino acids/sec to about 50,000 amino acids/sec, about 15,000 amino acids/sec to about 20,000 amino acids/sec, about 15,000 amino acids/sec to about 25,000 amino acids/sec, about 15,000 amino acids/sec to about 30,000 amino acids/sec, about 15,000 amino acids/sec to about 40,000 amino acids/sec, about 15,000 amino acids/sec to about 50,000 amino acids/sec, about 20,000 amino acids/sec to about 25,000 amino acids/sec, about 20,000 amino acids/sec to about 30,000 amino acids/sec, about 20,000 amino acids/sec to about 40,000 amino acids/sec, about 20,000 amino acids/sec to about 50,000 amino acids/sec, about 25,000 amino acids/sec to about 30,000 amino acids/sec, about 25,000 amino acids/sec to about 40,000 amino acids/sec, about 25,000 amino acids/sec to about 50,000 amino acids/sec, about 30,000 amino acids/sec to about 40,000 amino acids/sec, about 30,000 amino acids/sec to about 50,000 amino acids/sec, or about 40,000 amino acids/sec to about 50,000 amino acids/sec. [0311] In some embodiments, an average rate of translocation can comprise at least about 0.01 nm/s, at least about 0.1 nm/s, at least about 0.5 nm/s, at least about 0.6 nm/s, at least about 0.7 nm/s, at least about 0.8 nm/s, at least about 0.9 nm/s, at least about 1 amino acid/sec, at least about 2 nm/s, at least about 3 nm/s, at least about 4 nm/s, at least about 5 nm/s, at least about 6 nm/s, at least about 7 nm/s, at least about 8 nm/s, at least about 9 nm/s, at least about 10 nm/s, at least about 11 nm/s, at least about 12 nm/s, at least about 13 nm/s, at least about 14 nm/s, at least about 15 nm/s, at least about 16 nm/s, at least about 17 nm/s, at least about 18 nm/s, at least about 19 nm/s, at least about 20 nm/s, at least about 30 nm/s, at least about 40 nm/s, at least about 50 nm/s, at least about 60 nm/s, at least about 70 nm/s, at least about 80 nm/s, at least about 90 nm/s, at least about 100 nm/s, at least about 200 nm/s, at least about 300 nm/s, at least about 400 nm/s, at least about 500 nm/s, at least about 600 nm/s, at least about 700 nm/s, at least about 800 nm/s, at least about 900 nm/s, at least about 1000 nm/s, at least about 5000 nm/s, at least about 10000 nm/s, at least about 15000 nm/s, at least about 20000 nm/s, or greater than about 20000 nm/s. In some embodiments, an average rate of translocation can comprise at most about 20000 nm/s, at most about 15000 nm/s, at most about 10000 nm/s, at most about 5000 nm/s, at most about 1000 nm/s, at most about 900 nm/s, at most about 800 nm/s, at most about 700 nm/s, at most about 600 nm/s, at most about 500 nm/s, at most about 400 nm/s, at most about 300 nm/s, at most about 200 nm/s, at most about 100 nm/s, at most about 90 nm/s, at most about 80 nm/s, at most about 70 nm/s, at most about 60 nm/s, at most about 50 nm/s, at most about 40 nm/s, at most about 30 nm/s, at most about 20 nm/s, at most about 19 nm/s, at most about 18 nm/s, at most about 17 nm/s, at most about 16 nm/s, at most about 15 nm/s, at most about 14 nm/s, at most about 13 nm/s, at most about 12 nm/s, at most about 11 nm/s, at most about 10 nm/s, at most about 9 nm/s, at most about 8 nm/s, at most about 7 nm/s, at most about 6 nm/s, at most about 5 nm/s, at WSGR Docket Number: 64828-710.601 most about 4 nm/s, at most about 3 nm/s, at most about 2 nm/s, at most about 1 amino acid/sec, at most about 0.9 nm/s, at most about 0.8 nm/s, at most about 0.7 nm/s, at most about 0.6 nm/s, at most about 0.5 nm/s, at most about 0.1 nm/s, at most about 0.01 nm/s, or less than about 0.01 nm/s. [0312] In some embodiments, an average rate of translocation can be between about 0.1 nm/s to about 10000 nm/s. In some embodiments, an average rate of translocation can be between about 0.1 nm/s to about 100 nm/s. In some embodiments, an average rate of translocation can comprise between about 0.1 nm/s to about 0.5 nm/s, about 0.1 nm/s to about 1 amino acid/sec, about 0.1 nm/s to about 10 nm/s, about 0.1 nm/s to about 20 nm/s, about 0.1 nm/s to about 30 nm/s, about 0.1 nm/s to about 40 nm/s, about 0.1 nm/s to about 50 nm/s, about 0.1 nm/s to about 60 nm/s, about 0.1 nm/s to about 70 nm/s, about 0.1 nm/s to about 80 nm/s, about 0.1 nm/s to about 100 nm/s, about 0.5 nm/s to about 1 amino acid/sec, about 0.5 nm/s to about 10 nm/s, about 0.5 nm/s to about 20 nm/s, about 0.5 nm/s to about 30 nm/s, about 0.5 nm/s to about 40 nm/s, about 0.5 nm/s to about 50 nm/s, about 0.5 nm/s to about 60 nm/s, about 0.5 nm/s to about 70 nm/s, about 0.5 nm/s to about 80 nm/s, about 0.5 nm/s to about 100 nm/s, about 1 amino acid/sec to about 10 nm/s, about 1 amino acid/sec to about 20 nm/s, about 1 amino acid/sec to about 30 nm/s, about 1 amino acid/sec to about 40 nm/s, about 1 amino acid/sec to about 50 nm/s, about 1 amino acid/sec to about 60 nm/s, about 1 amino acid/sec to about 70 nm/s, about 1 amino acid/sec to about 80 nm/s, about 1 amino acid/sec to about 100 nm/s, about 10 nm/s to about 20 nm/s, about 10 nm/s to about 30 nm/s, about 10 nm/s to about 40 nm/s, about 10 nm/s to about 50 nm/s, about 10 nm/s to about 60 nm/s, about 10 nm/s to about 70 nm/s, about 10 nm/s to about 80 nm/s, about 10 nm/s to about 100 nm/s, about 20 nm/s to about 30 nm/s, about 20 nm/s to about 40 nm/s, about 20 nm/s to about 50 nm/s, about 20 nm/s to about 60 nm/s, about 20 nm/s to about 70 nm/s, about 20 nm/s to about 80 nm/s, about 20 nm/s to about 100 nm/s, about 30 nm/s to about 40 nm/s, about 30 nm/s to about 50 nm/s, about 30 nm/s to about 60 nm/s, about 30 nm/s to about 70 nm/s, about 30 nm/s to about 80 nm/s, about 30 nm/s to about 100 nm/s, about 40 nm/s to about 50 nm/s, about 40 nm/s to about 60 nm/s, about 40 nm/s to about 70 nm/s, about 40 nm/s to about 80 nm/s, about 40 nm/s to about 100 nm/s, about 50 nm/s to about 60 nm/s, about 50 nm/s to about 70 nm/s, about 50 nm/s to about 80 nm/s, about 50 nm/s to about 100 nm/s, about 60 nm/s to about 70 nm/s, about 60 nm/s to about 80 nm/s, about 60 nm/s to about 100 nm/s, about 70 nm/s to about 80 nm/s, about 70 nm/s to about 100 nm/s, or about 80 nm/s to about 100 nm/s. [0313] In some embodiments, an average rate of translocation can comprise between about 100 nm/sec to about 20,000 nm/sec. In some embodiments, an average rate of translocation can comprise between about 100 nm/sec to about 200 nm/sec, about 100 nm/sec to about 300 nm/sec, about 100 nm/sec to about 400 nm/sec, about 100 nm/sec to about 500 nm/sec, about 100 nm/sec to about 1,000 nm/sec, about 100 nm/sec to about 2,500 nm/sec, about 100 nm/sec to about 5,000 nm/sec, about 100 nm/sec to about 7,500 nm/sec, about 100 nm/sec to about 10,000 nm/sec, about 100 nm/sec to about 15,000 nm/sec, about 100 nm/sec to about 20,000 WSGR Docket Number: 64828-710.601 nm/sec, about 200 nm/sec to about 300 nm/sec, about 200 nm/sec to about 400 nm/sec, about 200 nm/sec to about 500 nm/sec, about 200 nm/sec to about 1,000 nm/sec, about 200 nm/sec to about 2,500 nm/sec, about 200 nm/sec to about 5,000 nm/sec, about 200 nm/sec to about 7,500 nm/sec, about 200 nm/sec to about 10,000 nm/sec, about 200 nm/sec to about 15,000 nm/sec, about 200 nm/sec to about 20,000 nm/sec, about 300 nm/sec to about 400 nm/sec, about 300 nm/sec to about 500 nm/sec, about 300 nm/sec to about 1,000 nm/sec, about 300 nm/sec to about 2,500 nm/sec, about 300 nm/sec to about 5,000 nm/sec, about 300 nm/sec to about 7,500 nm/sec, about 300 nm/sec to about 10,000 nm/sec, about 300 nm/sec to about 15,000 nm/sec, about 300 nm/sec to about 20,000 nm/sec, about 400 nm/sec to about 500 nm/sec, about 400 nm/sec to about 1,000 nm/sec, about 400 nm/sec to about 2,500 nm/sec, about 400 nm/sec to about 5,000 nm/sec, about 400 nm/sec to about 7,500 nm/sec, about 400 nm/sec to about 10,000 nm/sec, about 400 nm/sec to about 15,000 nm/sec, about 400 nm/sec to about 20,000 nm/sec, about 500 nm/sec to about 1,000 nm/sec, about 500 nm/sec to about 2,500 nm/sec, about 500 nm/sec to about 5,000 nm/sec, about 500 nm/sec to about 7,500 nm/sec, about 500 nm/sec to about 10,000 nm/sec, about 500 nm/sec to about 15,000 nm/sec, about 500 nm/sec to about 20,000 nm/sec, about 1,000 nm/sec to about 2,500 nm/sec, about 1,000 nm/sec to about 5,000 nm/sec, about 1,000 nm/sec to about 7,500 nm/sec, about 1,000 nm/sec to about 10,000 nm/sec, about 1,000 nm/sec to about 15,000 nm/sec, about 1,000 nm/sec to about 20,000 nm/sec, about 2,500 nm/sec to about 5,000 nm/sec, about 2,500 nm/sec to about 7,500 nm/sec, about 2,500 nm/sec to about 10,000 nm/sec, about 2,500 nm/sec to about 15,000 nm/sec, about 2,500 nm/sec to about 20,000 nm/sec, about 5,000 nm/sec to about 7,500 nm/sec, about 5,000 nm/sec to about 10,000 nm/sec, about 5,000 nm/sec to about 15,000 nm/sec, about 5,000 nm/sec to about 20,000 nm/sec, about 7,500 nm/sec to about 10,000 nm/sec, about 7,500 nm/sec to about 15,000 nm/sec, about 7,500 nm/sec to about 20,000 nm/sec, about 10,000 nm/sec to about 15,000 nm/sec, about 10,000 nm/sec to about 20,000 nm/sec, or about 15,000 nm/sec to about 20,000 nm/sec. [0314] A method may comprise detecting a current or change thereof. In some embodiments, a current or change thereof may be detected while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore). In some embodiments, a current or change thereof may be detected while at least a portion of an analyte translocates through a pore. In some embodiments, the method may comprise using a current or change thereof to determine one or more characteristics of an analyte (e.g., a non-nucleic acid based polymer analyte) and/or at least a portion of an analyte. In some embodiments, the current or change thereof may be used to determine a plurality of characteristics (e.g., at least about 1, 2, 3, 4, 5, 10, or more characteristics). The characteristics of at least a portion of the analyte may comprise characteristics of an analyte described herein. [0315] As an example, provided herein is a method for determining a characteristic of an analyte comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a mem-brane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, at WSGR Docket Number: 64828-710.601 least a portion of a peptide, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino ac-ids per second to about 35000 amino acids per second or (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s; (b) detecting (1) a current or change thereof, or (2) voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte. [0316] In some embodiments, the methods described herein may comprise translocating an additional analyte (e.g., an additional non-nucleic acid based polymer analyte) through a pore (e.g., a nanopore). The additional analyte may be translocated through a same pore as a first analyte. The additional analyte may be translocated through a different pore (e.g., an additional pore) as a first analyte. In some embodiments, an additional analyte comprises at least a portion of the additional analyte. The at least a portion of the additional analyte can comprise a non-nucleic acid based polymer analyte (e.g., at least a portion of an additional protein, at least a portion of an additional polypeptide, at least a portion of an additional peptide, or a combination thereof). In some embodiments, at least a portion of the additional analyte may translocate through a pore described herein. The additional analyte may translocate through a pore described herein with an average rate of translocation described herein. In some embodiments, an additional current or change thereof and/or an additional voltage or change thereof may be detected. In some embodiments, the additional current or change thereof and/or the additional voltage or change thereof may be detected while at least a portion of an additional analyte translocates through a pore. In some embodiments, the additional current or change thereof and/or the additional voltage or change thereof may be detected while at least a portion of an additional analyte resides in a pore (e.g., the additional analyte enters a pore from a first side and may not exit a pore on a second side). In some embodiments, the additional current or change thereof and/or the additional voltage or change thereof may be used to identify and/or determine one or more characteristics of at least a portion of the additional analyte. A characteristic of the additional analyte can comprise a shape of the additional analyte, a structure of the additional analyte, one or more mutations of the additional analyte, a sequence of the additional analyte, a surface charge of the additional analyte, one or more post-translation modifications of the additional analyte, or one or more ligands coupled to the additional analyte, or any combination thereof. [0317] In some aspects, provided herein is a method for characterizing an analyte (e.g., a non-nucleic acid based polymer analyte). The analyte may be translocated through a pore described herein. The pore (e.g., nanopore) may be disposed within a membrane. The pore may be part of a nanopore system described herein. In some embodiments, at least a portion of an analyte (e.g., a non-nucleic acid based polymer analyte) may be translocated through a pore. The at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or any combination thereof. A signal or change WSGR Docket Number: 64828-710.601 thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected. In some embodiments, a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected while there is no analyte in a pore described herein (e.g., the pore can comprise an open pore). In some embodiments, a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected while at least a portion of an analyte translocates through a pore. In some embodiments, a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) may be detected while at least a portion of an analyte resides in a pore. [0318] One or more characteristics may be assigned to at least a portion of an analyte (e.g., at least a portion of a non-nucleic acid based polymer analyte). The one or more characteristics may be assigned based on the signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof). The one or more characteristics may be assigned based on an electrical signal or change thereof, a database, or any combination thereof. The database may comprise one or more signals (e.g., reference signals). The reference signal can comprise a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) identified and/or determined for an analyte. In some embodiments, the reference signal may be for one or more analytes (e.g., one or more polypeptides, one or more proteins, or one or more peptides, or one or more fragments thereof, or one or more proteoforms thereof, or one or more variants thereof, or any combination thereof). In some embodiments, variants of the analyte (e.g., non-nucleic acid based polymer analyte) may comprise one or more post-translational modifications (PTMs) and/or one or more conjugations to the analyte (e.g., drug conjugate, barcode, polynucleotide, or leader construct, or any combination thereof). The one or more post-translational modifications may comprise naturally occurring PTMs, or non-naturally occurring PTMs, or any combination thereof. [0319] As an example, provided herein is a method for characterizing an analyte comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more fragments thereof, or one or more proteoforms thereof, or one or more variants thereof, or a combination thereof. [0320] In some embodiments, assigning can comprise measuring one or more characteristics, or quantitating one or more characteristics, or any combination thereof. In some embodiments, the one or more characteristics WSGR Docket Number: 64828-710.601 may be assigned by scoring a signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof). The signal or change thereof may be scored against the one or more reference signals and/or at least a portion of the one or more reference signals. In some embodiments, scoring may comprise aligning at least a portion of the signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof). In some embodiments, at least a portion of the signal or change thereof may be aligned with the one or more reference signals and/or at least a portion of the one or more reference signals. In some embodiments, the alignment may comprise soft alignment. [0321] In some embodiments, the methods provided herein may be repeated to generate a plurality of signals or changes thereof (e.g., electrical signals or changes thereof, currents or changes thereof, or any combination thereof). For example, a plurality of analytes may translocate through a plurality of nanopores. The plurality of analytes may translocate through a plurality of nanopores disposed in membranes. As the plurality of analytes translocates through the plurality of nanopores, a plurality of signals (e.g., electrical signals, current signals, or any combination thereof) may be generated. The plurality of signals (e.g., electrical signals, current signals, or any combination thereof) may be detected. One or more characteristics may be assigned to the plurality of analytes (e.g., the plurality of non-nucleic acid based polymer analytes). The one or more characteristics may be assigned based on the plurality of signals (e.g., electrical signals, current signals, or any combination thereof). The one or more characteristics may be assigned based on the plurality of signals, a database, or any combination thereof. [0322] In some aspects, provided herein is a method for sample analysis. A sample may be provided. In some embodiments, the sample can comprise an analyte. In some embodiments, the sample can comprise a plurality of analytes (e.g., a plurality of non-nucleic acid based polymer analytes). At least a subset of the plurality of analytes may undergo any of the methods and/or system disclosed herein. The plurality of analytes can comprise a first analyte and a second analyte. In some embodiments, the plurality of analytes may be translocated. The plurality of analytes may be translocated through a pore described herein (e.g., a pore disposed within a membrane). In some embodiments, at least a portion of the analyte (e.g., a first analyte and/or a second analyte) may be translocated through a pore. The at least a portion of the analyte may comprise at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or any combination thereof. In some embodiments, translocation of the analyte (e.g., the first analyte and/or the second analyte) through a pore may generate a signal or change thereof (e.g., a first current or change thereof and/or a second current or change thereof). The first current or change thereof and/or the second current or change thereof may be detected. In some embodiments, the first current or change thereof and/or the second current or change thereof may be used to determine a characteristic (e.g., a first characteristic and/or a second characteristic). In some embodiments, the first current or change thereof and/or the second current or change thereof may be used to determine a WSGR Docket Number: 64828-710.601 plurality of characteristics. At least a plurality of characteristics may be determined using the methods and/or system disclosed herein. In some embodiments, a sample may be analyzed by using the first characteristic and/or a second characteristic. For example, one or more properties of the sample may be characterized by using the first characteristic and/or a second characteristic. [0323] As an example, a method for sample analysis can comprise: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, at least a portion of a first peptide, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, at least a portion of a second peptide, or a combination thereof; (c) detecting (i) (1) a first current or change thereof, or (2) a first voltage or change thereof while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (3) a second current or change thereof, or (4) a second voltage or change thereof while the at least the portion of the second analyte is translocating through the second nanopore; (d) using (i) (1) the first current or change thereof, or (2) the first voltage or change thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (3) the second current or change thereof, or (4) the second voltage or change thereof to determine a second characteristic of the at least the portion of the second analyte; and (e) characterizing one or more properties of the sample using the first characteristic or the second characteristic determined in (d). [0324] Characterizing one or more properties of the sample may comprise determining a feature. The feature may be a feature of a proteome. The proteome may be associated with the sample. The proteome may refer to a set of proteins expressed by the organism from which the same may be derived. Characterizing one or more properties of the sample may comprise a proteome coverage of a proteome. The proteome coverage can refer to extrapolation of the number of protein discoveries by future measurements conditioned on a sequence of already performed measurements. Characterizing one or more properties of the sample may comprise a sequence coverage of the first analyte (e.g., a first polypeptide) and/or a second analyte (e.g., a second polypeptide). Sequence coverage can refer to a number of sequencing reads that may be uniquely mapped to a reference sequence and may be applied to a known part of the genome. [0325] In some embodiments, a first nanopore and a second nanopore may be the same nanopore. In some embodiments, a first nanopore and a second nanopore may be different nanopores. In some embodiments, a first membrane and a second membrane may be the same membrane. In some embodiments, a first membrane and a second membrane may be different membranes. WSGR Docket Number: 64828-710.601 [0326] One or more properties of a sample can comprise identification of a type of a sample (e.g., a type associated with the sample). The sample type can comprise any sample type described herein. One or more properties of a sample can comprise identification of an origin of a sample. The sample origin can comprise any sample origin described herein. In some embodiments, one or more properties may comprise one or more dynamic changes in a sample. Dynamic changes may be determined through continuous measurement of a sample (e.g., one or more analytes in a sample). Sensors of a nanopore system described herein may be exposed to the sample and detect changes as the sample changes over time in situ. Dynamic changes may be observed via continuous measurement of a sample. In some embodiments, dynamic changes may be observed via repeated measurements of a sample. An interaction may be determined. The interaction can comprise an interaction of a first analyte and/or second analyte of a sample. The interaction can comprise an interaction of a first analyte and/or second analyte with one or more molecules. A sample (e.g., a cell of a biological sample) may comprise a set of one or more interactions. A first analyte and/or a second analyte (e.g., a first non-nucleic acid based polymer analyte and a second non-nucleic acid based polymer analyte) may have one or more protein-protein interactions with one or more molecules of the sample. In some embodiments, a plurality of analytes may have one or more interactions with one or more molecules (e.g., one or more polypeptides, one or more proteins, one or more peptides, one or more nucleic acids, or one or more small molecules, or any combination thereof). For example, following translocation of an analyte through a nanopore, a characterized property may comprise an interaction between the analyte and a bound entity (e.g., a leader construct, a recognition element, a PTM, or a conjugation, or any combination thereof). The characterized property can provide information on the analyte (e.g., the bound entity) and the sample (e.g., the sample containing binding partners to at least the analyte). As another example, characterizing properties of the sample may be performed through binding partners (e.g., binding molecules) to bind to and/or isolate an analyte from a sample. The binding partners (e.g., binding molecules) may bind to an analyte via affinity binding. The binding may be measured by extracting the analyte from the sample and translocating through a nanopore described herein. PORES [0327] In some aspects, the present disclosure provides pores for analyzing analytes. [0328] In some embodiments, a pore can be a biological pore. In some embodiments, a biological pore comprises a biological molecule. In some embodiments, a pore comprises a protein. In some embodiments, a pore comprises an assembly of proteins. In some embodiments, a pore comprises an assembly of subunits. In some embodiments, a pore comprises an assembly of fused proteins. In some embodiments, a pore comprises DNA. In some embodiments, a pore comprises a DNA origami structure. In some embodiments, a pore comprises a hybrid of DNA and peptides. In some embodiments, a pore comprises a G-quadruplex. In some WSGR Docket Number: 64828-710.601 embodiments, a pore comprises a polymer or a covalent organic framework. In some embodiments, a pore comprises a helical self-assembling pore or a chemically synthesized pore. In some embodiments, a pore comprises silicon, carbon, metal, metallic oxide, a metal-organic-framework, or a MXene. In some embodiments, a DNA based pore carries a large surface charge that can be used to create strong ion-selective electro-osmotic gradients. In some embodiments, a pore can be comprised in a membrane. In some embodiments, a pore comprises a toroidal protein. In some embodiments, a pore comprises a non- transmembrane protein. In some embodiments, a pore comprises cyclic peptides. In some embodiments, a pore comprises an assembly of cell penetrating molecules. In some embodiments, a pore comprises cell penetrating peptides. In some embodiments, a pore comprises portions of phage portal complexes. In some embodiments, a pore comprises portions of cellular transmembrane transport complexes. [0329] In some aspects, the present disclosure provides pores for detecting and/or characterizing an analyte (e.g., a biopolymer). A pore may be a wild-type pore and/or a pore may be an engineered pore. In some embodiments, the pore (e.g., nanopore) comprises a transmembrane region. In some embodiments, the pore comprises a hydrophilic portion. In some embodiments, the pore comprises a hydrophobic portion. In some embodiments, the pore comprises a hydrophilic and a hydrophobic portion. In some embodiments, a pore comprises an opening (e.g., an entrance). In some embodiments, a pore comprises at least one opening. In some embodiments, a pore can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) openings. An entrance to a nanopore may be defined by a widest dimension (e.g., a measure from a first edge of an entrance to a second edge of the entrance). A pore may be measured by a diameter, or a circumference, or any combination thereof. [0330] A pore can comprise a channel through which an analyte may enter. Herein the terms “channel”, “lumen” and/or “vestibule” may be used interchangeably. The channel may be of the wild-type biological nanopore or the engineered biological nanopore. In some embodiments, an analyte may be trapped in the channel of the nanopore. In some embodiments, an analyte may translocate through the channel of the nanopore. In some embodiments, an analyte may partially translocate through the channel of the nanopore. The channel may be a same width through the entire channel or a channel may have two or more different widths through the entire channel. [0331] The channel may comprise at least one region. For example, the channel of the pore (e.g., the biological nanopore) may comprise a first region, a second region, and/or a third region. In some embodiments, the channel of the nanopore comprises a constriction (e.g., a constriction region). The constriction region may be a region of the channel different in size (e.g., width, length, diameter, circumference, or a widest dimension, or any combination thereof) than one or more other regions of the channel. The second region of the channel may have the constriction region. The first region and/or third region of the channel and the second region of the channel (e.g., comprising the constriction region) may be adjacent (e.g., immediately adjacent) to one another. In other WSGR Docket Number: 64828-710.601 cases, the first region and/or third region and second region of the channel may be separated by a distance of at most about 4.0 nm, at most about 3.0 nm, at most about 2.0 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, at most about 0.1 nm, or less than about 0.1 nm. In some embodiments, a first region and/or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance of at least about 0.001 nm, at least about 0.01 nm, at least about 0.05 nm, at least about 0.1 nm, at least about 0.5 nm, at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm. In some embodiments, a first region and/or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance of at most about 15 nm, at most about 10 nm, at most about 5 nm, at most about 4 nm, at most about 3 nm, at most about 2 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.1 nm, at most about 0.05 nm, at most about 0.01 nm, at most about 0.001 nm, or less than about 0.001 nm. In some embodiments, a first region and/or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance from about 0.001 nm to about 15 nm. In some embodiments, a first region and/or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance from at least about 0.001 nm. In some embodiments, a first region and/or third region of the channel and a second region of the channel (e.g., comprising the constriction region) are separated by a distance from about 0.001 nm to about 0.01 nm, about 0.001 nm to about 0.05 nm, about 0.001 nm to about 0.1 nm, about 0.001 nm to about 0.5 nm, about 0.001 nm to about 1 nm, about 0.001 nm to about 2 nm, about 0.001 nm to about 3 nm, about 0.001 nm to about 4 nm, about 0.001 nm to about 5 nm, about 0.001 nm to about 10 nm, about 0.001 nm to about 15 nm, about 0.01 nm to about 0.05 nm, about 0.01 nm to about 0.1 nm, about 0.01 nm to about 0.5 nm, about 0.01 nm to about 1 nm, about 0.01 nm to about 2 nm, about 0.01 nm to about 3 nm, about 0.01 nm to about 4 nm, about 0.01 nm to about 5 nm, about 0.01 nm to about 10 nm, about 0.01 nm to about 15 nm, about 0.05 nm to about 0.1 nm, about 0.05 nm to about 0.5 nm, about 0.05 nm to about 1 nm, about 0.05 nm to about 2 nm, about 0.05 nm to about 3 nm, about 0.05 nm to about 4 nm, about 0.05 nm to about 5 nm, about 0.05 nm to about 10 nm, about 0.05 nm to about 15 nm, about 0.1 nm to about 0.5 nm, about 0.1 nm to about 1 nm, about 0.1 nm to about 2 nm, about 0.1 nm to about 3 nm, about 0.1 nm to about 4 nm, about 0.1 nm to about 5 nm, about 0.1 nm to about 10 nm, about 0.1 nm to about 15 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 2 nm, about 0.5 nm to about 3 nm, about 0.5 nm to about 4 nm, about 0.5 nm to about 5 nm, about 0.5 nm to about 10 nm, about 0.5 nm to about 15 nm, about 1 nm to about 2 nm, about 1 nm to about 3 nm, about 1 nm to about 4 nm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 15 nm, about 2 nm to about 3 nm, about 2 nm to about 4 nm, about 2 nm to about 5 nm, about 2 nm to about 10 nm, WSGR Docket Number: 64828-710.601 about 2 nm to about 15 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3 nm to about 10 nm, about 3 nm to about 15 nm, about 4 nm to about 5 nm, about 4 nm to about 10 nm, about 4 nm to about 15 nm, about 5 nm to about 10 nm, about 5 nm to about 15 nm, or about 10 nm to about 15 nm. [0332] The constriction region of the nanopore may be a narrower region of the channel than another region of the channel. In some embodiments, the constriction region of the nanopore can contribute to the electrical resistance of the nanopore. A modulation of electrical resistance may allow the nanopore to differentiate between analytes in a complex sample. Therefore, modifying a constriction region of a nanopore to shift an electrical resistance may modulate the electro-osmotic force and/or may improve the ability of the nanopore to characterize an analyte. Characterization of an analyte may occur at the constriction region. In the constriction region, the current flow may be modulated most by the composition (e.g., local composition, e.g., amino acid composition) of the analyte within. The electro-osmotic flow (EOF) may be maximally created at a narrow region (e.g., a constriction region). The EOF may be maximally created at a constriction region due to a maximal electrostatic effect on cation or anion flux in the constrained dimensions of the constriction. [0333] In some embodiments, the nanopore comprises a shape (e.g., a geometry). For example, a nanopore may be cylindrical. In some embodiments, the nanopore can be conical shape. In some embodiments, the nanopore can be globular shape. In some embodiments, the nanopore can be hourglass shape. In some embodiments, the nanopore can be a toroidal shape, comprising a ring and a channel. In some embodiments, a nanopore comprises a biological nanopore or a solid state nanopore. The toroidal shape may comprise a toroidal polyhedral shape comprising a ring and a channel. The ring may comprise the protein or proteins that form the nanopore. The ring may comprise a cross sectional geometry similar to the protein or proteins that form the nanopore. The ring may be wider at a first side (e.g., a cis side) than a second side (e.g., a trans side), or wider at the second side (e.g., the trans side) than the first side (e.g., the cis side). The ring can comprise a portion comprising a conical geometry, a cylindrical geometry, or an amorphous geometry, or combinations thereof. The channel can comprise the central portion of the nanopore geometry that does not comprise the proteins or peptides of the nanopore. The channel may allow molecules to translocate through the nanopore (i.e. through the channel). [0334] A channel may restrict molecules from translocating through the nanopore. The restriction may be based on a width of the channel or a charge of the channel. The channel can comprise a channel length. The channel length can be the length of the channel as measured along a longitudinal axis of the channel. This longitudinal axis may run perpendicular to a membrane. The length may be measured perpendicular to the ring of the shape (e.g., the toroidal shape) of the geometry of the nanopore. The channel length can be measured as the distance along the longitudinal axis of the channel between the most distant points of the nanopore along the longitudinal axis of the channel. In some embodiment, a channel may have a start point on a first side (e.g., WSGR Docket Number: 64828-710.601 a cis side) of a nanopore, and an end point on a second side (e.g., a trans side) of a nanopore, or a start point on a second side (e.g., a trans side) of a nanopore, and an end point on a first side (e.g., a cis side) of a nanopore. In some embodiments a channel length can be less than a linear length or a contour length of an analyte. In some embodiments a channel length can be greater than a linear length or a contour length of an analyte. [0335] In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from an alpha-carbon position of an amino acid backbone. The dimension of the constriction region may be measured from a first alpha-carbon position to a second alpha-carbon position. In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position of at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about 1.0 nm, at least about 1.1 nm, at least about 1.2 nm, at least about 1.3 nm, at least about 1.4 nm, at least about 1.5 nm, at least about 1.6 nm, at least about 1.7 nm, at least about 1.8 nm, at least about 1.9 nm, at least about 2.0 nm, at least about 2.1 nm, at least about 2.2 nm, at least about 2.3 nm, at least about 2.4 nm, at least about 2.5 nm, at least about 2.6 nm, at least about 2.7 nm, at least about 2.8 nm, at least about 2.9 nm, at least about 3.0 nm, at least about 3.1 nm, at least about 3.2 nm, at least about 3.3 nm, at least about 3.4 nm, at least about 3.5 nm, at least about 3.6 nm, at least about 3.7 nm, at least about 3.8 nm, at least about 3.9 nm, at least about 4.0 nm, or greater than about 4.0 nm. In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position of at most about 4.0 nm, at most about 3.9 nm, at most about 3.8 nm, at most about 3.7 nm, at most about 3.6 nm, at most about 3.5 nm, at most about 3.4 nm, at most about 3.3 nm, at most about 3.2 nm, at most about 3.1 nm, at most about 3.0 nm, at most about 2.9 nm, at most about 2.8 nm, at most about 2.7 nm, at most about 2.6 nm, at most about 2.5 nm, at most about 2.4 nm, at most about 2.3 nm, at most about 2.2 nm, at most about 2.1 nm, at most about 2.0 nm, at most about 1.9 nm, at most about 1.8 nm, at most about 1.7 nm, at most about 1.6 nm, at most about 1.5 nm, at most about 1.4 nm, at most about 1.3 nm, at most about 1.2 nm, at most about 1.1 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, or less than about 0.2 nm. [0336] In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position from about 0.2 nm to about 4 nm. In some embodiments, a constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from a first alpha-carbon position to a second alpha-carbon position from about 0.2 nm WSGR Docket Number: 64828-710.601 to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1.5 nm, about 0.2 nm to about 2 nm, about 0.2 nm to about 2.5 nm, about 0.2 nm to about 3 nm, about 0.2 nm to about 3.5 nm, about 0.2 nm to about 4 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 1.5 nm, about 0.3 nm to about 2 nm, about 0.3 nm to about 2.5 nm, about 0.3 nm to about 3 nm, about 0.3 nm to about 3.5 nm, about 0.3 nm to about 4 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 1.5 nm, about 0.4 nm to about 2 nm, about 0.4 nm to about 2.5 nm, about 0.4 nm to about 3 nm, about 0.4 nm to about 3.5 nm, about 0.4 nm to about 4 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 1.5 nm, about 0.5 nm to about 2 nm, about 0.5 nm to about 2.5 nm, about 0.5 nm to about 3 nm, about 0.5 nm to about 3.5 nm, about 0.5 nm to about 4 nm, about 1 nm to about 1.5 nm, about 1 nm to about 2 nm, about 1 nm to about 2.5 nm, about 1 nm to about 3 nm, about 1 nm to about 3.5 nm, about 1 nm to about 4 nm, about 1.5 nm to about 2 nm, about 1.5 nm to about 2.5 nm, about 1.5 nm to about 3 nm, about 1.5 nm to about 3.5 nm, about 1.5 nm to about 4 nm, about 2 nm to about 2.5 nm, about 2 nm to about 3 nm, about 2 nm to about 3.5 nm, about 2 nm to about 4 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 3.5 nm, about 2.5 nm to about 4 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, or about 3.5 nm to about 4 nm. [0337] In some embodiments, a distance or dimension (e.g., diameter) may be measured from an atom to a nearest atom of the side chain of the amino acid residue. The side chain (e.g., atom of the side chain) may protrude into the constriction region of the channel and/or constriction-forming portion of the monomer. In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be at least about 0.0001 nm, at least about 0.0005 nm, at least about 0.001 nm, at least about 0.005 nm, at least about 0.01 nm, at least about 0.02nm, at least about 0.03 nm, at least about 0.04 nm, at least about 0.05 nm, at least about 0.06 nm, at least about 0.07 nm, at least about 0.08 nm, at least about 0.09 nm, at least about 0.1 nm, at least about 0.2 nm, at least about 0.3 nm, at least about 0.4 nm, at least about 0.5 nm, at least about 0.6 nm, at least about 0.7 nm, at least about 0.8 nm, at least about 0.9 nm, at least about 1.0 nm, at least about 1.1 nm, at least about 1.2 nm, at least about 1.3 nm, at least about 1.4 nm, at least about 1.5 nm, at least about 1.6 nm, at least about 1.7 nm, at least about 1.8 nm, at least about 1.9 nm, at least about 2.0 nm or greater than about 2.0 nm. In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be at most about 2.0 nm, at most about 1.9 nm, at most about 1.8 nm, at most about 1.7 nm, at most about 1.6 nm, at most about 1.5 nm, at most about 1.4 nm, at most about 1.3 nm, at most about 1.2 nm, at most about 1.1 nm, at most about 1.0 nm, at most about 0.9 nm, at most about 0.8 nm, at most about 0.7 nm, at most about 0.6 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, at most about 0.1 nm, at most about 0.09 nm, at most WSGR Docket Number: 64828-710.601 about 0.08 nm, at most about 0.07 nm, at most about 0.06 nm, at most about 0.05 nm, at most about 0.04 nm, at most about 0.03 nm, at most about 0.02 nm, at most about 0.01 nm, at most about 0.005 nm, at most about 0.001 nm, at most about 0.0005 nm, at most about 0.0001 nm, or less than about 0.0001 nm. [0338] In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be from about 0.0001 nm to about 2 nm. In some embodiments, a distance or dimension (e.g., diameter) of an atom to a nearest atom of an amino acid residue of an engineered monomer and/or engineered biological described herein may be from about 0.0001 nm to about 0.001 nm, about 0.0001 nm to about 0.005 nm, about 0.0001 nm to about 0.01 nm, about 0.0001 nm to about 0.05 nm, about 0.0001 nm to about 0.1 nm, about 0.0001 nm to about 0.2 nm, about 0.0001 nm to about 0.3 nm, about 0.0001 nm to about 0.4 nm, about 0.0001 nm to about 0.5 nm, about 0.0001 nm to about 1 nm, about 0.0001 nm to about 2 nm, about 0.001 nm to about 0.005 nm, about 0.001 nm to about 0.01 nm, about 0.001 nm to about 0.05 nm, about 0.001 nm to about 0.1 nm, about 0.001 nm to about 0.2 nm, about 0.001 nm to about 0.3 nm, about 0.001 nm to about 0.4 nm, about 0.001 nm to about 0.5 nm, about 0.001 nm to about 1 nm, about 0.001 nm to about 2 nm, about 0.005 nm to about 0.01 nm, about 0.005 nm to about 0.05 nm, about 0.005 nm to about 0.1 nm, about 0.005 nm to about 0.2 nm, about 0.005 nm to about 0.3 nm, about 0.005 nm to about 0.4 nm, about 0.005 nm to about 0.5 nm, about 0.005 nm to about 1 nm, about 0.005 nm to about 2 nm, about 0.01 nm to about 0.05 nm, about 0.01 nm to about 0.1 nm, about 0.01 nm to about 0.2 nm, about 0.01 nm to about 0.3 nm, about 0.01 nm to about 0.4 nm, about 0.01 nm to about 0.5 nm, about 0.01 nm to about 1 nm, about 0.01 nm to about 2 nm, about 0.05 nm to about 0.1 nm, about 0.05 nm to about 0.2 nm, about 0.05 nm to about 0.3 nm, about 0.05 nm to about 0.4 nm, about 0.05 nm to about 0.5 nm, about 0.05 nm to about 1 nm, about 0.05 nm to about 2 nm, about 0.1 nm to about 0.2 nm, about 0.1 nm to about 0.3 nm, about 0.1 nm to about 0.4 nm, about 0.1 nm to about 0.5 nm, about 0.1 nm to about 1 nm, about 0.1 nm to about 2 nm, about 0.2 nm to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 2 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 2 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 2 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 2 nm, or about 1 nm to about 2 nm. [0339] In some embodiments, a pore can be a non-biological pore. In some embodiments, a pore can be a solid state pore. [0340] In some embodiments, a pore can be a nanopore. In some embodiments, a pore comprises a width of at least about 0.5 nanometers (nm), 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises a width of at most about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, WSGR Docket Number: 64828-710.601 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises a diameter of at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises a diameter of at most about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises an outer diameter of at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises an outer diameter of at most about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm. In some embodiments, a pore comprises an inner diameter of at least about 0.1 nm, 0.2, nm, 0.3 nm, 0.4 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, a pore comprises an inner diameter of at most about 0.1 nm, 0.2, nm, 0.3 nm, 0.4 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm. In some embodiments, an inner diameter can be a diameter of one or more constrictions (e.g., constriction regions) of a pore. [0341] In some embodiments, a pore can function at a temperature of at least about 4, 10, 20, 30, 40, 50, 60, 70, 80, or about 90 degrees Celsius. In some embodiments, a pore can function at a temperature of at most about 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 degrees Celsius. [0342] In some embodiments, a pore can be disposed in a membrane. In some embodiments, a pore comprises transmembrane region. In some embodiments, a transmembrane region can be formed upon assembly of multiple transmembrane region sequences present in a plurality of subunits that together form a pore. In some embodiments the transmembrane region can be partially or fully composed of beta-strands. The beta strands can partially or fully comprise amphipathic surfaces that can interface with an amphipathic membrane. In some embodiments the transmembrane region can be partially or fully composed of alpha-helicases. The alpha- helicases can partially or fully comprise amphipathic surfaces that can interface with the amphipathic membranes. In some embodiments, a transmembrane region sequence comprises an alternation of hydrophobic residues and hydrophilic residues. In some embodiments, a pore comprises a hydrophobic portion and a hydrophilic portion. In some embodiments, a transmembrane region of the pore comprises a hydrophobic portion. In some embodiments, a ring portion of a pore comprises a hydrophilic portion. In some embodiments, a pore comprises a protein that controls the translocation of an analyte (e.g. polypeptide or polynucleotide) across the pore. In some embodiments, a molecular motor can cause the translocation of an analyte across the pore. In some embodiments, the translocation of an analyte across the pore can be NTP-driven or ATP-driven. In some embodiments, the translocation of an analyte across the pore does not depend on NTP or ATP. In some embodiments, the translocation of an analyte across the pore does not depend on a molecular motor. WSGR Docket Number: 64828-710.601 [0343] In some embodiments the pore can be a monomer. In some embodiments the pore can be formed from multiple monomeric units. In some embodiments, the monomers that comprise the oligomeric pore may be identical. In some embodiments the monomers that comprise the oligomeric pore may be different. [0344] In some embodiments, the nanopore may be an artificial biological nanopore. For example, the monomers of the nanopore may comprise proteins designed de novo (e.g., designed using machine learning algorithms). In some embodiments, portions of the nanopore (e.g., portions of the one or more monomers of the nanopore) may comprise proteins designed de novo (e.g., designed using machine learning algorithms). [0345] In some embodiments, the nanopore may comprise an assembly of monomers. The nanopore may comprise a number of monomers. Monomers may be arranged vertically, horizontally, and/or layered as rings to form a nanopore described herein. In some embodiments, a nanopore (e.g., biological nanopore) comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, or greater than 50 monomers. In some embodiments, a nanopore (e.g., biological nanopore) comprises at most about 50, 40, 30, 25, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 monomers. In some embodiments, a nanopore may comprise 1 monomer. In some embodiments, a nanopore (e.g., biological nanopore) comprises from about 3 monomers to about 40 monomers. In some embodiments, a nanopore (e.g., biological nanopore) comprises from about 3 monomers to about 4 monomers, about 3 monomers to about 5 monomers, about 3 monomers to about 6 monomers, about 3 monomers to about 7 monomers, about 3 monomers to about 8 monomers, about 3 monomers to about 9 monomers, about 3 monomers to about 10 monomers, about 3 monomers to about 15 monomers, about 3 monomers to about 20 monomers, about 3 monomers to about 30 monomers, about 3 monomers to about 40 monomers, about 4 monomers to about 5 monomers, about 4 monomers to about 6 monomers, about 4 monomers to about 7 monomers, about 4 monomers to about 8 monomers, about 4 monomers to about 9 monomers, about 4 monomers to about 10 monomers, about 4 monomers to about 15 monomers, about 4 monomers to about 20 monomers, about 4 monomers to about 30 monomers, about 4 monomers to about 40 monomers, about 5 monomers to about 6 monomers, about 5 monomers to about 7 monomers, about 5 monomers to about 8 monomers, about 5 monomers to about 9 monomers, about 5 monomers to about 10 monomers, about 5 monomers to about 15 monomers, about 5 monomers to about 20 monomers, about 5 monomers to about 30 monomers, about 5 monomers to about 40 monomers, about 6 monomers to about 7 monomers, about 6 monomers to about 8 monomers, about 6 monomers to about 9 monomers, about 6 monomers to about 10 monomers, about 6 monomers to about 15 monomers, about 6 monomers to about 20 monomers, about 6 monomers to about 30 monomers, about 6 monomers to about 40 monomers, about 7 monomers to about 8 monomers, about 7 monomers to about 9 monomers, about 7 monomers to about 10 monomers, about 7 monomers to about 15 monomers, about 7 monomers to about 20 monomers, about 7 monomers to about 30 monomers, about 7 monomers to about 40 monomers, about 8 monomers to about 9 WSGR Docket Number: 64828-710.601 monomers, about 8 monomers to about 10 monomers, about 8 monomers to about 15 monomers, about 8 monomers to about 20 monomers, about 8 monomers to about 30 monomers, about 8 monomers to about 40 monomers, about 9 monomers to about 10 monomers, about 9 monomers to about 15 monomers, about 9 monomers to about 20 monomers, about 9 monomers to about 30 monomers, about 9 monomers to about 40 monomers, about 10 monomers to about 15 monomers, about 10 monomers to about 20 monomers, about 10 monomers to about 30 monomers, about 10 monomers to about 40 monomers, about 15 monomers to about 20 monomers, about 15 monomers to about 30 monomers, about 15 monomers to about 40 monomers, about 20 monomers to about 30 monomers, about 20 monomers to about 40 monomers, or about 30 monomers to about 40 monomers. [0346] As the monomers of the nanopore arranged vertically, horizontally, and/or layered, the amino acid residues (e.g., positively-charged amino acid residues, negatively-charged amino acid residues, or neutral amino acid residues, or any combination thereof) may form one or more rings of charges. In some embodiments, a pore may be engineered to contain regions of separate rings of charges along the longitudinal length of the channel. For example, a nanopore may be engineered to contain regions of at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, or greater than about 20 separate rings of charges along the longitudinal length of the channel. A nanopore may be engineered to contain regions of at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or less than about 2 separate rings of charges along the longitudinal length of the channel. A nanopore may be engineered to contain regions from about 2 to about 20 separate rings of charges along the longitudinal length of the channel. A nanopore may be engineered to contain regions from about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2 to about 6, about 2 to about 7, about 2 to about 8, about 2 to about 9, about 2 to about 10, about 2 to about 15, about 2 to about 20, about 3 to about 4, about 3 to about 5, about 3 to about 6, about 3 to about 7, about 3 to about 8, about 3 to about 9, about 3 to about 10, about 3 to about 15, about 3 to about 20, about 4 to about 5, about 4 to about 6, about 4 to about 7, about 4 to about 8, about 4 to about 9, about 4 to about 10, about 4 to about 15, about 4 to about 20, about 5 to about 6, about 5 to about 7, about 5 to about 8, about 5 to about 9, about 5 to about 10, about 5 to about 15, about 5 to about 20, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 15, about 6 to about 20, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 15, about 7 to about 20, about 8 to about 9, about 8 to about 10, about 8 to about 15, about 8 to about 20, about 9 to about 10, about 9 to about 15, about 9 to about WSGR Docket Number: 64828-710.601 20, about 10 to about 15, about 10 to about 20, or about 15 to about 20 separate rings of charges along the longitudinal length of the channel. [0347] In some embodiments, a polypeptide can translocate through a pore at a rate of about 10 amino acids per second (aa/sec), 20 aa/sec, 30 aa/sec, 40 aa/sec, 50 aa/sec, 60 aa/sec, 70 aa/sec, 80 aa/sec, 90 aa/sec, 100 aa/sec, 110 aa/sec, 120 aa/sec, 130 aa/sec, 140 aa/sec, 150 aa/sec, 200 aa/sec, 250 aa/sec, 300 aa/sec, 350 aa/sec, 400 aa/sec, 450 aa/sec, or about 500 aa/sec. In some embodiments, a polypeptide can translocate through a pore at a rate greater than about 10 aa/sec, 20 aa/sec, 30 aa/sec, 40 aa/sec, 50 aa/sec, 60 aa/sec, 70 aa/sec, 80 aa/sec, 90 aa/sec, 100 aa/sec, 110 aa/sec, 120 aa/sec, 130 aa/sec, 140 aa/sec, 150 aa/sec, 200 aa/sec, 250 aa/sec, 300 aa/sec, 350 aa/sec, 400 aa/sec, 450 aa/sec, or about 500 aa/sec. In some embodiments, a polypeptide can translocate through a pore at a rate less than about 10 aa/sec, 20 aa/sec, 30 aa/sec, 40 aa/sec, 50 aa/sec, 60 aa/sec, 70 aa/sec, 80 aa/sec, 90 aa/sec, 100 aa/sec, 110 aa/sec, 120 aa/sec, 130 aa/sec, 140 aa/sec, 150 aa/sec, 200 aa/sec, 250 aa/sec, 300 aa/sec, 350 aa/sec, 400 aa/sec, 450 aa/sec, or about 500 aa/sec. [0348] In some embodiments, a pore can be configured to provide a condition for dominant electro-osmotic capture of an analyte. For example, for a nanopore system (e.g., an Aerolysin nanopore system or another nanopore system described herein), low pH conditions may increase the net positive charge inside the pore channel, resulting in (i) increased anion selectivity, (ii) a strong net anion-selective pore, or (iii) an increased electrostatic repulsion of mostly positively charged analytes, or (iv) any combination thereof. Low pH can comprise a pH of at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, or less than about 1. The resulting strong electro-osmotic flux through the pore can be exploited to capture analytes against the direction of the electrophoretic forces (EPF) acting upon them (e.g. with a positive applied potential at the trans electrode for a system with mostly positively charged peptides in the cis solution). It can be advantageous to exploit electro-osmotic forces to capture analytes since it can be less sensitive to charge composition. It can be advantageous for capturing and/or detecting a diverse composition of unlabeled peptides (e.g. neutral, net positive, net negative). [0349] In some embodiments, an electro-osmotic force (EOF) may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an electro-osmotic force may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an electrophoretic force may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an electrophoretic force may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an EOF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction and an EPF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. In some embodiments, an EOF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction and an EPF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an EOF may be in a second side (e.g., trans side) to WSGR Docket Number: 64828-710.601 first side (e.g., cis side) direction and an EPF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction. In some embodiments, an EOF may be in a second side (e.g., trans side) to first side (e.g., cis side) direction and an EPF may be in a first side (e.g., cis side) to second side (e.g., trans side) direction. [0350] The strength of electro-osmotic force (EOF) acting on the analytes can be tuned (e.g. by mutagenesis). A pore (e.g., nanopore) described herein can comprise one or more mutations. The mutation may be a substitution, an insertion, a deletion, or a chemical modification, or any combination thereof. The mutation may comprise a positively-charged amino acid residue, or a negatively-charged amino acid reside, or any combination thereof. For example, the electro-osmotic force can be reduced to increase the duration for which the analytes may be retained in the pore. For example, the anion ion-selectivity bias and resulting net anionic electro-osmotic flux that results from low pH conditions can be reduced by introducing acidic residues (e.g., by substitution adjacent to the aromatic mutations). Acidic mutation substitutions that reduce net positive charge can reduce electrostatic repulsion of mostly positively charge analytes. Net positive charge can also be reduced by replacing basic residues with neutral or acidic residues. Without wishing to be bound by theory, one or more mutations of negatively charged amino acid residues may increase a negative charge of a pore described herein. The increased negative charge may increase a duration that a positively-charged analyte resides in the pore. As another example, one or more mutations of positively charged amino acid residues may increase a positively charge of a pore described herein. The increased positive charge may increase a duration that a negatively- charged analyte resides in the pore. In some embodiments, one or more aromatic mutations (e.g., insertion of one or more aromatic amino acid residues and/or substitution of one or more aromatic amino acid residues) may affect a diameter of a nanopore described herein. The one or more aromatic mutations may decrease a diameter of a nanopore described herein due to the bulky side chain configuration of aromatic amino acid residues. Mutations of one or more amino acid residues in a constriction region and/or lumen-facing region of a nanopore described herein may provide for greater accuracy of determining one or more characteristics of an analyte. In some embodiments, one or more aromatic mutations (e.g., insertion of one or more aromatic amino acid residues and/or substitution of one or more aromatic amino acid residues) may affect one or more charges of a nanopore described herein. [0351] In some embodiments, a membrane can provide a partition for providing a voltage difference between a first side (e.g., cis side) and a second side (e.g., trans side) of a pore. In some embodiments, an EOF can result from a net ionic current flow cis to trans. In some embodiments, a cis to trans EOF results from a net ionic current flow cis to trans over a total ionic current flow, also referred to as a relative net current flow cis to trans, of greater than about 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, or about 0.99. In some embodiments, a cis to trans EOF results from a net ionic current flow trans to cis over a total ionic current flow, also referred WSGR Docket Number: 64828-710.601 to as a relative net current flow cis to trans, of less than about 0.0, -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, - 0.9, -0.95, or about -0.99. [0352] In some embodiments, the electro-osmotic force comprises a net ionic current flow from the first side (e.g., cis side) to the second side (e.g., trans side). In some embodiments, the electro-osmotic force can be modulated by a pH, a type of a salt, a concentration of a salt, an osmotic pressure across the membrane of the system, or a modification of the nanopore, or any combinations thereof. In some embodiments, the electro- osmotic force can be modulated by a modification of a charge of the nanopore. In some embodiments, the electro-osmotic force can be modulated by an asymmetric salt distribution between the first side (e.g., cis side) and second side (e.g., trans side) of the membrane. In some embodiments, the electro-osmotic force can be modulated by modification of a charge of the nanopore. [0353] In some embodiments, a pore can be configured to provide a dominant EOF in the direction cis to trans across the membrane of a pore system. In some embodiments, a pore can be configured to provide a dominant EOF in the direction trans to cis across the membrane of a pore system. In some embodiments, a pore can be configured to provide an EOF that acts against an electrophoretic force (EPF) across the membrane of a pore system. As shown in FIGs. 1A-1C, nanopores can have a strong net Electro-Osmotic Force (EOF) in the direction cis-to-trans across a membrane as indicated by the arrow. The Electrophoretic Forces (EPF) acting on the analyte can depend on the composition of charges on the analyte in the sections in and near the nanopore channel, and therefore can sometimes act in the net direction cis-to-trans or trans-to-cis. A strong and dominant cis-to-trans EOF can cause the capture, stretching, and efficient translocation of long polymer analytes from the cis compartment to the trans compartment regardless of the net direction of the EPF. The net flow can arise from a large cis-to-trans ion flow dominating over any trans-to-cis ion flows (e.g., a lower flow or counter- charged ions under an applied potential). In some embodiments, a positive charge may be applied to a compartment of the nanopore system (e.g., a trans compartment). With a positive voltage applied to a compartment (e.g., a trans compartment) across the membrane, nanopores with net positive internal charge may be used to limit the flow of cations from trans to cis. In some embodiments, a negative charge may be applied to a compartment of the nanopore system (e.g., a trans compartment). With a negative voltage applied to a compartment of the nanopore system (e.g., a trans compartment) across the membrane, nanopores with net negative internal charge may be used to limit the flow of anions from trans to cis. [0354] In some embodiments, a pore can be configured to provide an EOF that acts with an EPF across the membrane of a pore system. In some embodiments, a pore can comprise a relative ion selectivity P(+)/P(-) of greater than about 5.0 or less than about 0.1. In some embodiments, a pore can comprise a relative ion selectivity P(+)/P(-) of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0. In some embodiments, a pore can comprise a WSGR Docket Number: 64828-710.601 relative ion selectivity P(+)/P(-) of less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or about 5.0. [0355] In some embodiments, a pore can comprise a relative ion selectivity P(+)/P(-) of at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or greater than about 5 under an applied voltage difference across the membrane. In some embodiments, a pore can comprise a relative ion selectivity P(+)/P(-) of at most about 5, at most about 4, at most about 3, at most about 2, at most about 1, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, at most about 0.5, at most about 0.4, at most about 0.3, at most about 0.2, at most about 0.1, or less than about 0.1 under an applied voltage difference across the membrane. [0356] In some embodiments, a pore can comprise a relative ion selectivity P(+)/P(-) from about 0.1 to about 5 under an applied voltage difference across the membrane. In some embodiments, a pore can comprise a relative ion selectivity P(+)/P(-) from about 0.1 to about 0.2, about 0.1 to about 0.3, about 0.1 to about 0.4, about 0.1 to about 0.5, about 0.1 to about 1, about 0.1 to about 1.5, about 0.1 to about 2, about 0.1 to about 2.5, about 0.1 to about 3, about 0.1 to about 4, about 0.1 to about 5, about 0.2 to about 0.3, about 0.2 to about 0.4, about 0.2 to about 0.5, about 0.2 to about 1, about 0.2 to about 1.5, about 0.2 to about 2, about 0.2 to about 2.5, about 0.2 to about 3, about 0.2 to about 4, about 0.2 to about 5, about 0.3 to about 0.4, about 0.3 to about 0.5, about 0.3 to about 1, about 0.3 to about 1.5, about 0.3 to about 2, about 0.3 to about 2.5, about 0.3 to about 3, about 0.3 to about 4, about 0.3 to about 5, about 0.4 to about 0.5, about 0.4 to about 1, about 0.4 to about 1.5, about 0.4 to about 2, about 0.4 to about 2.5, about 0.4 to about 3, about 0.4 to about 4, about 0.4 to about 5, about 0.5 to about 1, about 0.5 to about 1.5, about 0.5 to about 2, about 0.5 to about 2.5, about 0.5 to about 3, about 0.5 to about 4, about 0.5 to about 5, about 1 to about 1.5, about 1 to about 2, about 1 to about 2.5, about 1 to about 3, about 1 to about 4, about 1 to about 5, about 1.5 to about 2, about 1.5 to about 2.5, about 1.5 to about 3, about 1.5 to about 4, about 1.5 to about 5, about 2 to about 2.5, about 2 to about 3, about 2 to about 4, about 2 to about 5, about 2.5 to about 3, about 2.5 to about 4, about 2.5 to about 5, about 3 to about 4, about 3 to about 5, or about 4 to about 5 under an applied voltage difference across the membrane. [0357] In some embodiments, a pore can comprise a relative ion selectivity P(+)/P(-) of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5 under an applied voltage difference across the membrane. [0358] In some embodiments, a pore comprises a lumen. In some embodiments, a nanopore lumen comprises a net charge of at least about 2 coulombs, at least about 3 coulombs, at least about 4 coulombs, at least about 5 coulombs, at least about 10 coulombs, at least about 15 coulombs, at least about 20 coulombs, at least about 25 coulombs, at least about 30 coulombs, at least about 35 coulombs, at least about 40 coulombs, at least about 45 WSGR Docket Number: 64828-710.601 coulombs, at least about 50 coulombs, at least about 55 coulombs, at least about 60 coulombs, at least about 70 coulombs, at least about 80 coulombs, at least about 90 coulombs, at least about 100 coulombs, at least about 150 coulombs, at least about 200 coulombs, or greater than about 200 coulombs. In some embodiments coulombs, a nanopore lumen comprises a net charge of at most about 200 coulombs, at most about 150 coulombs, at most about 100 coulombs, at most about 90 coulombs, at most about 80 coulombs, at most about 70 coulombs, at most about 60 coulombs, at most about 55 coulombs, at most about 50 coulombs, at most about 45 coulombs, at most about 40 coulombs, at most about 35 coulombs, at most about 30 coulombs, at most about 25 coulombs, at most about 20 coulombs, at most about 15 coulombs, at most about 10 coulombs, at most about 5 coulombs, at most about 4 coulombs, at most about 3 coulombs, at most about 2 coulombs, or less than about 2 coulombs. In some embodiments, a nanopore lumen comprises a net charge from about 2 to about 200 coulombs. In some embodiments, a nanopore lumen comprises a net charge from at most about 200. In some embodiments, a nanopore lumen comprises a net charge from about 2 to about 5, about 2 to about 10, about 2 to about 20, about 2 to about 30, about 2 to about 40, about 2 to about 50, about 2 to about 75, about 2 to about 100, about 2 to about 125, about 2 to about 150, about 2 to about 200, about 5 to about 10, about 5 to about 20, about 5 to about 30, about 5 to about 40, about 5 to about 50, about 5 to about 75, about 5 to about 100, about 5 to about 125, about 5 to about 150, about 5 to about 200, about 10 to about 20, about 10 to about 30, about 10 to about 40, about 10 to about 50, about 10 to about 75, about 10 to about 100, about 10 to about 125, about 10 to about 150, about 10 to about 200, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 20 to about 75, about 20 to about 100, about 20 to about 125, about 20 to about 150, about 20 to about 200, about 30 to about 40, about 30 to about 50, about 30 to about 75, about 30 to about 100, about 30 to about 125, about 30 to about 150, about 30 to about 200, about 40 to about 50, about 40 to about 75, about 40 to about 100, about 40 to about 125, about 40 to about 150, about 40 to about 200, about 50 to about 75, about 50 to about 100, about 50 to about 125, about 50 to about 150, about 50 to about 200, about 75 to about 100, about 75 to about 125, about 75 to about 150, about 75 to about 200, about 100 to about 125, about 100 to about 150, about 100 to about 200, about 125 to about 150, about 125 to about 200, or about 150 to about 200 coulombs. In some embodiments, a pore lumen comprises a net positive charge. In some embodiments, a pore lumen comprises a net negative charge. [0359] In some embodiments, a pore comprises a recognition region. A recognition region can be identified structurally by the dimensions of the central channel, e.g., X-ray diffraction structures, electron-microscopy structures, and/or computer modeling (e.g., molecular modeling, or homology modeling, or any combination thereof). A recognition region may be a region where electric field lines concentrate. A recognition region may be where a presence of an analyte disrupts the most the ionic current flowing through a pore, e.g., under an applied potential. A recognition region may comprise one or more narrow cross-sections of a pore channel, WSGR Docket Number: 64828-710.601 which can be, e.g., a diameter of less than 2 nanometers or 1 nanometer. In some embodiments, a pore can be engineered to have one or more narrow sections of an internal diameters (constrictions) within the recognition region, which can provide higher sensitivity/ionic current deflection to analytes. In some embodiments, a pore can be engineered to provide longer or shorter residence time of analytes. [0360] In some embodiments, a lumen-facing recognition region of a pore can be engineered (by one or more natural or non-natural amino acid substitutions, deletions, or modifications) to manipulate the internal dimensions, hydrophobicity, or aromaticity, or combinations thereof, of the pore. In some embodiments, engineering the lumen-facing recognition region of the pore increases the dwell time and/or resolution for peptides traversing the pore. In some embodiments, a lumen-facing recognition region of a pore can be engineered to decrease a translocation speed of an analyte through a pore. In some embodiments, a lumen- facing region of the pore can be engineered to reduce the analyte interacting or binding to the pore lumen. In some embodiments, a lumen-facing region of the pore can be engineered to increase the analyte interacting or binding to the pore lumen. In some embodiments, a lumen-facing recognition region of a pore can be engineered by modifying outwards facing residues to perturb the nearby lumen-facing residues. [0361] In some embodiments, a charge in a channel of a pore can adapted to alter the selectivity of the pore. In some embodiments, a pore may be modified by one or more mutations. In some embodiments, a mutation comprises one or more point mutations. In some embodiments, a point mutation can be at a non-conserved position. In some embodiments, a point mutation can be a lumen-facing mutation. In some embodiments, a point mutation can be a membrane-facing mutation. In some embodiments, a point mutation can alter a characteristic of a pore. In some embodiments, a point mutation can alter a pore channel charge, conductance at a set pH, ion selectivity, electro-osmotic flux, conductivity, shape, or structure, or combinations thereof. In some embodiments, a point mutation can allow for a conductance or analyte translation at a pH of less than about 1, 2, 3, 3.8, 4, 4.5, 6, 7, 8, 9, 1011, 12, 13, or about 14. In some embodiments, a point mutation can allow for a conductance or analyte translation at a pH of greater than about 1, 2, 3, 3.8, 4, 4.5, 6, 7, 8, 9, 1011, 12, 13, or about 14. In some embodiments, one or more point mutations may affect a diameter of a pore described herein. The one or more point mutations may modulate (e.g., widen or narrow) a diameter of a constriction region of a pore described herein. The constriction region of a nanopore described herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) measured from an alpha-carbon position of an amino acid backbone. The dimension of the constriction region may be measured from a first alpha-carbon position to a second alpha-carbon position. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter of at least about 0.2 nm, 0.3 nm, 0.4 nm, 0.5 nm, 1.0 nm, 1.5 nm, 2.0 nm, 2.5 nm, 3.0 nm, 3.5 nm, 4.0 nm, 4.5 nm, 5.0 nm, or greater than about 5.0 nm. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a WSGR Docket Number: 64828-710.601 diameter of at most about 5.0 nm, at most about 4.5 nm, at most about 4.0 nm, at most about 3.5 nm, at most about 3.0 nm, at most about 2.5 nm, at most about 2.0 nm, at most about 1.5 nm, at most about 1.0 nm, at most about 0.5 nm, at most about 0.4 nm, at most about 0.3 nm, at most about 0.2 nm, or less than about 0.2 nm. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter between about 0.2 nm to about 5 nm. In some embodiments, one or more point mutations may modulate a constriction region of a nanopore to comprise a diameter between about 0.2 nm to about 0.3 nm, about 0.2 nm to about 0.4 nm, about 0.2 nm to about 0.5 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1.5 nm, about 0.2 nm to about 2 nm, about 0.2 nm to about 2.5 nm, about 0.2 nm to about 3 nm, about 0.2 nm to about 3.5 nm, about 0.2 nm to about 4 nm, about 0.2 nm to about 5 nm, about 0.3 nm to about 0.4 nm, about 0.3 nm to about 0.5 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 1.5 nm, about 0.3 nm to about 2 nm, about 0.3 nm to about 2.5 nm, about 0.3 nm to about 3 nm, about 0.3 nm to about 3.5 nm, about 0.3 nm to about 4 nm, about 0.3 nm to about 5 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 1.5 nm, about 0.4 nm to about 2 nm, about 0.4 nm to about 2.5 nm, about 0.4 nm to about 3 nm, about 0.4 nm to about 3.5 nm, about 0.4 nm to about 4 nm, about 0.4 nm to about 5 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 1.5 nm, about 0.5 nm to about 2 nm, about 0.5 nm to about 2.5 nm, about 0.5 nm to about 3 nm, about 0.5 nm to about 3.5 nm, about 0.5 nm to about 4 nm, about 0.5 nm to about 5 nm, about 1 nm to about 1.5 nm, about 1 nm to about 2 nm, about 1 nm to about 2.5 nm, about 1 nm to about 3 nm, about 1 nm to about 3.5 nm, about 1 nm to about 4 nm, about 1 nm to about 5 nm, about 1.5 nm to about 2 nm, about 1.5 nm to about 2.5 nm, about 1.5 nm to about 3 nm, about 1.5 nm to about 3.5 nm, about 1.5 nm to about 4 nm, about 1.5 nm to about 5 nm, about 2 nm to about 2.5 nm, about 2 nm to about 3 nm, about 2 nm to about 3.5 nm, about 2 nm to about 4 nm, about 2 nm to about 5 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 3.5 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 5 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 5 nm, or about 4 nm to about 5 nm. [0362] In some embodiments, a charge in a channel of a pore can be adapted to alter an EOF. In some embodiments, a charge in a channel of a pore can be altered by changing the type of charged residue, the location of a charge, or the dimensions of the pore, or combinations thereof. In some embodiments, a pore can be modified to create a high overlap between Debye layers (alternatively termed Stern layers, the Gouy-Chapman diffuse layer or the electric double layer) or double layers to create energy barriers to limit the flow of a specific ion. In some embodiments, increased positive charge in the pore channel can increase transport of anionic species and/or decrease the transport of cationic species. In some embodiments, increased negative charge in the pore channel can increase transport of cationic species and/or decreases the transport of anionic species. This can, in turn, alter the net electro-osmotic flux of hydrated ions flowing through the pore under an applied WSGR Docket Number: 64828-710.601 potential. In some cases, electro-osmotic forces may act against an electrophoretic force during analyte capture. In some embodiments, electro-osmotic forces may dominate an electrophoretic force during analyte capture. In some embodiments which use electrophoretic mechanisms to sense polymers, it can be advantageous to reduce the net ion-selectivity and/or electro-osmotic flux to a level where electrophoretic forces dominate analyte capture. For example, the anion ion-selectivity bias and resulting net anionic electro-osmotic flux can be reduced by introducing acidic residues by substitution adjacent to the aromatic mutations. In another example, net positive charge can be also reduced by replacing basic residues with neutral or acidic residue(s), optionally by substitution with aromatic residue(s) that also separately and additively improve peptide capture and/or discrimination (e.g. CytK-K128F and Aer-K238F). In some embodiments, a pore may be engineered to contain regions of 1, 2, 3, 4, 5, 6, or more separate rings of charges along the longitudinal length of the channel. In some embodiments, the rings may be spaced 0.5 nm, 1.0 nm, 1.5 nm, 2.0 nm, 3.0 nm or further from each other. [0363] In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at least about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA. In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at least about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA at a pH of less than about 2, 3, 3.8, 4, 4.5, 7, 7.5, or about 8. In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at most about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA. In some embodiments, a pore comprising a point mutation described herein can have an open pore current of at most about 20, 30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or about 400 pA at a pH of less than about 2, 3, 3.8, 4, 4.5, 7, 7.5, or 8. [0364] In some embodiments, a pore comprises an aromatic amino acid within a lumen of the pore. In some embodiments, a pore comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 aromatic amino acids within a lumen of the pore. In some embodiments, a pore comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 aromatic amino acids within a lumen of the pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases an analyte interaction with a lumen of a pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases an analyte residence time withing a pore. In some embodiments, an aromatic amino acid within a lumen of a pore increases signal differences between different analytes. WSGR Docket Number: 64828-710.601 [0365] In some embodiments, a pore comprises a monomer. In some embodiments, a pore comprises a dimer, a trimer, a tetramer, pentamer, a hexamer, a heptamer, an octamer, a nonamer, a decamer, an undecamer, or a dodecamer . In some embodiments, a pore comprises an oligomer. In some embodiments, a pore comprise a homo-oligomer or a hetero-oligomer. In some embodiments, a pore can comprise a plurality of subunits. In some embodiments, a pore comprises several repeating subunits, such as at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, a pore comprises several repeating subunits, such as at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, a plurality of subunits of a pore may be axially aligned. In some embodiments, a plurality of subunits of a pore comprise an approximately central axis. In some embodiments, a pore comprises a channel through which an ion can flow. In some embodiments, a pore comprises a plurality of subunits. In some embodiments, a pore comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 subunits. In some embodiments, a pore comprises at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 subunits. In some embodiments, a pore comprises a ring of multiple identical mutations. In some embodiments, a pore comprises a ring of multiple identical mutations in a recognition region that can be co-planar with a membrane and orthogonal to the direction of analyte passage. In some embodiments, a pore comprises different mutations for its subunits to comprise a hetero-oligomeric assembly. In some embodiments, a pore comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more mutant monomers. The number of mutated units can be adapted to modulate the extent/magnitude of the change to the recognition region. In some embodiments, a pore comprises mutations on one or more beta-strands. In some embodiments, beta-strands can comprise mutations either in the down strand or in the up strand of a beta strand, or in both. In some embodiments the pore comprises mutations to one or more alpha-helices. In some embodiments a mutation can be inward or lumen facing. In some embodiments a mutation can be outward or membrane facing. [0366] In some embodiments, a pore comprises a plurality of protomers. In some embodiments, a plurality of protomers can be comprised in an assembly that forms at least a portion of the pore, wherein the protomers form a channel region of the pore. In some embodiments, a plurality of protomers can comprise identical sequences or different sequences. In some embodiments, a pore comprises a plurality of mixed protomers. In some embodiments, a plurality of protomers may be separated from a pore. In some embodiments, a plurality of protomers may be fused to a pore. In some embodiments, a pore lumen comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 charged amino acids pointing towards the lumen of the pore per protomer. In some embodiments, a pore lumen comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 charged amino acids pointing towards the lumen of the pore per protomer. In some embodiments, a pore lumen comprises a plurality of negatively charged amino acids. In some embodiments, a pore lumen comprises a plurality of positively charged amino acids. In some WSGR Docket Number: 64828-710.601 embodiments, a pore can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, or about 100 individual proteins. In some embodiments, a pore can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, or about 100 individual proteins. In some embodiments, a pore can comprise charged residues either in rings around the pore in plane with the membrane, and/or vertically up the pore channel perpendicular to plane of the membrane. In some embodiments, a pore comprises charged residues at a pore entry. In some embodiments, a pore comprises charged residues at a pore exit. In some embodiments, a pore comprises charged residues at a pore constriction. In some embodiments, charged residues can comprise Asp, Glu, Arg, Lys, His, or non-natural amino acids. [0367] In some embodiments, a pore can be configured to detect analytes larger than 40 kDa. The size of a pore opening, channel, or constriction region, or any combination thereof, may be large enough to accommodate a large analyte (e.g., an analyte larger than 40 kDa). In some embodiments, a pore can be configured to detect analytes larger than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or about 10000 kDa. In some embodiments, a pore can be configured to detect analytes smaller than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or about 10000 kDa. In some embodiments, a pore comprises a cylindrically-shaped region. In some embodiments, a pore comprises a cone-shaped region. In some embodiments, a pore comprises a cylindrical chamber on a second side (e.g., trans side). In some embodiments, a pore comprises a cone chamber on a first side (e.g., cis side). In some embodiments, a pore comprises a cylindrical chamber on a second side (e.g., trans side) and a cone chamber on a first side (e.g., cis side) separated by an inner constriction. In some embodiments a pore comprises an hourglass shape. In some embodiments a pore comprises a cone shape on a first side (e.g., cis side) and a cone shape on a second side (e.g., trans side), separated by an inner constriction. [0368] In some embodiments, a pore comprises an affinity tag, e.g., a His-tag or Strep-tag. In some embodiments, an affinity tag can be appended to a N- or C-terminus of a pore or a subunit thereof. In some embodiments, an affinity tag can be appended to a pore or a subunit thereof via a linker, e.g., a GSA linker. An affinity tag may comprise a Glutathione-S-transferase (GST) tag, a Maltose-Binding Protein (MBP) tag, a FLAG tag, a c-myc tag, a hemagglutinin (HA) tag, a T7 tag, a calmodulin-binding peptide (CBP) tag, a biotinylation tag, or any combination thereof. In some embodiments, a linker can comprise (GGGGS)n and/or (SG)n, where n may comprise any integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than 10). In some embodiments, a linker described herein may comprise (GGGGS)3, (Gly)8, (Gly)6, (EAAAK)3, (EAAAK)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE, WSGR Docket Number: 64828-710.601 AGNRVRRSVG, RRRRRRRRR, GFLG, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, disulfide bond, or cysteine linkages, or any combination thereof. [0369] Gated nanopores can comprise nanopores described herein that may act as rapid, closable channels to gate transport of one or more analytes across a membrane. In some embodiments, a nanopore may exhibit spontaneous gating, in which the channel may not be open for an analyte to enter, translocate through, or any combination thereof. In some embodiments, a pore may exhibit no spontaneous gating for a period of at least about 10, 20, 30, or 60 s at an applied potential of less than about -50 mV or greater than about 50mV. In some embodiments, a pore may exhibit no spontaneous gating for a period of at most about 10, 20, 30, or 60 s at an applied potential of -50 mV. In some embodiments, a pore may exhibit no spontaneous gating for a period of at least about 5, 10, 15, or 20s at an applied potential of -150 mV. In some embodiments, a pore may exhibit no spontaneous gating for a period of at most about 5, 10, 15, or 20s at an applied potential of -150 mV. [0370] In some embodiments, a pore comprises residues configured to anchor the pore to a membrane. In some embodiments, a pore comprises a mutation that increases the solubility of the pore or a subunit thereof in a lipid bilayer. In some embodiments, a pore comprises a mutation that increases the solubility of the pore or a subunit thereof in a lipid bilayer in an external water environment. In some embodiments, a pore comprises residues configured to improve insertion efficiency into a membrane. In some embodiments, a pore comprises residues configured to reduce spontaneous gating. In some embodiments, a pore comprises residues configured to reduce signal noise. The signal noise can be noise in a signal comprising ionic current, impedance, current rectification, potential, tunnelling, conductivity, light, or mechanical deformation. [0371] In some embodiments, a pore comprises a protease. In some embodiments, a protease can be configured to degrade a polypeptide into a fragment. In some embodiments, one or more proteases degrade a polypeptide into one or more fragments. In some embodiments, a fragment enters a channel of a pore. In some embodiments, a polypeptide fragment comprises a portion comprising a non-natural amino acid, polyethylene glycol, PNA, DNA, or RNA, or combinations thereof. In some embodiments, a protease can be active. In some embodiments, a protease can be inactive. In some embodiments, a polypeptide can be degraded into a fragment before translocation through a pore. [0372] In some embodiments, a pore comprises an unfoldase. In some embodiments, an unfoldase linearizes a polymer which enters the pore. In some embodiments, a polymer threads through a pore while it can be recognized by ionic currents. In some embodiments, an unfoldase comprises a prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, or Valosin-containing protein-like ATPase. In some embodiments, an unfoldase may be modulated by an adaptor protein or other accessory proteins or chaperones. In some embodiments, the AAA+ enzyme (e.g., AAA+ unfoldase) is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, WSGR Docket Number: 64828-710.601 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Msp1, SecA, and functional homologs, orthologs, paralogs thereof. [0373] In some embodiments, a pore comprises a ring-forming protein. In some embodiments, a ring-forming protein can be configured to transport a polymer, e.g. a polypeptide, across a transmembrane region of a pore. In some embodiments, a ring-forming protein comprises a toroidal or donut-shaped multi-subunit protein. In some embodiments, a ring-forming protein comprises a ring-forming multimeric protein, such as an octameric, heptameric or hexameric protein. In some embodiments, a ring-forming protein comprises a heptameric protein. In some embodiments, a heptameric protein include those submitted to the Protein Data Bank (PDB) under one of the following unique accession or identification code codes: lg31, 1h64, 1hx5, 1i4k, 1i5l, 1i8f, 1i81, 1iok, 1j2p, 1jri, 1lep, 1lnx, 1loj, 1mgq, 1n9s, 1ny6, 1p3h, 1tzo, 1wnr, 1xck, 2cb4, 2cby, 2yf2, 3bpd, 3cf0, 3j83, 3ktj, 3m0e, 3st9, 4b0f, 4emg, 4gm2, 4hnk, 4hw9, 4jcq, 4ki8, 4owk, 4qhs, 4xq3, 5jzh, 5msj, 5msk, 5mx5 and 5uw8e. [0374] In some embodiments, a transmembrane portion of a pore comprises a linker. The linker may be a helical linker, a non-helical linker, a flexible linker, or a cleavable linker, or any combination thereof. In some embodiments, a linker can by hydrophilic or mostly hydrophilic. In some embodiments, a transmembrane portion of a pore comprises a flexible hydrophilic linker. A flexible linker can comprise a linker rich in small and/or polar amino acids (e.g., glycine, serine, or threonine, or any combination thereof), which can provide good flexibility and solubility. For example, a transmembrane portion can comprise on the N-and/or C-terminal side a flexible hydrophilic linker of at least about 3, 4, 5, 10, 15, or 20 amino acids. A transmembrane portion can comprise on the N-and/or C-terminal side a flexible hydrophilic linker of at most about 3, 4, 5, 10, 15, or 20 amino acids. In some embodiments, a hydrophilic linker can interact with the charged head groups of membrane (phospho)lipids. For example, hydrophilic residues include serine, threonine, asparagine, glutamine, aspartate, glutamate, lysine and arginine. In some embodiments, a hydrophilic linker comprises at least a portion comprising non-hydrophilic or non-natural amino acids to tune the linker’s properties. [0375] In some embodiments, a pore comprises a protease. In some embodiments, a pore comprises a protease- unfoldase pair. In some embodiments, a protease-unfoldase pair can be attached on a first side (e.g., cis side) of a FraC pore. Then, cleaved peptides can be sequentially recognized and translocated across the pore. In some embodiments, a protease-unfoldase pair can comprise a barrel-shaped ATP-dependent ClpXP protease. In some embodiments, a protease can encase digested peptides, preventing their release in solution. [0376] In some embodiments, a pore can be configured to proteolytically cleave analytes. In some embodiments, a pore can be configured to not to proteolytically cleave analytes. The conditions of a pore and/or nanopore system may comprise ATP concentration, or buffer types, or any combination thereof. The conditions of the pore and/or nanopore system can be configured to cleave or not to cleave analytes. For example, VAT WSGR Docket Number: 64828-710.601 can be capable of feeding the polypeptide through the pore at a speed that can be tuned by the concentration of ATP. A transmembrane proteasome can simultaneously process and identify different analytes. In some embodiments, translocated peptides may be proteolytically degraded. In some embodiments, the pore can be employed with an inactivated protease/proteasome which recognizes proteins as they are linearized and transported across the pore at a controlled speed. In some embodiments, the activity of the protease/proteasome can be monitored at the single molecule level. In some embodiments, translocated peptides may not be proteolytically degraded. In some embodiments, a proteasome and/or a portion of a proteasome may comprise a protease domain, or a translocase domain, or any combination thereof. [0377] In some embodiments, a pore comprises natural or non-natural aromatic amino acid residues. In some embodiments, a non-natural aromatic amino acid can be selected from the group consisting of 3,4-dihydroxy- L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine, L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr). In some embodiments, a non-natural aromatic amino acid can be a D-amino acid, a Homo-amino acid (methylene), a Beta-homo-amino acid, a N-methyl amino acid, or an Alpha-methyl amino acid. In some embodiments, a non-natural aromatic amino acid can be a derivatized Phe/Tyr/Trp amino acid, e.g., a ring- substituted Phe/Tyr/Trp amino acids. In some embodiments, a non-natural aromatic amino acid can be a derivative of Phe, Tyr or Trp, substituted by, e.g., a halogen, -CH3, OH, -CH2NH3, -C(O)H, -CH2CH3,-CN, - CH2CH2CH3, -SH, or another group. Non-natural aromatic amino acids include, but are not limited to, O- methyl-L-tyrosine; 3-methyl- phenylalanine; a p-acetyl-L-phenylalanine; O-4-allyl-L-tyrosine; 4-propyl-L- tyrosine; fluorinated phenylalanine; isopropyl-L-phenylalanine; ap-azido-L- phenylalanine; a p-acyl-L- phenylalanine; a p-benzoyl-L-phenylalanine; a phosphonotyrosine; a p-iodo-phenylalanine; p- bromophenylalanine; p- amino-L-phenylalanine; an isopropyl-L-phenylalanine; an amino-, isopropyl-, or O- allyl-containing phenylalanine analogue; a p-(propargyloxy) phenylalanine; 3-nitro-tyrosine; 5-fluoro- tryptophan, 5-hydroxy-tryptophan, 5-methoxy- tryptophan, 5-methyl-tryptophan, trifluoromethyl-tryptamine ethyl ester. [0378] In some embodiments, a non-naturally-occurring amino acids may be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express a mutant monomer. In some embodiments, a non- naturally-occurring amino acids may be introduced by expressing a mutant monomer in E. coli that may be auxotrophic for specific amino acids in the presence of synthetic (i.e. non-naturally-occurring) analogues of those specific amino acids. In some embodiments, a non-naturally-occurring amino acids may be introduced using synthetic peptide chemistry methods. Monomeric units of the pores may be formed entirely from synthetic peptides constructed using conjugation methods, e.g., chemical ligation, or cysteine coupling. In some embodiments, monomers of the pore may comprise partially synthetic units coupled to naturally expressed peptide units using coupling methods. WSGR Docket Number: 64828-710.601 [0379] In some embodiments, a pore comprises an alpha-helical or beta-barrel oligomeric pore forming toxin or porin. A pore can comprise a beta-barrel pore forming protein and/or peptide and/or an alpha helical pore forming protein and/or peptide. In some embodiments, a pore can be selected from the group consisting of Aerolysin (Aer), Cytolysin K (CytK), Mycobacterium smegmatis porin A (MspA), alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, phage derived portal proteins (Phi29, or G20c, or any combination thereof) or a mutant thereof. In some embodiments, a pore comprises a phage portal complex, or a cellular transmembrane transport complex. In some embodiments, a pore comprises an alpha-helix bundle or channel. In some embodiments, a pore comprises a transmembrane protein pore derived from beta-barrel pores or alpha- helix bundle pores. In some embodiments, a beta-barrel pore or beta-barrel pore forming protein and/or peptide comprises a barrel or channel structure comprising beta-strands. In some embodiments, a pore comprises charged residues on both the “up” strands and the “down” of a beta-strand. In some embodiments, charged residues can be located at sequence positions that co-locate them approximately co-planar in a beta-barrel. [0380] In some embodiments, the nanopore comprises a pore-forming toxin. The nanopore can comprise an α- pore-forming toxin, or a β-pore-forming toxin, or any combination thereof. The nanopore can comprise a pore- forming toxin derived from a bacterium. The bacterium can be of a genus of bacteria including, but not limited to, Xenorhabdus, Yersinia, Providencia, Pseudomonas, Proteus, Morganella, or Photorhabdus. In some embodiments, the nanopore comprises a pore-forming toxin derived from a bacterial species selected from the group consisting of Escherichia coli, Mycobacterium smegmatis, Staphylococcus aureus, Salmonella typhi, P. aeruginosa, A. baumanii, Klebsiella oxytoca, Bacillus cereus, A. hydrophila, S. marcescens, V. cholerae, P. entomophila, C. perfringens, and Y. enterocolitica. In some embodiments, a nanopore described herein may comprise one or more monomers of a T7 pore, a PN pore, a stable protein 1 (SP1) pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (α-HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a cytolysin A (ClyA) pore, a FhuA pore, a PFO pore, a TMH4C4 pore, any combination thereof, or homolog, paralog, ortholog, or any combination thereof. [0381] In some embodiments, the nanopore (e.g., the biological nanopore) can comprise a conical geometry or a semi-conical geometry. A conical geometry can comprise a shape in which a nanopore tapers over a longitudinal axis, wherein a first entrance of a nanopore is larger (e.g., comprises a wider dimension) than a second entrance. In some embodiments, the nanopore (e.g., the biological nanopore) can comprise a straight geometry (e.g., a cylindrical geometry). A straight geometry may comprise a shape in which a channel of a nanopore can be the same width (e.g., diameter) over its longitudinal axis. In some embodiments, the nanopore can be a T7 pore, a PN pore, a stable protein 1 (SP1) pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (α- HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a cytolysin A (ClyA) pore, a FhuA pore, a PFO pore, or a TMH4C4 pore, or any combination thereof. In some WSGR Docket Number: 64828-710.601 embodiments, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, α-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or any combination thereof. In some embodiments, an engineered biological nanopore described herein may comprise one or monomers from a T7 pore, a PN pore, a SP1 pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (α- HL) pore, a SPP1 pore, a FraC pore, a MspA pore, a CsgG pore, an OmpG pore, an aerolysin pore, a ClyA pore, a FhuA pore, a PFO pore, or a TMH4C4 pore, or any combination thereof. In some embodiments, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, α-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or any combination thereof. In some embodiments, the nanopore (e.g., the biological nanopore) can comprise a vestibule geometry (e.g., a globular geometry or goblet geometry). The nanopore may comprise an alpha-hemolysin nanopore, or a curli specific gene G (CsgG) nanopore, or any combination thereof. [0382] In some embodiments, a pore comprises comprise beta-toxins, such as alpha-hemolysin, anthrax toxin and leukocidins. In some embodiments, a pore comprises outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, CsgG. In some embodiments, a pore comprises outer membrane porin F (OmpF). In some embodiments, a pore comprises outer membrane porin G (OmpG). In some embodiments, a pore comprises outer membrane phospholipase A. In some embodiments, a pore comprises outer membrane protein FhuA. In some embodiments, a pore comprises outer membrane protein A (OmpA). In some embodiments, a pore comprises Neisseria autotransporter lipoprotein (NalP). In some embodiments, a pore comprises lysenin. In some embodiments, a pore comprises bacterial nucleoside transporter Tsx. In some embodiments, a pore comprises inner membrane proteins and outer membrane proteins, such as WZA and FraC. In some embodiments, a pore comprises Aer, CytK, MspA, aHL, CsgG, or FraC or an engineered mutant thereof. In some embodiments, a pore comprises a transmembrane pore derived from or based on Msp, e.g. MspA, a-hemolysin (a-HL), lysenin, CsgG, ClyA, Spl or haemolytic protein fragaceatoxin C (FraC). In some embodiments, a pore comprises an oligomeric accessory protein coupled to a transmembrane domain of the pore. In some embodiments, a pore comprises alpha-helical or beta- barrel transmembrane regions. In some embodiments, a beta-barrel pore or beta-barrel pore forming protein and/or peptide can include, but may not be limited to, beta-toxins, such as alpha-hemolysins, aerolysins, lysenin, cytolysins, cytolysin K, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A (OMPLA), ferric hydroxamate uptake component A (FhuA), Curli production transport component CsgG, and Neisseria autotransporter lipoprotein (NalP). In some embodiments, an alpha-helix pore or alpha-helical forming protein and/or peptide can include, but may not be limited to, inner membrane proteins and outer membrane proteins, such as WSGR Docket Number: 64828-710.601 Actinoporins, the outer membrane core complex (OMCC) of H. pylori Cag T4SS particles, and the transmembrane domain of the E. coli polysaccharide transporter Wza. In some embodiments, a pore comprises CytK or a genetically engineered mutant thereof. In some embodiments, a pore can comprise a porin. In some embodiments, a pore can comprise OmpF, OmpG, or FhuA. In some embodiments, a pore comprises a pore forming protein (PFP). In some embodiments, a PFP comprises an α-PFP. In some embodiments, a PFP comprises a β-PFPs. In some embodiments, a PFP comprises a bundle of α-helices. In some embodiments, a PFP comprises a transmembrane β-barrel. [0383] In some embodiments, the nanopore comprises CytK or a genetically engineered mutant thereof. In some embodiments, the mutant CytK comprises one or more of the amino acid substitutions selected from the group consisting of K128D, K128F, K155D, S120D, Q122D, G122D and S151D. FIGs. 3A-3D depict representations of a CytK nanopore and one or more mutations. FIG. 3A shows a surface representation of a pore (e.g., a CytK pore) and FIG. 3B depicts a cartoon representation of a β-barrel region, with N-terminal strands depicted as dark gray and C-terminal strands depicted as light gray. Residues include E112, T116, S120, Q122, S126, K128, E139, T143, Q145, T147, S151, and K155 (charged residues in bold). FIG. 3C shows residues in each beta strand of the transmembrane beta-barrel region of wild-type CytK, marking water-facing residues of the down- and up- strands most suitable for mutagenesis. In some embodiments, as shown in FIG. 3D, a mutant pore (e.g., a mutant CytK pore) can be a high ion selectivity mutant (e.g., CytK-2E-4D). The mutant pore can be in 1 M KCl, pH 7.5 {(p(K)/p(Cl) of 4.04 ± 0.07, and p(K)/p(Cl) of 1.3 at pH 3.8)}. Substitutions of the CytK nanopore can comprise one or more amino acid substitutions comprising K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, S151D, K155Q, T116D, S126D, T143D, Q145D, T147D, or any combination thereof. [0384] In some embodiments, a nanopore described herein can comprise one or more CytK monomers. A CytK monomer may comprise a wildtype CytK monomer. A CytK monomer may comprise a mutant CytK monomer. A CytK monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107. In some embodiments, a CytK monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107. In some embodiments, a CytK monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution) in an amino acid sequence as set forth in SEQ ID NOs.: 8 or 107. WSGR Docket Number: 64828-710.601 [0385] In some embodiments, a CytK monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112. In some embodiments, a CytK monomer of a nanopore described here may comprise an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112. In some embodiments, a CytK monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in any one of SEQ ID NOs.: 9-26 or 108-112. [0386] In some embodiments, a pore comprises engineered pores. In some embodiments, a pore comprises an oligomeric accessory protein coupled to a transmembrane domain. In some embodiments, a pore can be derived from naturally existing nanopores. In some embodiments, a pore comprises a protein pore templated by a DNA structure. In some embodiments, a pore comprises a de novo pore based on de novo alpha-helical or beta-barrel transmembrane regions. In some embodiments, a pore can comprise a de novo pore based on de novo beta- barrel pore forming protein and/or peptide and/or alpha-helical pore forming protein and/or peptide. [0387] In some embodiments, a pore can be a portion of an existing pore or mutations thereof. In some embodiments, a pore comprises a combination of elements of distinct pores or mutations thereof. In some embodiments, a pore comprises an assembly of genetically engineered pleurotolysin (Ply) A and B subunits. In some embodiments, a pore comprises a protein of the Membrane Attack Complex PerForin/Cholesterol Dependent Cytolysin (MACPF/CDC) protein superfamily. In some embodiments, a pore comprises perforin, complement C9, pneumolysin, or lysteriolysin. In some embodiments, a pore comprises a recognition or a binding portion configured to recognize or bind to an analyte. In some embodiments, a pore comprises a transmembrane portion comprising a channel for an analyte to pass through. In some embodiments the pore comprises a molecular or proteinaceous adapter. In some embodiments the molecular or proteinaceous adapter acts as a recognition or binding site for analytes. In some embodiments, the molecular or proteinaceous adapter can be a cyclic adapter. The adapter can be bound covalently or non-covalently. [0388] In some embodiments, a pore comprises a proteasome. In some embodiments, the proteasome can be a S20 proteasome, a 26S proteasome, a 11S proteasome, a ClpXP proteasome, open reading frame number 854 in the M. mazei genome, or combinations thereof. In some embodiments, the proteasome comprises a subunit or components of a proteasome. In some embodiments, a proteasome can be a fused proteasome. In some embodiments, a C-terminus of a subunit of a ring-forming (multimeric) protein comprising a flanked transmembrane sequence can be genetically fused to a N-terminus of a proteasome subunit. In some WSGR Docket Number: 64828-710.601 embodiments, a ring-forming protein subunit can be fused to an N-terminally truncated proteasome subunit such that the proteasome gate can be left open towards. In some embodiments, a pore comprises a proteasome fused to the pore. In some embodiments, a pore comprises a proteasome fused to the pore such that the proteasome can be located on the first side (e.g., cis side) of the pore when the pore can be disposed in a membrane. In some embodiments, a proteasome can be coupled to a pore. In some embodiments, a proteasome can be coupled non-covalently to a pore. A proteasome may be fused and/or coupled to a pore by a linker described herein. The linker may be a helical linker, a non-helical linker, a flexible linker, a cleavable linker, or any combination thereof. In some embodiments, a proteasome can be coupled to a pore on a first side (e.g., cis side) of a membrane. In some embodiments, a proteasome can be coupled to a pore on a second side (e.g., trans side) of a membrane. In some embodiments, a pore comprises one or more proteasome subunits. In some embodiments, a pore comprises a proteasome α-subunit. In some embodiments, a pore comprises a proteasome β-subunit. In some embodiments, a pore comprises a ring of proteasome α-subunits. In some embodiments, a pore comprises a ring of proteasome β-subunits. In some embodiments, a pore comprises a catalytically active subunit. In some embodiments, a pore comprises a protease. In some embodiments, a pore comprises a protease having a trypsin-type or chymotrypsin-type of activity. [0389] In some embodiments, a pore comprises prokaryotic AAA+ unfoldase ClpX. ClpX can unfold analytes by NTP-driven translocation of the polypeptide chain through the central pore of its hexameric assembly. In complex with the ClpP peptidase, ClpX can carry out protein degradation by translocating unfolded analytes directly into the ClpP proteolytic chamber. In some embodiments, a pore comprises a multi-protein pore sensor complex comprising an artificial ClpP pore, e.g. by fusion to PA, which sensor complex further comprises ClpX or a homologous protein unfoldase. [0390] In some embodiments, a pore comprises an oligomeric Fragaceatoxin C (FraC) pore. In some embodiments, FraC can be a type II pore. In some embodiments, a type II FraC pore comprises an apparent heptameric stoichiometry, and/or a conductance of about 1.22-1.26 nS when assayed at pH 7.5 in a 1M NaCl solution or about 0.99-1.08 nS when assayed at pH 4.5 in a 1 M KC solution. Conductance values can be determined by collecting single channels under -50 mV applied potential using 1 M NaCl, 15 mM Tris pH 7.5, or 1 M KCl, 0.1 M citric acid, 180 mM Tris base pH 4.5. In some embodiments, a type II FraC pore can comprise a pore size (at the narrowest constriction) of about 1.1 nm, which can be determined from homology modeling. [0391] In some embodiments, FraC can be a type III pore. In some embodiments, a type III FraC pore comprises an apparent hexameric stoichiometry, and/or a conductance of about 0.37-0.43 nS when assayed at pH 4.5 in a 1M KC solution. In some embodiments, a type III FraC pore comprises a pore size (at the narrowest constriction) of about 0.8 nm, which can be determined from homology modeling. WSGR Docket Number: 64828-710.601 [0392] In some embodiments, a nanopore described herein can comprise one or more FraC monomers. A FraC monomer may comprise a wildtype FraC monomer. A FraC monomer may comprise a mutant FraC monomer. A FraC monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52. In some embodiments, a FraC monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52. In some embodiments, a FraC monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NOs.: 51 or 52. [0393] In some embodiments, a nanopore described herein can comprise one or more alpha-hemolysin (α- hemolysin) monomers. An alpha-hemolysin (α-hemolysin) monomer may comprise a wildtype alpha- hemolysin (α-hemolysin) monomer. An alpha-hemolysin (α-hemolysin) monomer may comprise a mutant alpha-hemolysin (α-hemolysin) monomer. An alpha-hemolysin (α-hemolysin) monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 106. In some embodiments, an alpha-hemolysin (α-hemolysin) monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 106. In some embodiments, an alpha-hemolysin (α- hemolysin) monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 106. [0394] In some embodiments, a nanopore described herein can comprise one or more CsgG monomers. A CsgG monomer may comprise a wildtype CsgG monomer. A CsgG monomer may comprise a mutant CsgG monomer. A CsgG monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 128. In some embodiments, a CsgG monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 128. In some embodiments, WSGR Docket Number: 64828-710.601 a CsgG monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 128. [0395] In some embodiments, a MspA monomer may comprise a wildtype MspA monomer. In some embodiments, a wildtype MspA monomer may comprise an amino acid sequence as set forth in MGLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEGTLELG YQIGFPWSLGVGINFSYTTPNILIDDGDITAPPFGLNSVITPNLFPGVSISADLGNGPGIQEVATFSVDVS GAEGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDSVTTYGEPWNMNGSAGSAWSHPQFEK (SEQ ID NO.: 171). In some embodiments, a nanopore described herein can comprise one or more MspA monomers. A MspA monomer may comprise a mutant MspA monomer. A MspA monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, a MspA monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, a MspA monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 171. [0396] In some cases, an MspA nanopore may comprise a mutation in a lumen-facing region, or constriction region, or any combination thereof. In some embodiments, a mutation of an MspA nanopore can comprise a in a lumen-facing region and/or constriction region from one or more amino acid residues to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively- charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), one or more sulfur-containing amino acid residues, or any combination thereof. [0397] In some embodiments, a monomer of a MspA nanopore may comprise a mutation at position D90, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, a monomer of a MspA nanopore may comprise a mutation at position D91, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171. In some embodiments, the mutation at position D90 and/or D91 of the MspA monomer can comprise one or more mutations (e.g., a substitution mutations) to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively- WSGR Docket Number: 64828-710.601 charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 171. [0398] In some embodiments, the MspA nanopore can comprise a mutation (e.g., a substitution mutation) at position D90 to an asparagine residue, wherein the mutation comprises D90N and wherein the residue numbering corresponds to SEQ ID NO: 171. In some embodiments, the MspA nanopore can comprise a mutation (e.g., a substitution mutation) at position D91 to an asparagine residue, wherein the mutation comprises D91N and wherein the residue numbering corresponds to SEQ ID NO: 171. [0399] In some embodiments, a monomer of a nanopore described herein (e.g., a MspA nanopore) may comprise a mutation at a position 83, 88, 103, 105, or 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 171. In some embodiments, a nanopore (e.g., a MspA nanopore) can comprise one or more mutations of one or more monomers. The mutations of one or more monomers may comprise one or more mutations at position S103, I105, N108, T83, or L88, or any combination thereof, to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof. In some embodiments, nanopore (e.g., MspA nanopore) comprises at least one mutation (e.g., a substitution mutation) in one or more monomers, wherein the mutation comprises S103E, I105E, N108E, L88E, or T83E, or any combination thereof, and wherein the residue numbering corresponds to SEQ ID NO: 171. In some embodiments, a monomer of a nanopore (e.g., MspA nanopore) described herein may comprise one or more mutations at positions D90, D91, D93, A96, T83, L88, S103, I105, or N108, or any combination thereof. [0400] In some embodiments, a nanopore described herein may comprise any combination of monomers comprising an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128. In some embodiments, a nanopore described herein may comprise one or more monomers comprising an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128. In some embodiments, a nanopore described herein may comprise one or more WSGR Docket Number: 64828-710.601 monomers comprising an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128. In some embodiments, a nanopore described herein may comprise one or more monomers comprising at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in any one of SEQ ID NOs.: 8-26, 51, 52, 106-112, or 128. [0401] In some embodiments, a CsgG monomer may comprise a wildtype CsgG monomer. In some embodiments, a wildtype CsgG monomer may comprise an amino acid sequence as set forth in CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVPQSATAML VTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSIIGYESNVKSG GVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVG YTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES (SEQ ID NO.: 211). In some embodiments, a nanopore described herein can comprise one or more CsgG monomers. A CsgG monomer may comprise a mutant CsgG monomer. A CsgG monomer may comprise an amino acid sequence with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a CsgG monomer of a nanopore described here may comprise an amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a CsgG monomer may comprise at least about one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a nanopore described herein can comprise an engineered CsgG nanopore. [0402] The CsgG pore may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more monomers. The CsgG pore may be a CsgG/F pore. The CsgG/F pore may comprise a CsgG nanopore with a channel region and constriction region. The pore may further comprise a CsgF protein (e.g., a CsgF peptide adapter). In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position Y51, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position N55, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position F56, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position F48, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, a monomer of a CsgG nanopore may comprise a mutation at position F58, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. In some embodiments, the WSGR Docket Number: 64828-710.601 mutation at position Y51, N55, F56, F48, F58, or any combination thereof of the CsgG monomer can comprise one or more mutations (e.g., insertion mutations, deletion mutations, substitution mutations) to one or more aromatic residues (e.g., tryptophan (W) residue, phenylalanine (F) residue, tyrosine (Y) residue, or histidine (H) residue, or any combination thereof), one or more negatively-charged amino acid residues, one or more positively-charged amino acid residues, one or more neutral-charged residues, one or more acidic amino acid residues, one or more amidic amino acid residues (e.g., asparagine (N) residue and/or glutamine (Q) residue), or one or more sulfur-containing amino acid residues, or any combination thereof, where the residue numbering corresponds to the amino acid sequence as set forth in SEQ ID NO.: 211. [0403] In some embodiments, a pore can be coupled or fused to one or more accessory partner proteins that aids the binding and/or functioning of a protein translocase. In some embodiments, a translocase comprises an unfoldase, a helicase, an exonuclease, a protease translocase, or a topoisomerase. In some embodiments, a pore can be coupled to an inactive ClpP. In some embodiments, a pore can be coupled to an active ClpP. [0404] Also provided herein are nucleic acid molecules encoding any one of the pore proteins disclosed herein. Also provided herein are expression vectors comprising nucleic acid molecules disclosed herein. Also provided herein are host cells comprising expression vectors disclosed herein. Also provided herein are some methods of inserting a pore disclosed herein into a membrane. [0405] In some embodiments, a nanopore may comprises a steric or electrostatic obstruction. The steric or electrostatic obstruction may be added to a natural or a mutant variant of a biological nanopore. An adaptor can provide a steric or electrostatic obstruction. An adaptor can be a separate entity from the nanopore. An adaptor can provide an additional constriction zone, or an additional recognition element, or combinations thereof. An adaptor can be proteinaceous or chemical. An adaptor can comprise at least a portion of a molecule that couples to a nanopore and modifies a steric or electrostatic arrangement of a nanopore channel. [0406] An adaptor can be coupled to a nanopore. The coupling can be covalent or non-covalent. In some cases, the covalent coupling can be a covalent bond. In some instances, the covalent bound can be a polar covalent bond or a nonpolar covalent bond. In some cases, the noncovalent coupling can be a noncovalent bond. Non- limiting examples of noncovalent bond include hydrogen bonds, electrostatic interactions, van der Waals interactions, hydrophobic interactions, and cysteine bonds. In some cases, the adaptor can be coupled to the channel of the nanopore via a cysteine bond, a hydrogen bond, or an electrostatic interaction, or combinations thereof. In some cases, the adaptor can be coupled to the nanopore via a linker. Non-limiting examples of linkers include (GGGGS)3, (SG)n, (GGGGS)n, (Gly)8, (Gly)6, (EAAAK)3, (EAAAK)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, disulfide bond, or cysteine linkages, and any combination thereof. In some embodiments, a linker can comprise any combination of amino WSGR Docket Number: 64828-710.601 acids. In some cases, the amino acids can be canonical amino acids. In some cases, the amino acids can be non- natural amino acids. In some cases, the linker can comprise any combination of canonical amino acids and non- natural amino acids. In some cases, the linker can be ethylene glycol. In some cases, the linker can be polyethylene glycol.. In some cases, the linker can be a cysteine linkage. [0407] In some embodiments, the adaptor can be a separate entity from the nanopore channel. In some examples, the adaptor can be a protein coupled to the channel of the nanopore. In some cases, the adaptor may not be a portion of the amino acid sequence of the nanopore channel. In some cases, the adaptor may not modify the sequence of the nanopore channel. In some examples, the adaptor may not modify the amino acid residue sequence of the nanopore channel. [0408] An adaptor can be coupled to a subunit, a monomer, a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer, or a nonamer of a nanopore. [0409] In some embodiments, at least one adaptor can be coupled to a nanopore. In some embodiments, between about one adaptor to about 10 adaptors can be coupled to a nanopore. In some cases, at least about one adaptor, at least two about adaptors, at least about three adaptors, at least about four adaptors, at least about five adaptors, at least about six adaptors, at least about seven adaptors, at least about eight adaptors, at least about nine adaptors, or more can be coupled to a nanopore. In some cases, at most about nine adaptors, at most about eight adaptor, at most about seven adaptors, at most about six adaptors, at most about five adaptors, at most about four adaptors, at most about three adaptors, at most about two adaptors, at most about one adaptor, or less can be coupled to a nanopore. In some cases, about one adaptor, about two adaptors, about three adaptors, about four adaptors, about five adaptors, about six adaptors, about seven adaptors, about eight adaptors, or about nine adaptors can be coupled to a nanopore. [0410] An adaptor can comprise one or multiple proteinaceous subunits. In some cases, the adaptor protein can be a protein complex. In some cases, the protein complex can comprise one or more proteins (e.g., proteinaceous subunits). In some embodiments, the adaptor can comprise between about one to about 50 proteinaceous subunits. In some embodiments, the adaptor can comprise between about one to about five proteinaceous subunits, between about five to about 10 proteinaceous subunits, between about 10 to about 15 proteinaceous subunits, between about 15 to about 20 proteinaceous subunits, between about 20 to about 25 proteinaceous subunits, between about 25 to about 30 proteinaceous subunits, between about 30 to about 35 proteinaceous subunits, between about 35 to about 40 proteinaceous subunits, between about 40 to about 45 proteinaceous subunits, or between about 45 to about 50 proteinaceous subunits. In some cases, the adaptor can comprise at least about one proteinaceous subunit, at least about five proteinaceous subunits, at least about ten proteinaceous subunits, at least about 15 proteinaceous subunits, at least about 20 proteinaceous subunits, at least about 25 proteinaceous subunits, at least about 30 proteinaceous subunits, at least about 35 proteinaceous WSGR Docket Number: 64828-710.601 subunits, at least about 40 proteinaceous subunits, at least about 45 proteinaceous subunits, at least about 50 proteinaceous subunits, or more. In some cases, the adaptor can comprise at most about 50 proteinaceous subunits, at most about 45 proteinaceous subunits, at most about 40 proteinaceous subunits, at most about 35 proteinaceous subunits, at most about 30 proteinaceous subunits, at most about 25 proteinaceous subunits, at most about 20 proteinaceous subunits, at most about 15 proteinaceous subunits, at most about 10 proteinaceous subunits, at most about 5 proteinaceous subunits, at most about one proteinaceous subunit. [0411] An adaptor can provide a steric obstruction, or an electrostatic change, or combinations thereof. An adaptor can obstruct flow of an analyte through a channel of the nanopore. An adaptor can selectively obstruct flow through a nanopore. In some cases, an adaptor can obstruct flow through the nanopore by modifying a charge of a nanopore or a geometry of a nanopore. In some examples, the adaptor can obstruct flow of the analyte through the channel nanopore by decreasing the circumference of the channel. In some examples, the adaptor can obstruct flow of the analyte through the channel nanopore by causing the nanopore channel to have a net negative charge. An adaptor can narrow a portion of the channel of the nanopore so as to selectively obstruct larger molecules from passing through the nanopore channel by providing a steric barrier. The selectivity can be based at least in part on a charge characteristic of the adaptor. In some examples, the adaptor can prevent translocation of a nucleic acid analyte by causing the nanopore channel to have a net negative charge. In some cases, an adaptor can increase a positive net charge so as to decrease a flow of positively charged molecules, such as cations, by providing an electrostatic barrier. In some cases, an adaptor can increase a positive net charge so as to increase a flow of negatively charged molecules, such as anions, by creating attractive electrostatic interactions. In some cases, an adaptor can decrease a positive net charge as to increase a flow of positively charged molecule by providing an electrostatic barrier. In some cases, an adaptor can increase a negative net charge so as to decrease a flow of negatively charged molecules, such as anions or nucleic acid molecules, by providing an electrostatic barrier. In some cases, an adaptor can increase a negative net charge so as to increase a flow of positively charged molecules, such as cations, by creating attractive electrostatic interactions. In some cases, an adaptor can decrease a negative net charge so as to increase a flow of negatively charged molecules by providing an electrostatic barrier. A charge characteristic can be positive, negative, or neutral. [0412] In some cases, the adaptor can modify a net charge of the channel of the nanopore. In some cases, the adaptor can modify a net charge of the channel of the nanopore to be a positive net charge. In some examples, the adaptor can comprise surface exposed positively charge amino acid residues (e.g., lysine, arginine). In some cases, the adaptor can modify a net charge of the channel of the nanopore to be a negative net charge. In some examples, the adaptor can comprise surface exposed negatively charge amino acid residues (e.g., aspartic acid, glutamic acid). WSGR Docket Number: 64828-710.601 [0413] In some embodiments, the adaptor can extend at least a portion of a channel of a nanopore. In some embodiments, the adaptor can extend at least about one subunit of the nanopore. In some embodiments, the adaptor can extend at least about one subunit of the nanopore, at least about two subunits of the nanopore, at least about three subunits of the nanopore, at least about four subunits of the nanopore, at least about five subunits of the nanopore, at least about six subunits of the nanopore, at least about seven subunits of the nanopore, at least about eight subunits of the nanopore, or at least about nine subunits of the nanopore. In some cases, the adaptor can extend at most about nine subunits of the nanopore, at most about eight subunits of the nanopore, at most about seven subunits of the nanopore, at most about six subunits of the nanopore, at most about five subunits of the nanopore, at most about four subunits of the nanopore, at most about three subunits of the nanopore, at most about two subunits of the nanopore, at most about one subunits of the nanopore, or less. In some cases, the adaptor can extend about one subunit of the nanopore, about two subunits of the nanopore, about three subunits of the nanopore, about four subunits of the nanopore, about five subunits of the nanopore, about six subunits of the nanopore, about seven subunits of the nanopore, about eight subunits of the nanopore, or about nine subunits of the nanopore. [0414] An adaptor can constrict at least a portion of a channel of a nanopore. In some embodiments, the adaptor can decrease the circumference of the channel of the nanopore. In some embodiments, the adaptor can constrict at least about one subunit of the nanopore. In some embodiments, the adaptor can constrict at least about one subunit of the nanopore, at least about two subunits of the nanopore, at least about three subunits of the nanopore, at least about four subunits of the nanopore, at least about five subunits of the nanopore, at least about six subunits of the nanopore, at least about seven subunits of the nanopore, at least about eight subunits of the nanopore, or at least about nine subunits of the nanopore. In some cases, the adaptor can constrict at most about nine subunits of the nanopore, at most about eight subunits of the nanopore, at most about seven subunits of the nanopore, at most about six subunits of the nanopore, at most about five subunits of the nanopore, at most about four subunits of the nanopore, at most about three subunits of the nanopore, at most about two subunits of the nanopore, at most about one subunits of the nanopore, or less. In some cases, the adaptor can constrict about one subunit of the nanopore, about two subunits of the nanopore, about three subunits of the nanopore, about four subunits of the nanopore, about five subunits of the nanopore, about six subunits of the nanopore, about seven subunits of the nanopore, about eight subunits of the nanopore, or about nine subunits of the nanopore. [0415] In some embodiments, the adaptor can block a portion of the nanopore channel. In some cases, blocking a portion of the nanopore channel can reduce the ability of an analyte to move through the nanopore channel. In some cases, blocking a portion of the nanopore channel can reduce the ability of one or more salts to move through the nanopore channel. In some cases, blocking a portion of the nanopore channel can reduce the ability of one or more ions to move through the nanopore channel. In some cases, blocking a portion of the nanopore WSGR Docket Number: 64828-710.601 channel can reduce the ability of an analyte, one or more salts, and one or more ions to move through the nanopore channel. [0416] In some embodiments, the adaptor can be a proteinaceous adaptor. In some cases, the proteinaceous adaptor can be a protein. In some cases, the proteinaceous adaptor can be a monomer. In some cases, the proteinaceous adaptor can comprise more than one protein subunits. In some cases, the proteinaceous adaptor can comprise at least about 1 protein subunit, at least about 2 protein subunits, at least about 3 protein subunits, at least about 4 protein subunits, at least about 5 protein subunits, at least about 6 protein subunits, at least about 7 protein subunits, at least about 8 protein subunits, at least about 9 protein subunits, at least about 10 protein subunits, at least about 12 protein subunits, at least about 15 protein subunits, at least about 18 protein subunits, at least about 20 protein subunits, at least about 25 protein subunits, at least about 30 protein subunits, at least about 35 protein subunits, at least about 40 protein subunits, at least about 45 protein subunits, at least about 50 protein subunits, or greater than about 50 protein subunits. In some cases, the proteinaceous adaptor can comprise at most about 50 protein subunits, at most about 45 protein subunits, at most about 40 protein subunits, at most about 35 protein subunits, at most about 30 protein subunits, at most about 25 protein subunits, at most about 20 protein subunits, at most about 18 protein subunits, at most about 15 protein subunits, at most about 12 protein subunits, at most about 10 protein subunits, at most about 9 protein subunits, at most about 8 protein subunits, at most about 7 protein subunits, at most about 6 protein subunits, at most about 5 protein subunits, at most about 4 protein subunits, at most about 3 protein subunits, at most about 2 protein subunits, at most about 1 protein subunit, or less than about 1 protein subunit. [0417] In some embodiments, the adaptor can be at least about 0.2 kDa in size. In some cases, the adaptor can be between about 0.2 kDa to about 100 kDa in size. In some cases, the adaptor can be between about 0.2 kDa to about 1 kDa, between about 1 kDa to about 5 kDa, between about 5 kDa to about 10 kDa, between about 10 kDa to about 15 kDa, between about 15 kDa to about 20 kDa, between about 20 kDa to about 25 kDa, between about 25 kDa to about 30 kDa, between about 30 kDa to about 35 kDa, between about 35 kDa to about 40 kDa, between about 40 kDa to about 45 kDa, between about 45 kDa to about 50 kDa, between about 50 kDa to about 55 kDa, between about 55 kDa to about 60 kDa, between about 60 kDa to about 65 kDa, between about 65 kDa to about 70 kDa, between about 70 kDa to about 75 kDa, between about 75 kDa to about 80 kDa, between about 80 kDa to about 85 kDa, between about 85 kDa to about 90 kDa, between about 90 kDa to about 95 kDa, or between about 95 kDa to about 100 kDa in size. [0418] In some embodiments, the proteinaceous adaptor can comprise one or more protein subunits. In some cases, the one or more protein subunits can be the same. In some cases, the one or more protein subunits can be different. In some cases, the one or more different subunits can be from different proteins. WSGR Docket Number: 64828-710.601 [0419] A proteinaceous adaptor can comprise a CsgF subunit, a CsgF subunit truncation, or CsgF subunit homologs, or any combination thereof. In some cases, the proteinaceous adaptor can be a monomeric adaptor. In some cases, the proteinaceous adaptor can be an oligomeric adaptor. In some cases, the proteinaceous oligomeric adaptor can comprise one or more protein subunits. In some cases, the one or more protein subunits can be identical. In some cases, the one or more protein subunits can be different. In some cases, the one or more different protein subunits can be derived from different proteins. [0420] In some embodiments, the adaptor can be a chemical adaptor. In some cases, the chemical adaptor can comprise a non-protein based molecule. In some cases, the chemical adaptor can comprise a non-peptide based molecule. In some cases, a non-peptide based molecule can comprise cyclodextrin, cucurbituril, or a cyclic peptide, or any combination thereof. In some embodiments, the chemical adaptor can comprise a macrocyclic molecule. In some cases, the macrocyclic molecule can comprise crown ethers, calixarenes, porphyrins, cyclosporines, cyclems, or cyclams, or any combination thereof. A chemical adaptor can comprise a cyclodextrin, a cyclic peptide, a cucurbituril, crown ethers, calixarenes, porphyrins, cyclosporines, cyclems, or cyclams, or any combination thereof. [0421] Additional details of the pores (e.g., nanopores) described herein are disclosed in the International Patent Application No. PCT/NL2023/050568, International Patent Application No. PCT/NL2023/050570, International Patent Application No. PCT/NL2023/050633, and Greek Patent Application No. 20240100311, each of which is entirely incorporated herein by reference. TRANSLOCASE [0422] In some aspects, the present disclosure provides translocases for translocating analytes. [0423] In some embodiments, a pore comprises a molecular motor. In some embodiments, a molecular motor comprises a translocase. In some embodiments, a molecular motor comprises a protein translocase. In some embodiments, a pore comprises a protein translocase. In some embodiments, a protein translocase can be active or inactive. In some embodiments, a pore comprises a translocase-analyte complex on a first side (e.g., cis side). In some embodiments, a pore comprises a translocase- analyte complex on a second side (e.g., trans side). In some embodiments, a pore can be coupled to a translocase. In some embodiments, a pore may not be coupled to a translocase. In some embodiments, an electro-osmotic force pulls a translocase to a pore. In some embodiments, an electro-osmotic force pulls a translocase:analyte complex to a pore. In some embodiments, an electro-osmotic force pulls a translocase to a pore in a direction counter to an electrophoretic force. In some embodiments, a translocase comprises a protease domain. In some embodiments, a proteolytic activity of the protease components can be disabled. In some embodiments, a translocase does not comprise a protease domain. In some embodiments, a proteolytic activity of the protease components can be active or inactive. In some WSGR Docket Number: 64828-710.601 embodiments, a translocase moves an analyte through a pore. In some embodiments, a molecular motor can be ATP or NTP driven. In some embodiments, a molecular motor’s rate of translocation can be ATP-dependent or NTP-dependent. In some embodiments, a translocase increases a rate of translocation through a pore relative to translocation by an electrophoretic force alone. In some embodiments, a translocase increases a rate of translocation through a pore relative to translocation by an electro-osmotic force alone. In some embodiments, a translocase decreases a rate of translocation through a pore relative to translocation by an electrophoretic force alone. In some embodiments, a translocase decreases a rate of translocation through a pore relative to translocation by an electro-osmotic force alone. In some embodiments, a translocase increases a rate of translocation through a pore relative to translocation by an electro-osmotic force in combination with an electrophoretic force. In some embodiments, a translocase decreases a rate of translocation through a pore relative to translocation by an electro-osmotic force in combination with an electrophoretic force. [0424] Provided herein are some methods of forming a complex between a translocase and an analyte. The ratio of translocase to an analyte may be about 1 to 1 (translocase to analyte). The ratio of translocase to an analyte may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to 1 (translocase to analyte). The ratio of translocase to an analyte may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 to 1 (translocase to analyte). The ratio of translocase to an analyte may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to 1 (analyte to translocase). The ratio of translocase to an analyte may be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to 1 (analyte to translocase). [0425] As shown in FIG.2, a translocase can assist in translocating an analyte (e.g., a mixture of analytes, for example mixed amino-acid composition proteins) through a pore. In some embodiments, in a system comprising a nanopore in a membrane, a polymer analyte can be translocated through the nanopore from the cis compartment to the trans compartment with the aid of a translocase motor that progresses along the polymer analyte in the direction of the subset arrow (moving away from termini PA towards termini PB of polymer analyte). Depending on the charge composition of the portion of the polymer analyte within or near the nanopore central channel at any one time, and on the direction of the applied voltage, the net direction of the EPF acting on the polymer may be either cis-to-trans or trans-to-cis or effectively zero as the polymer progresses through the nanopore (as indicated by the dotted arrow labelled EPF). [0426] In some embodiments, a pore comprises a protein translocase on a first side (e.g., cis side). In some embodiments, a pore comprises a protein translocase on a second side (e.g., trans side). In some embodiments, a pore comprises a protein translocase on a first side (e.g., cis side) and/or a second side (e.g., trans side). In some embodiments, a pore comprises a protein translocase and its accessory protease on a first side (e.g., cis side) and/or a second side (e.g., trans side). In some embodiments, an analyte can be added to a first side (e.g., cis side) to be fed through a pore via a translocase on the first side (e.g., cis side), and a translocase on the WSGR Docket Number: 64828-710.601 second side (e.g., trans side) aids the progression of the analyte through the pore and/or prevents the folding of protein. [0427] In some embodiments, a translocase binds an analyte added to a first side (e.g., cis side) after it has been captured and partially translocated through a pore cis-to-trans, then pulls the analyte through the pore from cis-to-trans. In some embodiments, a translocase added to a second side (e.g., trans side) binds an analyte added to a second side (e.g., trans side) after it has been captured and partially translocated through a pore trans-to-cis, then pulls the analyte through the pore from trans-to-cis. In some embodiments, one or more unfoldases, translocases, unfoldase domains, or translocase domains, or any combination thereof can be positioned proximal to the nanopore. In some embodiments, one or more unfoldases, translocases, unfoldase domains, or translocase domains, or any combination thereof can be positioned proximal to the nanopore upon a binding with an analyte (e.g., a polypeptide, peptide, or protein, or fragments thereof, or any combination thereof). In some embodiments, a motor protein described herein may comprise at least a portion of a translocase (e.g., a translocase domain). A translocase domain can be a portion of a larger protein complex. [0428] In some embodiments, a translocase binds an analyte and unwinds the analyte prior to translocation through a pore. In some embodiments, more than one translocase can bind to an analyte. In some embodiments, a translocase binds an analyte and an electro-osmotic force pulls on the analyte as the analyte translocates through the pore. In some embodiments, the analyte transmits the electro-osmotic force up to the bound translocase. In some embodiments, the transmitted force keeps the translocase adjacent to the pore during translocation of the analyte. In some embodiments, the translocase unfolds a structure it encounters. In some embodiments, unfolding a structure can increase capture of the analyte by the pore. In some embodiments, the translocase modulates a movement of the analyte into the pore such that analyte-dependent changes in ionic current, conductivity, or impedance can be measured and/or characterized. In some embodiments, a translocase pulls an analyte in the cis to trans direction. In some embodiments, a translocase pulls an analyte in a trans to cis direction. [0429] In some embodiments, a translocase can be coupled to an opening of a pore. In some embodiments, a plurality of translocases can be coupled to an opening of a pore. In some embodiments, a central cavity of a translocase can be aligned with an entrance to a pore. In some embodiments, a translocase can be genetically fused to a protomer of a pore. In some embodiments, a translocase can be fused to a protomer of a pore via chemical attachment (e.g. via cysteines), enzymatic attachment, or via hybridization of complementary DNA tags that may be attached to the top of the pure and/or the translocase. [0430] In some embodiments, a translocase can be bound to an analyte prior to contacting with a pore, which may be referred to as “preloading” herein in appropriate contexts. In one embodiment one or more translocases are bound and/or loaded onto the target analyte (e.g., target protein) prior to addition to the nanopore system of WSGR Docket Number: 64828-710.601 the invention. The preloading can be performed under conditions that favor high efficiency of binding and/or loading, and/or optimal translocase movement along the target protein (whether modified or unmodified). For example, in one embodiment the translocase and one or more target proteins can be incubated at relatively higher concentration, and then diluted when added to the nanopore system of the invention. The preloading may be performed in conditions closer to the optimal binding conditions than employed in the nanopore system of the invention. For example, preloading can be performed in solutions that are closer to the optimal salt concentration and salt types, the optimal pH, the optimal temperature, and in the presence of optimal co-factors (e.g. NTP, or M2+ ions, or any combination thereof). Preloading may also be performed in combination with accessory cofactors that aid in binding. In other embodiments the preloading can be performed under conditions that enable multiple translocases to bind to a single target protein. This can be advantageous to provide better controlled movement of the analyte (e.g., the non-nucleic acid based polymer analyte) through the nanopore in the methods of the invention, and/or to improve the ability of the translocase to progress through more problematic regions of proteins (e.g., regions of very stable structure, regions with bulky modifications of the side chains, or regions of low traction such those composed of a high percentage of glycines). In one aspect, preloading to load multiple translocases can be performed under relatively high ratio of translocase to target protein. This may be performed in combination with target proteins that are modified to optimally bind multiple translocases, for example through attachment of sufficiently long leaders to the terminus(i) of the target proteins. For example, leaders can be designed to have sufficiently long binding and/or stall motifs to accommodate the footprint of the multiple translocases and/or stall the multiple translocases respectively. [0431] In another embodiment the translocases can be topologically closed around the leader and/or target protein to reduce or prevent unbinding, for example especially when the translocase:target-protein can be added to nanopore systems that employ conditions relatively unfavorable to binding (e.g. high salt concentration). Alternatively, oligomers can be connected by covalent coupling, e.g. by cross reaction between suitably placed cysteines between subunits. In some embodiments, genetic fusion and chemical coupling can be used in combination. [0432] In some embodiments, the preloading solution can be dilution prior to adding to one or more analytes. In some cases, the preloading solution can be diluted in solvent. In some cases, the preloading solution:solvent dilution can comprise from about 1:1 to about 1:100. In some cases, the preloading solution:solvent dilution can be at least about 1:1, at least about 1:2, at least about 1:5, at least about 1:10, at least about 1:20, at least about 1:25, at least about 1:30, at least about 1:40, at least about 1:50, at least about 1:60, at least about 1:70, at least about 1:75, at least about 1:80, at least about 1:90, at least about 1:100, or more than 1:100. In some cases, the preloading solution:solvent dilution can be at most about 1:100, at most about 1:90, at most about 1:80, at most about 1:75, at most about 1:70, at most about 1:60, at most about 1:50, at most about 1:40, at most about WSGR Docket Number: 64828-710.601 1:30, at most about 1:25, at most about 1:20, at most about 1:10, at most about 1:5, at most about 1:2, at most about 1:1, or less than 1:1. In some cases, the preloading solution:solvent dilution can comprise about 1:1, about 1:2, about 1:5, about 1:10, about 1:20, about 1:25, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:75, about 1:80, about 1:90, or about 1:100. [0433] In some embodiments, the preloading solution can comprise one or more translocases. In some cases, an analyte can be contacted with the preloading solution to form an analyte-translocase complex. In some cases, the analyte-translocase complex can be added to the nanopore system. In some cases, the analyte-translocase complex can be added to the first side (e.g., cis side) of the nanopore system. In some cases, the analyte- translocase complex can be added to the second side (e.g., trans side) of the nanopore system. In some embodiments, an EOF of a nanopore system described herein may maintain a translocase at a nanopore. A first side to second side electro-osmotic force can maintain the translocase of a complex at a first side entrance of a channel of a nanopore. In some embodiments, an EOF may maintain the translocase at the first entrance of the nanopore with feeding one or more analytes with an EOF (e.g., translocating one or more analytes in a same direction as the EOF). In some embodiments, an EOF may maintain the translocase at the first entrance of the nanopore with feeding one or more analytes against an EOF (e.g., translocating one or more analytes in an opposite direction as the EOF). [0434] In some embodiments, the preloading solution can further comprise one or more factors (e.g., cofactors). In some embodiments, the one or more cofactors can comprise NTP, M2+, NblA/B, ClpS, ClpF, Hsp10, Hsp60, calnexin, ERp29, ERp57, polyethylene glycol, dextran, Ficoll, iron manganese, cobalt, copper, penicillamine, trientine, sodium calcium edetate, ethylenediaminetetraacetic acid, and any combinations thereof. In some embodiments, the preloading solution can have between about 1 to about 100 cofactors. In some cases, the preloading solution can have at least about 1 cofactor, at least about 10 cofactors, at least about 20 cofactors, at least about 30 cofactors, at least about 40 cofactors, at least about 50 cofactors, at least about 60 cofactors, at least about 70 cofactors, at least about 80 cofactors, at least about 90 cofactors, at least about 100 cofactors, or more. In some cases, the preloading solution can have at most about 100 cofactors, at most about 90 cofactors, at most about 80 cofactors, at most about 70 cofactors, at most about 60 cofactors, at most about 50 cofactors, at most about 40 cofactors, at most about 30 cofactors, at most about 20 cofactors, at most about 10 cofactors, at most about one cofactor, or less. In some cases, the preloading solution can have about one cofactor, about 10 cofactors, about 20 cofactors, about 30 cofactors, about 40 cofactors, about 50 cofactors, about 60 cofactors, about 70 cofactors, about 80 cofactors, about 90 cofactors, or about 100 cofactors. [0435] In some embodiments, the one or more cofactors or the one or more chemicals enhance binding of the analyte to the one or more translocases. In some cases, the one or more chemicals can comprise chaperone molecules. In some cases, the chaperone molecules can comprise heat shock proteins. In some cases, the heat WSGR Docket Number: 64828-710.601 shock proteins can comprise Hsp10, Hsp 60, Hsp70, Hsp40, Hsp90, or Hsp100, or any combination thereof. In some cases, the chaperone molecules can comprise phage growth defect, overcome by mutation in page gene E, large subunit protein (GroEL). In some cases, the chaperone molecules can comprise Hsp10, Hsp60, Hsp70, Hsp40, Hsp90, Hsp100, GroEL, GRP78/BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase, peptidyl prolyl cis-trans isomerase, prolyl isomerase, or ERp57, or any combination thereof. In some cases, the chaperone molecule can keep the analyte in a folded structure. In some cases, the one or more chemicals can comprise crowding agents. In some cases, the crowding agents can reduce the volume of solvent available for other components of the preloading solution. In some cases, the crowding agents can stabilize analytes in the preloading solution. In some cases, the crowding agents can comprise polyethylene glycol, dextran, or Ficoll, or any combination thereof. In some cases, the one or more chemicals can comprise one or more metal cofactors. In some cases, the one or more metal cofactors can comprise iron, magnesium, manganese, cobalt, copper, zinc, or molybdenum, or any combination thereof. In some cases, the one or more chemicals can comprise one or more chelating agents. In some cases, the one or more chelating agents can comprise deferoxamine, deferiprone, deferasirox, dimercapto succinic acid (DMSA), penicillamine, trientine, sodium calcium edetate, or ethylenediaminetetraacetic acid, or any combination thereof. In some embodiments, the one or more chemicals can comprise Hsp10, Hsp60, Hsp70, Hsp40, Hsp90, Hsp100, GroEL, GRP78/BiP, GRP94, GRP170, calnexin, calreticulin, HSP47, ERp29, protein disulfide isomerase, peptidyl prolyl cis-trans isomerase, prolyl isomerase, ERp57, nucleotide triphosphates, glycine betaine, glycerol, dithiothreitol (DTT), iron, magnesium, manganese, cobalt, copper, zinc, molybdenum, Tris(2-carboxyethyl)phosphine (TCEP), glutathione, polyethylene glycol, dextran, Ficoll, deferoxamine, deferiprone, deferasirox, dimercapto succinic acid (DMSA), penicillamine, trientine, sodium calcium edetate, or ethylenediaminetetraacetic acid, or any combination thereof. [0436] In some embodiments, the one or more cofactors or the one or more chemicals increases binding of the analyte to the one or more translocases by between about 0.1% to about 500% compared to the binding of the analyte to the one or more translocases without the one or more cofactors or the one or more chemicals. In some embodiments, the one or more cofactors or the one or more chemicals increases binding of the analyte to the one or more translocases by between about 0.1% to about 1%, between about 10% to about 100%, or between about 100% to about 500%. In some embodiments, the one or more cofactors or the one or more chemicals increases binding of the analyte to the one or more translocases by at least about 0.1%, at least about 1%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or more compared to the binding of the analyte to the one or more translocases without the one or more cofactors or the one or more chemicals. In some WSGR Docket Number: 64828-710.601 embodiments, the one or more chemicals increases binding of the analyte to the one or more translocases by at most about 500%, at most about 400%, at most about 300%, at most about 200%, at most about 100%, at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, at most about 1%, at most about 0.1%, or less compared to the binding of the analyte to the one or more translocases without the one or more cofactors or the one or more chemicals. [0437] In some embodiments, the preloading solution may further comprise a solvent. The solvent can comprise water, phosphate buffer solution (PBS), Tris buffer, Tris-HCL, Tricine buffer, Bicarbonate buffer, MOPS buffer, Bis-tris methan buffer, Bicine buffer, HEPES buffer, MES buffer, or CAPS, or any combination thereof. [0438] In some embodiments, the preloading solution can be introduced to the analyte prior to introduction of the analyte to the nanopore system. In some cases, the preloading solution and the analyte can be combined together in a container separate from the nanopore system. In some cases, the container can be a microcentrifuge tube, a centrifuge tube, a test tube, a beaker, a flask, a pipette, or a culture tube, or any combination thereof. [0439] In some embodiments, the preloading solution and the analyte can be combined together to form a translocase-analyte complex. In some cases, the translocase-analyte complex can be formed in a container separate from the nanopore system. In some cases, once the translocase-analyte complex can be formed, the translocase-analyte complex can be added to the nanopore system. [0440] In some embodiments, the translocase-analyte complex can be added to the first side (e.g., cis side) of the nanopore system. In some embodiments, the translocase-analyte complex can be added to the second side (e.g., trans side) of the nanopore system. In some embodiments, the translocase-analyte complex can be added to the first side (e.g., cis side) and the second side (e.g., trans side) of the nanopore system. In some cases, the translocase-analyte complex may not be added to the first side (e.g., cis side) of the nanopore system. In some cases, the translocase-analyte complex may not be added to the second side (e.g., trans side) of the nanopore system. In some cases, the translocase-analyte complex can be added to the first side (e.g., cis side) of the nanopore system and may not be added to the second side (e.g., trans side) of the nanopore system. In some instances, the translocase-analyte complex can be added to the second side (e.g., trans side) of the nanopore system and may not be added to the first side (e.g., cis side) of the nanopore system. [0441] In some embodiments, the preloading solution can comprise one or more translocases. In some embodiments, the preloading solution can comprise one or more leader constructs. In some embodiments, the preloading solution can comprise one or more analytes. In some cases, the preloading solution can comprise one or more translocases and one or more leader constructs. In some cases, the preloading solution can comprise one or more translocases and one or more analytes. In some cases, the preloading solution can comprise one or WSGR Docket Number: 64828-710.601 more leader constructs and one or more analytes. In some cases, the preloading solution can comprise one or more translocases, one or more leader constructs, and one or more analytes [0442] In some embodiments, preloading can be performed under conditions that favor high efficiency of binding and/or loading, and/or optimal translocase movement along an analyte. In some embodiments, a translocase and an analyte may be incubated at a higher concentration, and then diluted to a lower concentration when being contacted with a pore. In some embodiments, preloading can be performed in solutions that may be closer to the optimal salt concentration and salt types, the optimal pH, the optimal temperature, and/or in the presence of optimal co-factors (e.g. NTP, or M2+ ions, or any combination thereof) that favor binding between a translocase and an analyte. In some embodiments, preloading can be performed in combination with an accessory cofactor that aids in binding. In some embodiments, an accessory cofactor comprises analyte adaptors, e.g., NblA/B, ClpS, ClpF. In some embodiments, an accessory cofactor comprises engineered binding cofactors such as ones derived from antibodies, nanobodies, or affimers. [0443] In some embodiments, preloading can be performed under conditions sufficient for multiple translocases to bind to a single analyte. In some embodiments, preloading with multiple translocases can be performed under a higher ratio of translocase to analyte. In some embodiments, preloading with multiple translocases can be performed in combination with an analyte that can be configured to bind multiple translocases, for example, through attachment of one or more leaders to a terminus of the analyte. [0444] In some embodiments, one or more translocases can be topologically closed around a leader and/or an analyte to reduce or prevent unbinding. In some embodiments, one or more translocases and an analyte can be added to a solution, to contact with a pore, that comprises conditions unfavorable to binding. In some embodiments, multiple units of translocases can be fused together by genetic fusion. In some embodiments, multiple units of translocases can be fused together by covalent coupling. [0445] In some embodiments, a translocase can be a member of the AAA+ superfamily. In some embodiments, the protein translocase comprises an NTP-driven unfoldase. In some embodiments, the protein translocase comprises an ATP-driven unfoldase. The term “translocase” and “unfoldase” may be used interchangeably. An unfoldase may be used to control a speed of an analyte through a pore. An unfoldase may be used to increase a speed of an analyte through a pore. An unfoldase may be used to decrease a speed of an analyte through a pore. In some embodiments, a translocase comprises Caseinolytic mitochondrial matrix peptidase chaperone subunit X (ClpX), Caseinolytic mitochondrial matrix peptidase chaperone subunit A (ClpA), proteasome-activating nucleotidase (PAN), LON, Valosin-containing protein-like ATPase of Thermoplasma acidophilium (VAT), AMA, 854, membrane-bound AAA (MBA), small archaeal ubiquitin-like modifier protein (SAMP), Caseinolytic mitochondrial matrix peptidase chaperone subunit C (ClpC), Caseinolytic mitochondrial matrix peptidase chaperone subunit E (ClpE), HsIU, Caseinolytic mitochondrial matrix peptidase chaperone subunit WSGR Docket Number: 64828-710.601 Y (ClpY), LonA, LonB, FtsH, Mpa, Cdc48-like protein of actinobacteria (Cpa), Msp1, SecA, or fragments thereof, or functional homologs thereof, or functional orthologs thereof, or functional paralogs thereof. In some embodiments, a translocase comprises a prokaryotic AAA+ unfoldase ClpX. For example, ClpX can unfold analytes by ATP-driven translocation of a polypeptide through a central pore of its hexameric assembly. In complex with the ClpP peptidase, ClpX can carry out protein degradation by translocating unfolded analytes directly into the ClpP proteolytic chamber. [0446] In some embodiments, a translocase comprises Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT). In some embodiments, a translocase comprises a proteasome-activating nucleotidase (PAN). In some embodiments, a translocase comprises PAN of Methanococcus jannaschii, which can be a complex of relative molecular mass 650,000 that can be homologous to the ATPases in the eukaryotic 26S proteasome. In some embodiments, a translocase comprises AMA, an AAA protein from Archaeoglobus and methanogenic archaea. In some embodiments, a translocase comprises the open reading frame number 854 in the M. mazei genome. In some embodiments, a translocase comprises MBA (membrane-bound AAA) or SAMPs (small archaeal modifier proteins). In some embodiments, a translocase comprises ClpA, a member of the two-domain AAA ATPases, from Escherichia coli. In some embodiments, a translocase comprises ClpC, an AAA protein from Staphylococcus Aureus. In some embodiments, a translocase comprises ClpE, a member of the two-domain AAA ATPases from Bacillus subtilis. In some embodiments, a translocase comprises HslU/ClpY, an AAA protein from Escherichia coli. In some embodiments, a translocase comprises Lon, a protease from Escherichia coli containing an AAA ATPase domain. In some embodiments, a translocase comprises FtsH, a membrane bound protease from Escherichia coli containing an AAA ATPase domain. In some embodiments, a translocase comprises ARC/Mpa, an AAA ATPase from Mycobacterium tuberculosis. In some embodiments, a translocase comprises Msp1, a membrane associated AAA ATPase from Saccharomyces cerevisiae. In some embodiments, a translocase comprises CDC48, an AAA ATPase from Saccharomyces cerevisiae. In some embodiments, a translocase comprises Cpa, a CDC48 homologue from actinobacteria. In some embodiments, a translocase comprises SecA, a protein translocase from Escherichia coli. [0447] In some embodiments, a pore comprises a translocase. In some embodiments, a translocase comprises Thermoplasma VCP-like ATPase. In some embodiments, a translocase comprises a proteasome-activating nucleotidase (PAN). In some embodiments, a translocase comprises AMA, an AAA protein from Archaeoglobus and methanogenic archaea. [0448] In some embodiments, a translocase comprises an open reading frame number 854 in the M. mazei genome. In some embodiments, a translocase comprises MBA (membrane-bound AAA) or SAMPs (small WSGR Docket Number: 64828-710.601 archaeal modifier proteins). In some embodiments, a translocase comprises helicases (e.g. gp4), exonucleases (lambda exonuclease), proteases translocases (e.g. Ftsk), or topoisomerases (e.g. topoisomerase II). [0449] In some embodiments, a translocase can translocate a polypeptide in the N-to-C direction, the C-to-N direction, or both. In some embodiments, a translocase may translate a polypeptide in the C-to-N or N-to-C direction. In some embodiments, a translocase binds to the N-terminus or the C-terminus of a polypeptide. [0450] In some embodiments, a translocase may translocate a biopolymer with a step size between about 0.5 amino acids per step to about 50 amino acids per step. In some embodiments, a translocase may translocate a biopolymer with a step size between about 0.5 amino acids per step to about 1 amino acid per step, about 0.5 amino acids per step to about 2 amino acids per step, about 0.5 amino acids per step to about 3 amino acids per step, about 0.5 amino acids per step to about 4 amino acids per step, about 0.5 amino acids per step to about 5 amino acids per step, about 0.5 amino acids per step to about 10 amino acids per step, about 0.5 amino acids per step to about 15 amino acids per step, about 0.5 amino acids per step to about 20 amino acids per step, about 0.5 amino acids per step to about 30 amino acids per step, about 0.5 amino acids per step to about 40 amino acids per step, about 0.5 amino acids per step to about 50 amino acids per step, about 1 amino acid per step to about 2 amino acids per step, about 1 amino acid per step to about 3 amino acids per step, about 1 amino acid per step to about 4 amino acids per step, about 1 amino acid per step to about 5 amino acids per step, about 1 amino acid per step to about 10 amino acids per step, about 1 amino acid per step to about 15 amino acids per step, about 1 amino acid per step to about 20 amino acids per step, about 1 amino acid per step to about 30 amino acids per step, about 1 amino acid per step to about 40 amino acids per step, about 1 amino acid per step to about 50 amino acids per step, about 2 amino acids per step to about 3 amino acids per step, about 2 amino acids per step to about 4 amino acids per step, about 2 amino acids per step to about 5 amino acids per step, about 2 amino acids per step to about 10 amino acids per step, about 2 amino acids per step to about 15 amino acids per step, about 2 amino acids per step to about 20 amino acids per step, about 2 amino acids per step to about 30 amino acids per step, about 2 amino acids per step to about 40 amino acids per step, about 2 amino acids per step to about 50 amino acids per step, about 3 amino acids per step to about 4 amino acids per step, about 3 amino acids per step to about 5 amino acids per step, about 3 amino acids per step to about 10 amino acids per step, about 3 amino acids per step to about 15 amino acids per step, about 3 amino acids per step to about 20 amino acids per step, about 3 amino acids per step to about 30 amino acids per step, about 3 amino acids per step to about 40 amino acids per step, about 3 amino acids per step to about 50 amino acids per step, about 4 amino acids per step to about 5 amino acids per step, about 4 amino acids per step to about 10 amino acids per step, about 4 amino acids per step to about 15 amino acids per step, about 4 amino acids per step to about 20 amino acids per step, about 4 amino acids per step to about 30 amino acids per step, about 4 amino acids per step to about 40 amino acids per step, about 4 amino acids per step to about 50 amino acids per step, WSGR Docket Number: 64828-710.601 about 5 amino acids per step to about 10 amino acids per step, about 5 amino acids per step to about 15 amino acids per step, about 5 amino acids per step to about 20 amino acids per step, about 5 amino acids per step to about 30 amino acids per step, about 5 amino acids per step to about 40 amino acids per step, about 5 amino acids per step to about 50 amino acids per step, about 10 amino acids per step to about 15 amino acids per step, about 10 amino acids per step to about 20 amino acids per step, about 10 amino acids per step to about 30 amino acids per step, about 10 amino acids per step to about 40 amino acids per step, about 10 amino acids per step to about 50 amino acids per step, about 15 amino acids per step to about 20 amino acids per step, about 15 amino acids per step to about 30 amino acids per step, about 15 amino acids per step to about 40 amino acids per step, about 15 amino acids per step to about 50 amino acids per step, about 20 amino acids per step to about 30 amino acids per step, about 20 amino acids per step to about 40 amino acids per step, about 20 amino acids per step to about 50 amino acids per step, about 30 amino acids per step to about 40 amino acids per step, about 30 amino acids per step to about 50 amino acids per step, or about 40 amino acids per step to about 50 amino acids per step. [0451] In some embodiments, a translocase may translocate a biopolymer with a step size of at least about 0.5 nanometers per step (nm/step), 1 nm/step, 2 nm/step, 3 nm/step, 4 nm/step, 5 nm/step, 10 nm/step, 20 nm/step, 30 nm/step, or greater than about 30 nm/step. In some embodiments, a translocase may translocate a biopolymer with a step size of at most about 30 nm/step, 20 nm/step, 10 nm/step, 5 nm/step, 4 nm/step, 3 nm/step, 2 nm/step, 1 nm/step, 0.5 nm/step, or less than about 0.5 nm/step. [0452] In some embodiments, a translocase may translocate a biopolymer with a step size between about 0.5 nanometers per step (nm/step) to about 50 nm/step. In some embodiments, a translocase may translocate a biopolymer with a step size between about 0.5 nm/step to about 1 nanometer per step, about 0.5 nm/step to about 2 nm/step, about 0.5 nm/step to about 3 nm/step, about 0.5 nm/step to about 4 nm/step, about 0.5 nm/step to about 5 nm/step, about 0.5 nm/step to about 10 nm/step, about 0.5 nm/step to about 15 nm/step, about 0.5 nm/step to about 20 nm/step, about 0.5 nm/step to about 30 nm/step, about 0.5 nm/step to about 40 nm/step, about 0.5 nm/step to about 50 nm/step, about 1 nanometer per step to about 2 nm/step, about 1 nanometer per step to about 3 nm/step, about 1 nanometer per step to about 4 nm/step, about 1 nanometer per step to about 5 nm/step, about 1 nanometer per step to about 10 nm/step, about 1 nanometer per step to about 15 nm/step, about 1 nanometer per step to about 20 nm/step, about 1 nanometer per step to about 30 nm/step, about 1 nanometer per step to about 40 nm/step, about 1 nanometer per step to about 50 nm/step, about 2 nm/step to about 3 nm/step, about 2 nm/step to about 4 nm/step, about 2 nm/step to about 5 nm/step, about 2 nm/step to about 10 nm/step, about 2 nm/step to about 15 nm/step, about 2 nm/step to about 20 nm/step, about 2 nm/step to about 30 nm/step, about 2 nm/step to about 40 nm/step, about 2 nm/step to about 50 nm/step, about 3 nm/step to about 4 nm/step, about 3 nm/step to about 5 nm/step, about 3 nm/step to about 10 nm/step, about 3 nm/step to about WSGR Docket Number: 64828-710.601 15 nm/step, about 3 nm/step to about 20 nm/step, about 3 nm/step to about 30 nm/step, about 3 nm/step to about 40 nm/step, about 3 nm/step to about 50 nm/step, about 4 nm/step to about 5 nm/step, about 4 nm/step to about 10 nm/step, about 4 nm/step to about 15 nm/step, about 4 nm/step to about 20 nm/step, about 4 nm/step to about 30 nm/step, about 4 nm/step to about 40 nm/step, about 4 nm/step to about 50 nm/step, about 5 nm/step to about 10 nm/step, about 5 nm/step to about 15 nm/step, about 5 nm/step to about 20 nm/step, about 5 nm/step to about 30 nm/step, about 5 nm/step to about 40 nm/step, about 5 nm/step to about 50 nm/step, about 10 nm/step to about 15 nm/step, about 10 nm/step to about 20 nm/step, about 10 nm/step to about 30 nm/step, about 10 nm/step to about 40 nm/step, about 10 nm/step to about 50 nm/step, about 15 nm/step to about 20 nm/step, about 15 nm/step to about 30 nm/step, about 15 nm/step to about 40 nm/step, about 15 nm/step to about 50 nm/step, about 20 nm/step to about 30 nm/step, about 20 nm/step to about 40 nm/step, about 20 nm/step to about 50 nm/step, about 30 nm/step to about 40 nm/step, about 30 nm/step to about 50 nm/step, or about 40 nm/step to about 50 nm/step. [0453] In some embodiments, an analyte (e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof) may move through (e.g., translocate) a nanopore at a rate. A rate of translocation may be of a free analyte (e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof). A free analyte (e.g., freely-moving analyte) may refer to an analyte (e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof) translocating without the use of a motor protein. The rate of translocation may be of an analyte (e.g., a peptide, a polypeptide, or a protein, or fragments thereof, or any combination thereof) translocating with a motor protein (e.g., a translocase). In some embodiments, an average rate of translocation with a motor protein can be at least about 1 amino acid/sec, at least about 2 amino acids/sec, at least about 3 amino acids/sec, at least about 4 amino acids/sec, at least about 5 amino acids/sec, at least about 10 amino acids/sec, at least about 20 amino acids/sec, at least about 30 amino acids/sec, at least about 40 amino acids/sec, at least about 50 amino acids/sec, at least about 75 amino acids/sec, at least about 100 amino acids/sec, or greater than about 100 amino acids/sec. In some embodiments, an average rate of translocation with a motor protein can be at most about 100 amino acids/sec, at most about 75 amino acids/sec, at most about 50 amino acids/sec, at most about 40 amino acids/sec, at most about 30 amino acids/sec, at most about 20 amino acids/sec, at most about 10 amino acids/sec, at most about 5 amino acids/sec, at most about 4 amino acids/sec, at most about 3 amino acids/sec, at most about 2 amino acids/sec, at most about 1 amino acid/sec, or less than about 1 amino acid/sec. [0454] In some embodiments, an average rate of translocation with a motor protein can be between about 1 amino acid/sec to about 100 amino acids/sec. In some embodiments, an average rate of translocation with a motor protein can be between about 1 amino acid/sec to about 2 amino acids/sec, about 1 amino acid/sec to about 3 amino acids/sec, about 1 amino acid/sec to about 4 amino acids/sec, about 1 amino acid/sec to about 5 WSGR Docket Number: 64828-710.601 amino acids/sec, about 1 amino acid/sec to about 10 amino acids/sec, about 1 amino acid/sec to about 20 amino acids/sec, about 1 amino acid/sec to about 30 amino acids/sec, about 1 amino acid/sec to about 40 amino acids/sec, about 1 amino acid/sec to about 50 amino acids/sec, about 1 amino acid/sec to about 75 amino acids/sec, about 1 amino acid/sec to about 100 amino acids/sec, about 2 amino acids/sec to about 3 amino acids/sec, about 2 amino acids/sec to about 4 amino acids/sec, about 2 amino acids/sec to about 5 amino acids/sec, about 2 amino acids/sec to about 10 amino acids/sec, about 2 amino acids/sec to about 20 amino acids/sec, about 2 amino acids/sec to about 30 amino acids/sec, about 2 amino acids/sec to about 40 amino acids/sec, about 2 amino acids/sec to about 50 amino acids/sec, about 2 amino acids/sec to about 75 amino acids/sec, about 2 amino acids/sec to about 100 amino acids/sec, about 3 amino acids/sec to about 4 amino acids/sec, about 3 amino acids/sec to about 5 amino acids/sec, about 3 amino acids/sec to about 10 amino acids/sec, about 3 amino acids/sec to about 20 amino acids/sec, about 3 amino acids/sec to about 30 amino acids/sec, about 3 amino acids/sec to about 40 amino acids/sec, about 3 amino acids/sec to about 50 amino acids/sec, about 3 amino acids/sec to about 75 amino acids/sec, about 3 amino acids/sec to about 100 amino acids/sec, about 4 amino acids/sec to about 5 amino acids/sec, about 4 amino acids/sec to about 10 amino acids/sec, about 4 amino acids/sec to about 20 amino acids/sec, about 4 amino acids/sec to about 30 amino acids/sec, about 4 amino acids/sec to about 40 amino acids/sec, about 4 amino acids/sec to about 50 amino acids/sec, about 4 amino acids/sec to about 75 amino acids/sec, about 4 amino acids/sec to about 100 amino acids/sec, about 5 amino acids/sec to about 10 amino acids/sec, about 5 amino acids/sec to about 20 amino acids/sec, about 5 amino acids/sec to about 30 amino acids/sec, about 5 amino acids/sec to about 40 amino acids/sec, about 5 amino acids/sec to about 50 amino acids/sec, about 5 amino acids/sec to about 75 amino acids/sec, about 5 amino acids/sec to about 100 amino acids/sec, about 10 amino acids/sec to about 20 amino acids/sec, about 10 amino acids/sec to about 30 amino acids/sec, about 10 amino acids/sec to about 40 amino acids/sec, about 10 amino acids/sec to about 50 amino acids/sec, about 10 amino acids/sec to about 75 amino acids/sec, about 10 amino acids/sec to about 100 amino acids/sec, about 20 amino acids/sec to about 30 amino acids/sec, about 20 amino acids/sec to about 40 amino acids/sec, about 20 amino acids/sec to about 50 amino acids/sec, about 20 amino acids/sec to about 75 amino acids/sec, about 20 amino acids/sec to about 100 amino acids/sec, about 30 amino acids/sec to about 40 amino acids/sec, about 30 amino acids/sec to about 50 amino acids/sec, about 30 amino acids/sec to about 75 amino acids/sec, about 30 amino acids/sec to about 100 amino acids/sec, about 40 amino acids/sec to about 50 amino acids/sec, about 40 amino acids/sec to about 75 amino acids/sec, about 40 amino acids/sec to about 100 amino acids/sec, about 50 amino acids/sec to about 75 amino acids/sec, about 50 amino acids/sec to about 100 amino acids/sec, or about 75 amino acids/sec to about 100 amino acids/sec. WSGR Docket Number: 64828-710.601 [0455] In some embodiments, a translocase can function at a temperature of at least about 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 10 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, about 90 °C, 100 °C, or greater than about 100°C. In some embodiments, a translocase can function at a temperature of at most about 100 °C, 90 °C, 80 °C, 70 °C, 60 °C, 50 °C, 40 °C, 30 °C, 20 °C, 10 °C, 5 °C, 4 °C, 3 °C, 2 °C, 1 °C, or less than about 1 °C. [0456] Also provided herein are nucleic acid molecules encoding any one of the translocase proteins disclosed herein. Also provided herein are expression vectors comprising nucleic acid molecules disclosed herein. Also provided herein are host cells comprising expression vectors disclosed herein. [0457] In some embodiments, the translocase can be capable to moving an analyte. In some embodiments, the translocase may not be capable of separating the strands of double-stranded nucleic acids. In some cases, the translocase may not be a helicase. In some embodiments, the translocase may not be capable of replicating nucleic acids. In some cases, the translocase may not be a nucleic acid polymerase. In some cases, the translocase may not be an DNA polymerase or an RNA polymerase. In some embodiments, the translocase may not be involved in nucleic acid replication. In some embodiments, the translocase may not be capable of cleaving the analyte. In some embodiments, the translocases may not be topoisomerase. [0458] In some embodiments, the translocase may be coupled to a nanopore. The translocase may be coupled covalently (e.g., genetically fused) or non-covalently (e.g., by a recognition element). In some embodiments, the translocase may not be coupled to the nanopore. In some cases, the translocase may not be coupled to the opening of the nanopore. In some cases, the translocase may not be coupled to the membrane adjacent to the nanopore. In some embodiments the translocase may not be bound to the nanopore. In some cases, the translocase may not be bound to the opening of the nanopore. In some case, the translocase may not be bound to the membrane adjacent to the nanopore. [0459] In some embodiments, the electro-osmotic force can capture a translocase-analyte complex (e.g., a translocase-analyte complex. The capture can be the result of pulling a portion of the analyte that may not be within the translocase of the translocase-analyte complex into the nanopore. The capture of a portion of the analyte by the nanopore channel can cause a portion of the analyte of the translocate-analyte complex to be translocated through the nanopore as the analyte may be pulled further into the nanopore channel by the electro- osmotic force. The translocation may occur in opposition to an electrophoretic force, or in conjunction with an electrophoretic force. The translocation of the analyte portion can bring the translocase of the translocase- analyte complex adjacent to the nanopore channel as portion of the analyte being translocated approaches the portion of the analyte within the translocase. The translocase can be brought adjacent to the nanopore on the first side (e.g., cis side) of the nanopore channel if the electro-osmotic force acts in a cis to trans direction or the second side (e.g., trans side) of the nanopore channel if the electro-osmotic force acts in a trans to cis direction. The electro-osmotic force can hold the translocase of the translocase-analyte complex adjacent to the WSGR Docket Number: 64828-710.601 nanopore of the nanopore channel, for example by continuing to draw the analyte through the nanopore and transferring the force of the electro-osmotic force to the attached translocase. Adjacent to the nanopore channel can be at an opening of the nanopore channel, also referred to as “on top” of the nanopore, or near the portion of the nanopore that may not be within the membrane. [0460] In some embodiments, the EOF can hold the translocase on the top of the nanopore without an analyte. In some cases, a portion of the translocase can be captured in the nanopore. In some cases, a portion of the translocase can be captured in the nanopore due to the electro-osmotic force. In some cases, a portion of the translocase can be captured in the nanopore due to the electrophoretic force. In some cases, a portion of the translocase can be captured in the nanopore due to the electro-osmotic force and the electrophoretic force. In some cases, the portion of the translocase captured in the nanopore can be a charged linker or a peptide extension of the translocase. [0461] When held adjacent to a nanopore channel, the translocase of the translocase-analyte complex can be held oriented such that a channel of the translocase can be adjacent to the channel of the nanopore. This orientation may be provided by the analyte being drawn into the nanopore by the electro-osmotic force, which can bring the point of connection of the analyte to the translocase (e.g., the translocase channel) adjacent to the nanopore channel and the translocase channel are aligned. In some embodiments, the translocase can control the rate of analyte translocation. The rate of translocation can be the result of the translocase acting on the analyte as a molecular motor. In some embodiments, the rate of translocation can be from about 0.1 amino acids per second (aa/s) to about 1,000 aa/s. In some cases, the rate of translocation can be at least about 0.1 aa/s, at least about 0.5 aa/s, at least about 1 aa/s, at least about 5 aa/s, at least about 10 aa/s, at least about 50 aa/s, at least about 100 aa/s, at least about 500 aa/s, at least about 1,000 aa/s, or more than 1,000 aa/s. In some cases, the rate of translocation can at least most about 1,000 aa/s, at most about 500 aa/s, at most about 100 aa/s, at most about 50 aa/s, at most about 10 aa/s, at most about 5 aa/s, at most about 1 aa/s, at most about 0.5 aa/s, at most about 0.1 aa/s, or less than 0.1 aa/s. In some cases, the rate of translocation can be about 0.1 aa/s, about 0.5 aa/s, about 1 aa/s, about 5 aa/s, about 10 aa/s, about 50 aa/s, about 100 aa/s, about 500 aa/s, or about 1,000 aa/s. [0462] The translocation orientation can be such that a feed direction of the translocase aligns with the channel of the nanopore. The feed direction can be oriented cis to trans or trans to cis. There may or may not be a gap between the lumen of the translocase channel and the lumen of the nanopore channel. The translocase can be held such that the translocase can feed the analyte through the nanopore in the direction of the electro-osmotic force, or it can be held such that the translocase pulls the analyte through the nanopore against the electro- osmotic force. WSGR Docket Number: 64828-710.601 [0463] The translocase can feed the analyte through the nanopore in the direction of the electroosmotic force such that it translocates at a rate faster or slower than the analyte that translocates with the electro-osmotic force alone. [0464] In some embodiments, the rate of translocation of the analyte through the nanopore with the translocase can be faster than a rate of translocation of the analyte through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 500% faster than a rate of translocation of the analytes through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about 180% to about 190%, between about 190% to about 200%, between about 200% to about 210%, between about 210% to about 220%, between about 220% to about 230%, between about 230% to about 240%, between about 240% to about 250%, between about 250% to about 260%, between about 260% to about 270%, between about 270% to about 280%, between about 280% to about 290%, between about 290% to about 300%, between about 300% to about 310%, between about 310% to about 320%, between about 320% to about 330%, between about 330% to about 340%, between about 340% to about 350%, between about 350% to about 360%, between about 360% to about 370%, between about 370% to about 380%, between about 380% to about 390%, between about 390% to about 400%, between about 400% to about 410%, between about 410% to about 420%, between about 420% to about 430%, between about 430% to about 440%, between about 440% to about 450%, between about 450% to about 460%, between about 460% to about 470%, between about 470% to about 480%, between about 480% to about 490%, or between about 490% to about 500% faster than a rate of translocation of the analytes through the nanopore without the translocase. [0465] In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be at least about 0.1%, at least about 0.5%, at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, WSGR Docket Number: 64828-710.601 at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least about 300%, at least about 310%, at least about 320%, at least about 330%, at least about 340%, at least about 350%, at least about 360%, at least about 370%, at least about 380%, at least about 390%, at least about 400%, at least about 410%, at least about 420%, at least about 430%, at least about 440%, at least about 450%, at least about 460%, at least about 470%, at least about 480%, at least about 490%, at least about 500%, or more than 500% faster than a rate of translocation of the analytes through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be at most about 500%, at most about 490%, at most about 480%, at most about 470%, at most about 460%, at most about 450%, at most about 440%, at most about 430%, at most about 420%, at most about 410%, at most about 400%, at most about 390%, at most about 380%, at most about 370%, at most about 360%, at most about 350%, at most about 340%, at most about 330%, at most about 320%, at most about 310%, at most about 300%, at most about 290%, at most about 280%, at most about 270%, at most about 260%, at most about 250%, at most about 240%, at most about 230%, at most about 220%, at most about 210%, at most about 200%, at most about 190%, at most about 180%, at most about 170%, at most about 160%, at most about 150%, at most about 140%, at most about 130%, at most about 120%, at most about 110%, at most about 100%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 1%, at most about 0.5%, at most about 0.1%, or less than 0.1% faster than a rate of translocation of the analytes through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% faster than a rate of translocation of the analytes through the nanopore without the translocase. WSGR Docket Number: 64828-710.601 [0466] In some embodiments, the rate of translocation of the analyte through the nanopore with the translocase can be slower than a rate of translocation of the analyte through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 500% slower than a rate of translocation of the analytes through the nanopore without the translocase. In some cases, the rate of translocation of the analyte through the nanopore with the translocase can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about 180% to about 190%, between about 190% to about 200%, between about 200% to about 210%, between about 210% to about 220%, between about 220% to about 230%, between about 230% to about 240%, between about 240% to about 250%, between about 250% to about 260%, between about 260% to about 270%, between about 270% to about 280%, between about 280% to about 290%, between about 290% to about 300%, between about 300% to about 310%, between about 310% to about 320%, between about 320% to about 330%, between about 330% to about 340%, between about 340% to about 350%, between about 350% to about 360%, between about 360% to about 370%, between about 370% to about 380%, between about 380% to about 390%, between about 390% to about 400%, between about 400% to about 410%, between about 410% to about 420%, between about 420% to about 430%, between about 430% to about 440%, between about 440% to about 450%, between about 450% to about 460%, between about 460% to about 470%, between about 470% to about 480%, between about 480% to about 490%, or between about 490% to about 500% slower than a rate of translocation of the analytes through the nanopore without the translocase. [0467] In some embodiments, the main force in the translocation of the analyte through the nanopore can be the EOF. In some embodiments, the translocation of the analyte through the nanopore can occur using the EOF. In some cases, the translocation of the analyte through the nanopore can occur in the absence of a translocase. In some cases, the translocation of the analyte through the nanopore can occur using the EOF and in the absence of a translocase. [0468] In some embodiments, the translocation of the analyte through the nanopore may not occur in the absence of the EOF. In some cases, the translocation of the analyte through the nanopore may not occur in the WSGR Docket Number: 64828-710.601 presence of a translocase. In some cases, the translocation of the analyte through the nanopore may not occur in the absence of the EOF and in the presence of a translocase. [0469] The translocase can be held at the cis or second side (e.g., trans side) of the nanopore without needing to be coupled to the nanopore. The electro-osmotic force can hold the translocase adjacent to the nanopore without additional coupling to the nanopore channel. The electro-osmotic force can hold the translocase adjacent to the nanopore so that the nanopore can couple with the translocase. The translocase can be held adjacent to the nanopore channel while the analyte translocates through the nanopore. After the analyte has fully translocate through the nanopore, the translocase may continue to be held adjacent to the nanopore, or the translocase may be released from the position adjacent to the nanopore. A released translocase may then form a translocase-analyte complex with another analyte. In some embodiments, the translocase may not be coupled to the nanopore. In some cases, the translocase may not be coupled adjacent to the nanopore. In some cases, the translocase may not be coupled to the membrane adjacent to the nanopore. [0470] Alternatively, in some embodiments, the translocase can be coupled to the first side (e.g., cis side) or the second side (e.g., trans side) of the nanopore. In some cases, the translocase can be coupled to the nanopore via a covalent bond. In some instances, the covalent bond can be a polar covalent bond. In some instances, the covalent bond can be a non-polar covalent bond. In some cases, the translocase can be coupled to the nanopore via a non-covalent bond. In some cases, the non-covalent bonds can comprise electrostatic interactions, hydrogens bonds, van der Waals interactions, or hydrophobic interactions, or any combination thereof. In some cases, the translocase can be coupled to the nanopore via a linker. In some cases, the linkers can comprise (GGGGS)3, (GGGGS)n, (SG)n, (Gly)8, (Gly)6, (EAAAK)3, (EAAAK)n, VSQTSKLTRAETVFPDV, PLGLWA, RVLAEA, EDVVCCSNSY, GGIEGRGS, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRRR, GFLG, A(EAAAK)4ALEA(EAAAK)4A, PAPAP, AEAAAKEAAAKA, (Ala-Pro)n, disulfide bond, or cysteine linkages, or any combination thereof. In some embodiments, a linker can comprise any combination of amino acids. In some cases, the amino acids can be canonical amino acids. In some cases, the canonical amino acids can comprise alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine, or any combination thereof. In some cases, the amino acids can be non-natural amino acids. In some cases, the non-natural amino acids can comprise hydroproline, beta-alanine, citrulline, ornithine, norleucine, 3- nitrotyrosine, nitroarginine, pyroglutamic acid, naphtylalanine, Abu, DAB, methionine sulfoxide, methionine sulfone, α-amino-n-butyric acid, norvaline, alloisoleucine, t-leucine, α-amino-n-heptanoic acid, pipecolic acid, allothreonine, homocysteine, homoserine, α,β-diaminopropionic acid, α,γ-diaminobutyric acid, β-alanine, β- amino-n-butyric acid, β-aminoisobutyric acid, β-aminoisobutyric acid, γ-aminobutyric acid, α-aminoisobutyric acid, isovaline, sarcosine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N- WSGR Docket Number: 64828-710.601 ethylalanine, N-methyl-β-alanine, N-ethyl-β-alanine, isoserine, α-hydroxy-γ-aminobbutyric acid, or any combinations thereof. In some cases, the linker can comprise any combination of canonical amino acids and non-natural amino acids. In some cases, the linker can be ethylene glycol. In some cases, the linker can be polyethylene glycol. In some cases, the linker can be biotin. In some cases, the linker can be streptavidin. In some cases, the linker can be cysteine linkages. In some case the linker can be formed using Spytag/Spycatcher, Halo-tag, Snap-tag or other bioconjugation methods. In some cases the linker can be formed from click chemistry. In some cases the linker attaches to non-natural amino acids. IONS AND MEMBRANES [0471] In some aspects, the present disclosure provides membranes for separating a chamber, providing an interface or a partition between two fluids, providing a pore, and/or containing a pore. [0472] In some embodiments, a method comprises using an electro-osmotic force (EOF) to feed an analyte from a first side (e.g., cis side) of a pore to a second side (e.g., trans side) of the pore. An EOF can pull on a polymer as it translocates through a pore, and can allow electrical signals, which depends on the structure and the composition of the polymer, to be measured and/or characterized. [0473] A schematic of a nanopore system is shown in FIG. 22. The schematic shows an example of one type of system that can be used with nanopore sensors for the electrical detection of analytes. Other types of systems may be also suitable, such as arrays of nanopore sensors on microchips for example. The schematic shows a chamber consisting of two compartments made of Delrin, separated by a Teflon film containing a 100 μm hole. Both compartments were filled with buffer and an electrode (e.g., Ag/AgCl electrode) can be connected to each chamber to facilitate electrical detection. A lipid membrane can be formed over the hole inside the Teflon film using the Langmuir-Blodgett method to separate the two compartments. Nanopores may be added from the cis chamber and allowed to insert into the membrane. Analytes may be added to the cis chamber for detection. In some aspects, the present disclosure provides a sensor system comprising a pore (e.g., a nanopore). In some aspects, the present disclosure provides a sensor system comprising a nanopore embedded in a membrane. In some cases, the membrane can be an amphipathic membrane. In some cases, the membrane can be a hydrophobic membrane. In some cases, the membrane can separate a chamber into a first side and a second side. In some embodiments, the chamber can be a fluid filled chamber. In some cases, the membrane can comprise at least one nanopore. Disclosed herein is a sensor system comprising a proteinaceous nanopore embedded in an amphipathic or hydrophobic membrane separating a fluid filled chamber into at least two sides (e.g., chambers). In some embodiments, one side (e.g., a first side) of a fluid filled chamber can be a first side (e.g., cis side) and another side (e.g., a second side) of a fluid filled chamber can be a second side (e.g., trans side). In some embodiments, the nanopore can be a conical shaped proteinaceous nanopore. In some WSGR Docket Number: 64828-710.601 embodiments, the nanopore can be a cylindrical shaped proteinaceous nanopore. In some embodiments, the nanopore can be a vestibule shaped proteinaceous nanopore. The nanopore may comprise an opening on a first side (e.g., a cis side) of a fluid filled chamber (e.g., a cis opening). The nanopore may comprise an opening on a second side (e.g., a trans side) of a fluid filled chamber (e.g., a trans opening). [0474] According to the invention, a sensor system comprises a nanopore (e.g., an engineered biological nanopore) embedded in an amphipathic or hydrophobic membrane. In some aspects, the present disclosure provides a sensor system comprising a pore. In some embodiments, the pore can be a nanopore. The nanopore can be conical shaped. The nanopore can be cylindrical shaped. The nanopore can be vestibule shaped. The term "membrane" used herein in its conventional sense can refer to a thin, film-like structure that separates the chamber of the system into a first side (e.g., a cis side or cis compartment) and a second side (e.g., a trans side or trans compartment). The membrane separating the first and second sides can comprise at least one pore (e.g., a biological nanopore). The pore may be a nanopore. The nanopore may be an engineered biological nanopore as described herein. The nanopore may have enhanced cation-selectivity. Membranes can be generally classified into synthetic membranes and biological membranes. Any membrane may be used in accordance with the invention. Multiple nanopores may be present in one membrane. In some embodiments, a membrane of a nanopore system described herein may comprise at least about, at most about, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000 nanopores, or any number of nanopores between two of these values. [0475] The membrane can be an amphiphilic layer. An amphiphilic layer can refer to a layer formed from amphiphilic molecules, such as phospholipids, which have both at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic molecules may be synthetic or naturally occurring. In some embodiments, the membrane may comprise multiple layers. In some embodiments, the membrane may be functionalized. In some embodiments, the membrane may be functionalized with a thiol group, a peptide, a nucleic acid, or a biomolecule, or combinations thereof. Non- naturally occurring amphiphiles which form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). The block copolymers can comprise decane and show low ionic conductance and increased longevity of use. Membranes comprising block- copolymers may comprise mixtures of block copolymers by length, chemistry of the units, number of units, polydispersity, etc. Membranes formed of block copolymers may include di-block or tri-block copolymers for example, or mixtures thereof. Block copolymers for use in the invention may comprise units of Polyethylene Oxide (PEO), Polypropylene Oxide (PPO), Poly(N-isopropylacrylamide) (PNIPAM), Polylactic Acid (PLA), Polycaprolactone (PCL), PMOXA, Polystyrene (PS), Poly(methyl methacrylate) (PMMA), Polyvinyl Alcohol (PVA), or Polyvinyl Pyrrolidone (PVP), or variants thereof, or any combination thereof. WSGR Docket Number: 64828-710.601 [0476] In some embodiments, a membrane of a system described herein may comprise a thickness. In some embodiments, a membrane may be at least about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 14 nm, 16 nm, 18 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, or greater than about 150 nm thick. In some embodiments, a membrane comprise a thickness from about 0.5 nm to about 100 nm. In some embodiments, a membrane comprise a thickness from about 0.5 nm to about 1 nm, about 0.5 nm to about 2 nm, about 0.5 nm to about 3 nm, about 0.5 nm to about 4 nm, about 0.5 nm to about 5 nm, about 0.5 nm to about 10 nm, about 0.5 nm to about 20 nm, about 0.5 nm to about 30 nm, about 0.5 nm to about 40 nm, about 0.5 nm to about 50 nm, about 0.5 nm to about 100 nm, about 1 nm to about 2 nm, about 1 nm to about 3 nm, about 1 nm to about 4 nm, about 1 nm to about 5 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 100 nm, about 2 nm to about 3 nm, about 2 nm to about 4 nm, about 2 nm to about 5 nm, about 2 nm to about 10 nm, about 2 nm to about 20 nm, about 2 nm to about 30 nm, about 2 nm to about 40 nm, about 2 nm to about 50 nm, about 2 nm to about 100 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3 nm to about 10 nm, about 3 nm to about 20 nm, about 3 nm to about 30 nm, about 3 nm to about 40 nm, about 3 nm to about 50 nm, about 3 nm to about 100 nm, about 4 nm to about 5 nm, about 4 nm to about 10 nm, about 4 nm to about 20 nm, about 4 nm to about 30 nm, about 4 nm to about 40 nm, about 4 nm to about 50 nm, about 4 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 100 nm, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 100 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 100 nm, about 40 nm to about 50 nm, about 40 nm to about 100 nm, or about 50 nm to about 100 nm. [0477] The nanopore system typically comprises a first side (e.g., cis side) comprising a first conductive liquid medium in liquid communication with a second side (e.g., trans side) comprising a second conductive liquid medium. [0478] In some embodiments, a method comprises translocating a polymer through a system comprising a pore. In some embodiments, the pore can be comprised in a membrane. In some embodiments, the membrane separates a fluidic chamber into a first side (e.g., cis side) and a second side (e.g., trans side). In some embodiments, the membrane insulates a first side (e.g., cis side) and a second side (e.g., trans side). In some embodiments, a sample can be added to a first side (e.g., cis side). In some embodiments, a sample can be added to a first side (e.g., cis side) in a solution of one or more electrolytes. A system can comprise a cis to trans EOF. An EOF can be generated from a net ionic current flow from a first side (e.g., cis side) to a second side (e.g., WSGR Docket Number: 64828-710.601 trans side) of a membrane. In some embodiments, a sample can be added to a second side (e.g., trans side). In some embodiments, a sample can be added to a second side (e.g., trans side) in a solution of one or more electrolytes. A system can comprise a trans to cis EOF. An EOF can be generated from a net ionic current flow from a second side (e.g., trans side) to a first side (e.g., cis side) of a membrane. [0479] In some embodiments, a system can have a cis to trans EOF resulting from a net ionic current flow cis to trans over total ionic current flow (herein also referred to as Irel) of at least about -0.5, at least about -0.4, at least about -0.3, at least about -0.2, at least about -0.1, at least about 0, at least about 0.05, at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, at least about 0.5, or greater than about 0.5. In some embodiments, a system can have a cis to trans EOF resulting from a net ionic current flow cis to trans over total ionic current flow of at most about 0.5, at most about 0.45, at most about 0.4, at most about 0.35, at most about 0.3, at most about 0.25, at most about 0.2, at most about 0.15, at most about 0.1, at most about 0.05, at most about 0, at most about -0.1, at most about -0.2, at most about -0.3, at most about -0.4, at most about -0.5, or less than about -0.5. In some embodiments, a system can have a trans to cis EOF resulting from a net ionic current flow (Irel) of at least about -0.5, at least about -0.4, at least about -0.3, at least about -0.2, at least about -0.1, at least about 0, at least about 0.05, at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, at least about 0.5, or greater than about 0.5. In some embodiments, a system can have a cis to trans EOF resulting from a net ionic current flow trans to cis over total ionic current flow of at most about 0.5, at most about 0.45, at most about 0.4, at most about 0.35, at most about 0.3, at most about 0.25, at most about 0.2, at most about 0.15, at most about 0.1, at most about 0.05, at most about 0, at most about -0.1, at most about -0.2, at most about -0.3, at most about -0.4, at most about -0.5, or less than about -0.5. [0480] In some embodiments, the system can comprise an ion-selectivity P(+)/P(-) of at least about 0.1, at least about 0.2, at least about 0.3, at least about 0.4, at least about 0.5, at least about 0.6, at least about 0.7, at least about 0.8, at least about 0.9, at least about 1.0, at least about 1.5, at least about 2.0, at least about 2.5, at least about 3.0, at least about 3.5, at least about 4.0, at least about 5.0, or greater than about 5.0. In some embodiments, the system can comprise an ion-selectivity P(+)/P(-) of at most about 5.0, at most about 4.0, at most about 3.5, at most about 3.0, at most about 2.5, at most about 2.0, at most about 1.5, at most about 1.0, at most about 0.9, at most about 0.8, at most about 0.7, at most about 0.6, at most about 0.5, at most about 0.4, at most about 0.3, at most about 0.2, at most about 0.1, or less than about 0.1. The cis to trans EOF can be against the trans to cis electrophoretic force (EPF) acting on the analyte. In some embodiments, the pore system described herein can have an ion selectivity P(+)/P(-) of greater than about 3.0 or less than about 0.3 under an applied voltage across the membrane. Also provided is a pore-membrane system for translocating an analyte through a pore, the system comprising a pore comprised in a membrane separating a fluidic chamber of the pore system into a first WSGR Docket Number: 64828-710.601 side (e.g., cis side) and a second side (e.g., trans side) and wherein the analyte is to be added to the first side (e.g., cis side), wherein the pore system has a cis to trans electro-osmotic force (EOF) resulting from a net ionic current flow cis-to-trans, so that the analyte can be captured in the pore. The dominant cis to trans EOF can result, for example, from a net ionic current flow cis-to-trans over total ionic current flow of at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, at least about 0.5, or greater than about 0.5. In some embodiments, a dominant cis to trans EOF can result from a net ionic current flow cis-to-trans over total ionic current flow between about 0.01 to about 1. In some embodiments, a dominant cis to trans EOF can result from a net ionic current flow cis-to- trans over total ionic current flow between about 0.01 to about 0.1, about 0.01 to about 0.15, about 0.01 to about 0.2, about 0.01 to about 0.25, about 0.01 to about 0.3, about 0.01 to about 0.35, about 0.01 to about 0.4, about 0.01 to about 0.45, about 0.01 to about 0.5, about 0.01 to about 0.75, about 0.01 to about 1, about 0.1 to about 0.15, about 0.1 to about 0.2, about 0.1 to about 0.25, about 0.1 to about 0.3, about 0.1 to about 0.35, about 0.1 to about 0.4, about 0.1 to about 0.45, about 0.1 to about 0.5, about 0.1 to about 0.75, about 0.1 to about 1, about 0.15 to about 0.2, about 0.15 to about 0.25, about 0.15 to about 0.3, about 0.15 to about 0.35, about 0.15 to about 0.4, about 0.15 to about 0.45, about 0.15 to about 0.5, about 0.15 to about 0.75, about 0.15 to about 1, about 0.2 to about 0.25, about 0.2 to about 0.3, about 0.2 to about 0.35, about 0.2 to about 0.4, about 0.2 to about 0.45, about 0.2 to about 0.5, about 0.2 to about 0.75, about 0.2 to about 1, about 0.25 to about 0.3, about 0.25 to about 0.35, about 0.25 to about 0.4, about 0.25 to about 0.45, about 0.25 to about 0.5, about 0.25 to about 0.75, about 0.25 to about 1, about 0.3 to about 0.35, about 0.3 to about 0.4, about 0.3 to about 0.45, about 0.3 to about 0.5, about 0.3 to about 0.75, about 0.3 to about 1, about 0.35 to about 0.4, about 0.35 to about 0.45, about 0.35 to about 0.5, about 0.35 to about 0.75, about 0.35 to about 1, about 0.4 to about 0.45, about 0.4 to about 0.5, about 0.4 to about 0.75, about 0.4 to about 1, about 0.45 to about 0.5, about 0.45 to about 0.75, about 0.45 to about 1, about 0.5 to about 0.75, about 0.5 to about 1, or about 0.75 to about 1. [0481] In some embodiments, a method comprises controlling a movement of analytes through a pore. In some embodiments, the method comprises using a cis-to-trans electro-osmotic flow (EOF). In some embodiments, the EOF can be generated by a cis-to-trans excess of ions flowing through a pore. The EOF can feed a wide range of analytes, including elongated, complex polymeric analytes, from cis to trans through a pore. In some embodiments, the EOF can feed against the direction of an electrophoretic force (EPF) acting on a polymer. [0482] In some embodiments, cations such as K+, Na+, NH4+ or other suitably small and/or highly mobile cation may be on a first side (e.g., cis side) of a pore to provide the majority of the ionic flux through the pore from cis to trans under negative applied voltage to trans. In some embodiments, anionic salts of glutamate, acetate or other large and/or less mobile anions may be on a second side (e.g., trans side) of a pore to limit anionic flux through the pore from trans to cis under negative applied voltage to trans. WSGR Docket Number: 64828-710.601 [0483] In some embodiments, the one or more salts can comprise sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc chloride hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr’s salt, sodium potassium sulphate, potassium permanganate, tetra amino cupric sulphate, zinc chloride hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, magnesium nitrate, potassium glutamate, sodium ferricyanide, sodium ferrocyanide, potassium ferricyanide, or potassium ferrocyanide, or any combination thereof. [0484] In some embodiments, the one or more salts on the first side (e.g., cis side) of the membrane can comprise sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc chloride hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr’s salt, sodium potassium sulphate, potassium permanganate, tetra amino cupric sulphate, zinc chloride hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, or magnesium nitrate, or any combination thereof. In some embodiments, the one or more salts on the second side (e.g., trans side) of the membrane can comprise sodium chloride, sodium carbonate, ammonium chloride, sodium acetate, potassium cyanide, zinc chloride hydroxide, potassium chlorate, calcium phosphate, sodium nitrate, potassium cerium fluoride, Mohr’s salt, sodium potassium sulphate, potassium permanganate, tetra amino cupric sulphate, zinc chloride hydroxide monohydrate, monosodium glutamate, copper sulfate, calcium chloride, potassium chloride, magnesium sulfate, magnesium chloride, sodium acetate, or magnesium nitrate, or any combination thereof. [0485] In some embodiments, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be between about 0.1% to about 500% lower than the concentration of one or more salts on the second side (e.g., trans side) of the membrane. In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about 180% to about 190%, between about 190% to about 200%, between about 200% to about 210%, between about 210% to about 220%, between about 220% to about 230%, between about 230% WSGR Docket Number: 64828-710.601 to about 240%, between about 240% to about 250%, between about 250% to about 260%, between about 260% to about 270%, between about 270% to about 280%, between about 280% to about 290%, between about 290% to about 300%, between about 300% to about 310%, between about 310% to about 320%, between about 320% to about 330%, between about 330% to about 340%, between about 340% to about 350%, between about 350% to about 360%, between about 360% to about 370%, between about 370% to about 380%, between about 380% to about 390%, between about 390% to about 400%, between about 400% to about 410%, between about 410% to about 420%, between about 420% to about 430%, between about 430% to about 440%, between about 440% to about 450%, between about 450% to about 460%, between about 460% to about 470%, between about 470% to about 480%, between about 480% to about 490%, or between about 490% to about 500% lower than the concentration of one or more salts on the second side (e.g., trans side) of the membrane. [0486] In some embodiments, the concentration of one or more salts on the second side (e.g., trans side) of the membrane can be between about 0.1% to about 500% higher than the concentration of one or more salts on the first side (e.g., cis side) of the membrane. In some cases, the concentration of one or more salts on the second side (e.g., trans side) of the membrane can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about 180% to about 190%, between about 190% to about 200%, between about 200% to about 210%, between about 210% to about 220%, between about 220% to about 230%, between about 230% to about 240%, between about 240% to about 250%, between about 250% to about 260%, between about 260% to about 270%, between about 270% to about 280%, between about 280% to about 290%, between about 290% to about 300%, between about 300% to about 310%, between about 310% to about 320%, between about 320% to about 330%, between about 330% to about 340%, between about 340% to about 350%, between about 350% to about 360%, between about 360% to about 370%, between about 370% to about 380%, between about 380% to about 390%, between about 390% to about 400%, between about 400% to about 410%, between about 410% to about 420%, between about 420% to about 430%, between about 430% to about 440%, between about 440% to about 450%, between about 450% to about 460%, between about 460% to about 470%, between about 470% WSGR Docket Number: 64828-710.601 to about 480%, between about 480% to about 490%, or between about 490% to about 500% higher than the concentration of one or more salts on the first side (e.g., cis side) of the membrane. [0487] In some embodiments, a method comprises providing a cis-to-trans electro-osmotic flow, or vice versa. The EOF can arise from a net flow of ions (e.g. cis to trans) that creates a strong force on the solvent itself (water) sufficient to move the fluid, which further imposes a significant force on any molecules within the flux. Electroosmosis can either compete or cooperate with an electrophoretic force (EPF). [0488] In some embodiments, a cis-to-trans EOF dominates over EPF. In some embodiments, complex and/or charged analytes can be captured and translocated through a pore, under dominant cis-to-trans EOF acting against trans-to-cis EPF. Without being bound to a particular theory, dominant EOF may capture the analyte in the pore with relatively high residence time, wherein the EOF pulls on the analyte directly through a pore. In some embodiments, a non-nucleic acid based polymer analyte can be translocated using an electro-osmotic force. [0489] In some embodiments, the EOF can be greater than the EPF. In some cases, the EOF can be between about 0.1% to about 500% greater than the EPF. In some cases, the EOF can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about 180% to about 190%, between about 190% to about 200%, between about 200% to about 210%, between about 210% to about 220%, between about 220% to about 230%, between about 230% to about 240%, between about 240% to about 250%, between about 250% to about 260%, between about 260% to about 270%, between about 270% to about 280%, between about 280% to about 290%, between about 290% to about 300%, between about 300% to about 310%, between about 310% to about 320%, between about 320% to about 330%, between about 330% to about 340%, between about 340% to about 350%, between about 350% to about 360%, between about 360% to about 370%, between about 370% to about 380%, between about 380% to about 390%, between about 390% to about 400%, between about 400% to about 410%, between about 410% to about 420%, between about 420% to about 430%, between about 430% to about 440%, between about 440% to about 450%, between about 450% to about 460%, between about 460% WSGR Docket Number: 64828-710.601 to about 470%, between about 470% to about 480%, between about 480% to about 490%, or between about 490% to about 500% longer greater than the EPF. [0490] In some embodiments, the translocation of the analyte through the nanopore occurs in the direction of the EOF. In some embodiments, the translocation of the analyte through the nanopore occurs in the direction of the EPF. In some embodiments, the translocation of the analyte through the nanopore occurs in the direction of the EOF and the direction of the EPF. [0491] Alternatively, in some embodiments, the translocation of the analyte through the nanopore occurs in the direction of the EPF. In some embodiments, the translocation of the analyte through the nanopore occurs in the opposite direction of the EOF. In some embodiments, the translocation of the analyte through the nanopore occurs in the direction of the EPF and opposite the direction of the EOF. In some embodiments, an EPF can be provided. The EPF can be acting in an opposite direction to an EOF. In some embodiments, an electro-osmotic force may translocate at least a portion of an analyte from the first side through the nanopore to the second side against an electro-phoretic force acting in a direction opposite the electro-osmotic force. In some embodiments, use of a motor protein described herein may provide for translocation of one or more analytes against a prevailing EOF. [0492] Alternatively, in some embodiments, the EPF can be greater than the EOF. In some embodiments, the EPF can be greater than the EOF. In some cases, the EPF can be between about 0.1% to about 500% greater than the EOF. [0493] As used herein, cis can be used to designate a side to which an analyte can be added. As used herein, trans can be used to designate a side to which an analyte can be pulled through a pore. The terms “trans” and “cis” may be used, merely as a matter of notation and convention determined by electronics/voltage polarity at the trans electrode. For example, the first side (e.g., cis side) can be designated as a reference voltage (e.g., ground) and the applied transmembrane potential can be designated as the potential on the second side (e.g., trans side). As an example, a pore system (e.g., a nanopore system) described herein may comprise a first side (e.g., a cis side) and a second side (e.g., a second side (e.g., trans side). As another example, a pore system (e.g., a nanopore system) described herein may comprise a first side (e.g., a trans side) and a second side (e.g., a cis side). A positive current can be designated as the current in which positive charge (e.g. K+ ions) moves through the pore from the trans to the first side (e.g., cis side), or negative charge (e.g. Cl− ions) from the cis to the second side (e.g., trans side). Thus, the signs and directions of an EOF and/or EPF can depend on the polarity of an applied voltage, the charge of an analyte, the relative conditions in a first side (e.g., cis side) and a second side (e.g., trans side), ion selectivity of a pore, as well as the notation or convention used. An analyte can also be added to the second side (e.g., trans side) to perform trans-to-cis translocation of the analyte, e.g., in a method where the EOF can be created trans-to-cis. WSGR Docket Number: 64828-710.601 [0494] Ion selective ion flux across membranes can be described by the Goldman-Hodgkin-Katz (GHK) flux equation, which can be used to determine the ionic current (I(S)) of ion species S across the membrane as a function of the applied potential (Vm):
Figure imgf000126_0001
[0496] where P(S) can be the membrane permeability of ion species S, zs the valency of the ion, F the Faraday constant, R the gas constant, T the temperature and [S]cis and [S]trans the cis and trans concentrations of ion species S, respectively. The GHK flux equation can therefore be used to determine the separate current flow contributions (e.g. I(S1), I(S2), I(S3), or any combination thereof) of various ion species (e.g. S1, S2, S3, or any combination thereof) in a system, flowing either cis-to-trans or trans-to-cis. [0497] The separate ionic current contributions can be combined to determine the measured current (I) (which accounts for the direction of the ionic flows relative to the polarity of the applied voltage): [0498] ^ = ^(^^) + ^(^^) + ^(^^) …. [0499] which can approximately match the ionic current that can be measured experimentally across the pore system under an applied voltage (ignoring any access resistance from the bulk solution). [0500] The total absolute ionic current (Itotal) flowing through the pore regardless of direction can be given by the sum of absolute component currents:
Figure imgf000126_0002
[0502] When accounting for direction of flow, the separate ionic currents (e.g. I(S1), I(S2), I(S3), or any combination thereof) can also be combined to determine the separate components of the net ionic current flowing cis-to-trans (^^→^) and net ionic current flowing trans-to-cis (^^→^).
Figure imgf000126_0003
[0505] These in turn can be used to determine the net ion current flow cis-to-trans
Figure imgf000126_0004
: [0506] ^∆^→^ = ^^→^ − ^^→^ [0507] To provide a relative magnitude of the net current flow cis-to-trans as a proportion of the total current flowing through the pore, the net cis-to-trans can be divided by the total amount of current flowing to obtain:
Figure imgf000126_0005
[0509] where Irel is the relative net current flow cis-to-trans. [0510] In a balanced pore system, under an applied voltage the cis-to-trans current
Figure imgf000126_0006
is can be balanced by an equal trans-to-cis current (It→c), so that
Figure imgf000126_0007
≈ 0 and Irel ≈ 0. In a pore system where the net current WSGR Docket Number: 64828-710.601 flowing can be cis-to-trans then Irel > 1 up to a maximum of Irel = 1 when all the current can be flowing cis-to- trans. Vice versa, in a pore system where the current flows trans-to-cis then Irel = -1. Therefore, Irel can vary between -1 and 1, and the further away from 0 the stronger the net current flowing through the pore in one direction is, and hence the stronger the resulting EOF can be in that direction. [0511] In some embodiments, analyte capture and translocation can be performed with a large net cis-to-trans current >> 0 arising from a large relative difference between the cis-to-trans current and the trans-to-cis current (I∆t→c), or vice versa (I∆t→c >> 0 from Irel < 1). In some embodiments, Irel can be greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, or greater than 0.4 or less than -0.4. In some embodiments, Irel can be greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or about 0.9. In some embodiments, Irel can be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1.0. In some embodiments, Irel can be less than about -0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, or about -0.9. In some embodiments, Irel can be greater than about - 0.1, -0.2, -0.3, -0.4, -0.5, -0.6, -0.7, -0.8, -0.9, or about -1.0. [0512] The GHK flux equation can indicate the conditions under which to create a large net cis-to-trans current by altering one or more of its variables, including the system ion-selectivity P(+)/P(-), the mixtures of salts used, the salt concentrations and salt asymmetries, and the applied voltage. The GHK indicates that at least three methods can generate a net total ion flux across the membrane: 1) an asymmetry in electrolyte concentration (e.g.1 M KCl buffer in cis and 0.1 M KCl buffer in trans), 2) an asymmetry in electrolyte compositions with different permeabilities (e.g.1 M KCl in cis and 1 M KGlutamate in trans), 3) the use of ion-selective membrane channels. These methods can be used individually or in any combination. [0513] In some embodiments, a highly ion-selective pore system provides a directional net flow of water (the EOF) across a membrane, even without salt asymmetry. In some embodiments, a pore can be ion selective by charge selectivity (e.g., the charge in the inner surface of a pore can repel a particular sign of charge). [0514] The ion selectivity of a pore can be quantified by measuring the current-voltage (I-V) relationship under asymmetric electrolyte conditions. Under asymmetric electrolyte conditions, a net flow of ions can occur when no voltage can be applied (Vm = 0 mV). However, when a specific reversal potential (Vr) can be applied, the flux of positive and negative ions can be equal in magnitude and direction, and no net current may be measured across the system. Thus, the GHK flux equation can be solved at 0 pA for both species of ions to yield the ion- selectivity ratio:
Figure imgf000127_0001
[0516] wherein P(X+) and P(Y-) denote the permeability of the pore system for cation species X and anion species Y respectively. [^^ ^]and [^^ ^] are the activity of ion Y and X respectively in the indicated compartment, and can be calculated by multiplying the concentration with the mean ion activity coefficient (which may be WSGR Docket Number: 64828-710.601 tabulated for most electrolytes, see, e.g., Lide, D. R., 2003, CRC handbook of chemistry and physics, 84th edition, Handb. Chem. Phys.53, 2616). The latter can correct for the presence of other ions in concentrated electrolyte solutions. The empirical ion-selectivity ratio (P(X+)/P(Y-)) can be inserted back into the GHK flux equations in combination with experimental measurements of ionic current versus applied voltage (I-V curves) for a pore system containing the XY salts on both cis and trans to determine the absolute values of P(X+) and P(Y-). Thus, permeability P(S) can be determined for any ion species S employed in the pore system, and then used in the GHK flux equations to determine the underlying ionic current flows for pore systems containing mixtures of two or more ion species (e.g. asymmetric salt types). [0517] Under symmetric salt conditions, in a system comprised primarily of two ions X+ and Y- in both compartments, the ion-selectivity ratio (P(X+)/P(Y-)) can determine the relative ion flux that may flow across the membrane cis-to-trans and trans-to-cis. Thus, if P(X+)/P(Y-) > 1, the cation species dominate the ion flux and the EOF can be directed towards the negative electrode, whereas the EOF can be directed towards the positive electrode when P(X+)/P(Y-) < 1. Pores with larger (P(X+)/P(Y-)) ratios may have a larger net ion flux and hence a larger EOF. [0518] When mixtures of salts are employed, to a first approximation the ion-selectivity ratio can be given by:
Figure imgf000128_0001
[0520] ^^^ ( ^^ ) average ions in the indicated compartment, where average permeability can be calculated using ^ ^^ ( ^^ ) = (P(1)[P(1)] + P(2)[P(2)] + ...)/[total] for the indicated polarity ions in the indicated compartment, where P(s) and [P(s)] are the permeability and concentration respectively for species S = 1, 2, ... or any combination thereof in given compartment, and [total] is the total concentration of the same ions. [0521] In some embodiments, a method uses an ion-selectivity ratio^ ^^ ( ^ ^ ^^ )/^^^ ( ^ ^ ^^ ) > 2.0 or < 0.5, > 2.5 or < 0.4, > 3.0 or < 0.33, > 3.5 or < 0.29, in combination with a symmetrical salt system can be sufficient to drive capture and translocation of complex analytes against a prevailing EPF under an applied voltage across the membrane of 20 mV to 1 V, 50 mV to 300 mV, or 75 mV to 200 mV. The ion-selectivity ratio can be at least about 1.1, 1.5, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or about 10. The ion-selectivity ratio can be at most about 1.1, 1.5, 2.0, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9 or about 10. The ion-selectivity ratio can be at least about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or about 0.40. The ion-selectivity ratio can be at most about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, or about 0.40. The applied voltage can be at least about 10 mV, 20 mV, 30 mV, 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, 90 mV, 100 mV, 150 mV, 200 mV, 250 mV, 300 mV, 350 mV, 400 mV, 450 mV, 500 mV, 600 mV, 700 mV, 800 mV, 900 mV, or about 1000 mV in magnitude. The applied voltage can be at most about 10 mV, 20 mV, 30 mV, 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, 90 mV, 100 mV, 150 mV, 200 mV, 250 mV, 300 mV, 350 mV, WSGR Docket Number: 64828-710.601 400 mV, 450 mV, 500 mV, 600 mV, 700 mV, 800 mV, 900 mV, or about 1000 mV in magnitude. In some embodiments, an applied voltage described herein can refer to one or more applied voltages. For example, an applied voltage may refer to a first applied voltage and/or a second applied voltage. A first applied voltage, or a second applied voltage, or any combination thereof can be applied to any side of a nanopore system described herein. For example, (i) a first applied voltage may be applied to a first side and a second applied voltage may be applied to a second side, (ii) a first applied voltage may be applied to a first side and a second applied voltage may be applied to a first side, and/or (iii) a first applied voltage may be applied to a second side and a second applied voltage may be applied to a second side. [0522] In some embodiments, an EOF can be modulated by genetically engineering or selecting a pore structure which provides a specific steric and/or the electrostatic conditions within its inner channel that adjusts the preference for translocating one ion over another. The net charge of the channel, or the geometry of the channel, or any combination thereof, can influence a flow of molecules through the channel. The flowing molecules can be analytes, ions, water, or other molecules, or any combination thereof on a first side (e.g., cis side) or a second side (e.g., trans side) of a nanopore. The flowing molecules can generate an ionic current from a flow of ions. Without wishing to be bound by thereof, as an analyte translocates through a pore, other molecules (such as ions) can be obstructed from translocating through the pore. This obstruction in translocation of other molecules (e.g., ions) can change the ionic current by changing the rate of flow of ions. This change in current can be measured, for example, by a pair of electrodes configured to measure a current from a first side (e.g., cis side) to a second side (e.g., trans side) across the nanopore. A nanopore of a nanopore system described herein may employ alternative means of measuring the voltage-current properties of the nanopore system, such as those that employ fluorescence probes of ionic flux or field effect transistor systems than measure changes in voltage. However, there are also other suitable detection methods, such as tunneling, surface enhanced raman, plasmonics, and other spectroscopic methods that do not measure the ionic current and instead measure the properties of the target analyte in the nanopore directly. In some embodiments, the change in current can be measured by a pair of electrodes configured to measure a current from a first side (e.g., cis side) to a second side (e.g., trans side) across a membrane of which the nanopore may be disposed. In some cases, a narrow geometry of the channel can slow a progression of an analyte through a pore. A change to a net charge or a geometry of a channel of a nanopore can change the flow of molecules through the pore. As an example, changing (e.g., mutating) a channel to have a more negative net charge can reduce a flow of a negatively charged molecule (e.g., a chloride ion). As another example, changing (e.g., mutating) a channel to have a more positive net charge can reduce a flow of a positively charged molecule (e.g., a potassium ion). In some embodiments, changing a channel to have a wider geometry can increase a flow of a larger molecule (e.g., a glucose molecule or a peptide analyte). In some embodiments, changing a channel to have a more negative net charge and a WSGR Docket Number: 64828-710.601 narrower geometry can reduce a flow of a large, negatively charged molecule (e.g., a glutamate ion). The net charge of the channel can influence the flow of charged molecules through the nanopore. The net charge of the channel can influence the flow of charged molecules through the nanopore. In some embodiments, a shift in the net charge can make some charged molecules translocate more easily through the pore. In some embodiments, a shift in the net charge can make some charged molecules translocate with more difficulty through the pore. In some embodiments, one or more monomers of the nanopore described herein may be modified to change a net charge. A nanopore described herein may be genetically engineered to comprise one or more negatively- charged amino acid residues, or one or more positively-charged amino acid residues, or any combination thereof. In some embodiments, at least about 1 amino acid residue, 2 amino acid residues, 3 amino acid residues, 4 amino acid residues, 5 amino acid residues, 10 amino acid residues, or greater than about 10 amino acid residues may be mutated in a nanopore described herein. In some embodiments, at most about 10 amino acid residues, 5 amino acid residues, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues, 1 amino acid residue, or less than about 1 amino acid residue may be mutated in a nanopore described herein. A nanopore may comprise a mutation (e.g., an insertion, deletion, substitution, or chemical modification, or any combination thereof) of one or more amino acid residues to one or more positively-charged amino acid residues, one or more negatively-charged amino acid residues, one or more aromatic amino acid residues, one or more acidic amino acid residues, one or more neutrally-charged amino acid residues, one or more basic amino acid residues, one or more amidic amino acid residues, or one or more sulfur-containing amino acid residues, or any combination thereof. [0523] In some embodiments, an EOF of the nanopore described herein comprising a a genetically engineered negatively charged region of the channel may comprise an EOF of at least about 1.1-fold, at least about 1.2- fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 7.0-fold, at least about 8.0-fold, at least about 9.0-fold, or at least about 10.0-fold greater than an EOF generated by a wild-type nanopore. In some embodiments, an EOF of the nanopore described herein comprising a a genetically engineered negatively charged region of the channel may comprise an EOF of at most about 10.0-fold, at most about 9.0-fold, at most about 8.0-fold, at most about 7.0-fold, at most about 6.0-fold, at most about 5.0-fold, at most about 4.5-fold, at most about 3.5-fold, at most about 3.0-fold, at most about 2.5-fold, at most about 2.0-fold, at most about 1.5-fold, at most about 1.4-fold, at most about 1.3-fold, at most about 1.2-fold, or at most about 1.1-fold greater than an EOF generated by a wild- type nanopore. [0524] For example, the net charge of the channel of a pore can be increased so as to electrostatically limit the flux of one species of ions in one direction across the pore, while retaining/enhancing the flux of an oppositely WSGR Docket Number: 64828-710.601 charged species of ions flowing in the opposite direction under an applied voltage. The EOF can be enhanced by either adding more charges to the residues lining the walls of the channel, or narrowing the channel dimensions, or a combination thereof. Charge and/or steric barriers to the flow of specific ions can also be created by chemical modification of the inner surface of a pore. For example, cysteine residues can be reacted with derivates of maleimide or iodoacetate. A wide array of chemical modifications and/or reaction types can be used to modulate ion selectivity, including but not limited to modification of cysteines, modification of lysines, incorporation of unnatural amino acids, modification of unnatural amino acids with click chemistry groups, and the like. Charge and/or steric barriers to the flow of specific ions can also be created by use of proteinaceous or chemical adapters inside the pore channel. For example, circular chemical adapters such as cyclodextrins or cucurbiturils can be incorporated into the pore. In some embodiments, protein based adapters can be employed, such as the CsgF subunit of the CsgG pore, which can separately be mutated and/or engineered to create steric and/or electrostatic barriers. The protein or chemical adapters can be attached either by non-covalent docking or by covalent approaches. Charge and/or steric barriers can be engineered into a pore channel by the addition of amino acids into the sequence in and around the regions that comprise the channel (e.g. into the beta-barrel transmembrane region of a beta-barrel pore such as alpha-hemolysin) to create a loop, turn, constriction or other extrusion that reduces the diameter of the pore. In some embodiments, charge and/or steric barriers can be created at either the cis or trans entrance to the pore channel, and away from the narrowest parts of the pore where analyte discrimination can be strongest, to create a locally depleted regions of charge that alter the ion-selectivity through the pore. Ion-selectivity biases can also be generated by altering the system conditions or adding additives that change the properties of the water-facing residues in the channel of the pore. For example, the pH of the system can be adjusted, either on both side of the membrane or just one side of the membrane, to change the protonation state of the pore. For example, low pH can be employed (e.g. preferably <6.0, most preferably <4.0) to increase the net positive charge inside the pore, to increase the bias towards anion flow. In some embodiments, high pH can be used (e.g. >8.0, or >10.5) to increase the net negative charge inside the pore to increase the bias towards cation flow. In some embodiments, additives that interact with the water- facing residues can be added to the solution to change the ionic or steric properties of the water-facing residues inside the pore. EOF can be generated by providing different salt type(s), providing salt asymmetries, providing a certain pH, providing additives such as Guanidinium chloride or guanidine hydrochloride (abbreviated GdmCl and sometimes GdnHCl or GuHCl, GuCl) and osmotics. [0525] In some embodiments, EOF can be generated with a chemical gradient of an ion between two sides of a pore. For example, EOF can be generated with a strong asymmetric ion flow where there can be an asymmetric ion concentration between two sides of a pore. Low salt concentration conditions can be used in the compartment from which it can be desired to have low ionic transfer, relative to higher salt concentration in the WSGR Docket Number: 64828-710.601 compartment from which high ionic flux can be desired. For example, for setting up a system with a strong cis-to-trans EOF, a low concentration of salt can be employed in the trans compartment to limit the flow of ions from trans to cis. For example, a pore system can be set up with 1 M KGlu (potassium glutamate) in the cis compartment and 0.2 M KGlu in the trans compartment. The salt gradient between the compartments can be at least about 0.001 M, 0.01 M, 0.05 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, or greater than about 1.0 M. In some embodiments, a salt gradient between the compartments can be at most about 1.0 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.05 M, 0.01 M, 0.001 M, or less than about 0.001 M. In some embodiments, a salt gradient between the compartments can be between about 0.01 M to about 1 M. In some embodiments, a salt gradient between the compartments can be between about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.2 M, about 0.01 M to about 0.3 M, about 0.01 M to about 0.4 M, about 0.01 M to about 0.5 M, about 0.01 M to about 0.6 M, about 0.01 M to about 0.7 M, about 0.01 M to about 0.9 M, about 0.01 M to about 0.9 M, about 0.01 M to about 1 M, about 0.05 M to about 0.1 M, about 0.05 M to about 0.2 M, about 0.05 M to about 0.3 M, about 0.05 M to about 0.4 M, about 0.05 M to about 0.5 M, about 0.05 M to about 0.6 M, about 0.05 M to about 0.7 M, about 0.05 M to about 0.9 M, about 0.05 M to about 0.9 M, about 0.05 M to about 1 M, about 0.1 M to about 0.2 M, about 0.1 M to about 0.3 M, about 0.1 M to about 0.4 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.6 M, about 0.1 M to about 0.7 M, about 0.1 M to about 0.9 M, about 0.1 M to about 0.9 M, about 0.1 M to about 1 M, about 0.2 M to about 0.3 M, about 0.2 M to about 0.4 M, about 0.2 M to about 0.5 M, about 0.2 M to about 0.6 M, about 0.2 M to about 0.7 M, about 0.2 M to about 0.9 M, about 0.2 M to about 0.9 M, about 0.2 M to about 1 M, about 0.3 M to about 0.4 M, about 0.3 M to about 0.5 M, about 0.3 M to about 0.6 M, about 0.3 M to about 0.7 M, about 0.3 M to about 0.9 M, about 0.3 M to about 0.9 M, about 0.3 M to about 1 M, about 0.4 M to about 0.5 M, about 0.4 M to about 0.6 M, about 0.4 M to about 0.7 M, about 0.4 M to about 0.9 M, about 0.4 M to about 0.9 M, about 0.4 M to about 1 M, about 0.5 M to about 0.6 M, about 0.5 M to about 0.7 M, about 0.5 M to about 0.9 M, about 0.5 M to about 0.9 M, about 0.5 M to about 1 M, about 0.6 M to about 0.7 M, about 0.6 M to about 0.9 M, about 0.6 M to about 0.9 M, about 0.6 M to about 1 M, about 0.7 M to about 0.9 M, about 0.7 M to about 0.9 M, about 0.7 M to about 1 M, about 0.9 M to about 0.9 M, about 0.9 M to about 1 M, or about 0.9 M to about 1 M. [0526] In some embodiments, a system described herein may comprise a fluidic chamber; and a membrane comprising a nanopore that separates the fluidic chamber into a first side (e.g., cis side) and a second side (e.g., trans side). The first side can comprise a first solution. The second side can comprise a second solution. In some embodiments, the first solution or concentration thereof and/or the second solution or concentration thereof may generate an EOF. The first solution may comprise a concentration of a solute (e.g., a first solute). A second solution may comprise a concentration of a solute (e.g., a second solute). A solute may comprise one or more WSGR Docket Number: 64828-710.601 salts, electrolytes, osmolytes, ions, or any combination thereof. In some embodiments, the first solution comprises a first concentration of a solute and/or the second solution comprises a second concentration of a solute. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute may generate an electro-osmotic force (EOF). In some embodiments, the electro- osmotic force comprises a net ionic current flow from the first side (e.g., cis side) to the second side (e.g., trans side). In some embodiments, the electro-osmotic force comprises a net ionic current flow from the second side (e.g., trans side) to the first side (e.g., cis side). [0527] In some embodiments, a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be at least about 0.01 M, 0.05 M, 0.10 M, 0.20 M, 0.30 M, 0.40 M, 0.50 M, 0.60 M, 0.70 M, 0.80 M, 0.90 M, 1.00 M, 1.10 M, 1.25 M, 1.50 M, 1.75 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, 4.5 M, 5 M, or greater than about 5 M. In some embodiments, a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be at most about 5 M, 4.5 M, 4 M, 3.5 M, 3 M, 2.5 M, 2 M, 1.75 M, 1.5 M, 1.25 M, 1.10 M, 1.0 M, 0.9 M, 0.8 M, 0.7 M, 0.6 M, 0.5 M, 0.4 M, 0.3 M, 0.2 M, 0.1 M, 0.05 M, 0.01 M, or less than about 0.01 M. In some embodiments, a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be between about 0.01 M to about 5 M. In some embodiments, a difference in salt or electrolyte concentrations between a first side (e.g., cis side) and a second side (e.g., trans side) can be between about 0.01 M to about 0.05 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.25 M, about 0.01 M to about 0.5 M, about 0.01 M to about 0.75 M, about 0.01 M to about 1 M, about 0.01 M to about 2 M, about 0.01 M to about 3 M, about 0.01 M to about 4 M, about 0.01 M to about 5 M, about 0.05 M to about 0.1 M, about 0.05 M to about 0.25 M, about 0.05 M to about 0.5 M, about 0.05 M to about 0.75 M, about 0.05 M to about 1 M, about 0.05 M to about 2 M, about 0.05 M to about 3 M, about 0.05 M to about 4 M, about 0.05 M to about 5 M, about 0.1 M to about 0.25 M, about 0.1 M to about 0.5 M, about 0.1 M to about 0.75 M, about 0.1 M to about 1 M, about 0.1 M to about 2 M, about 0.1 M to about 3 M, about 0.1 M to about 4 M, about 0.1 M to about 5 M, about 0.25 M to about 0.5 M, about 0.25 M to about 0.75 M, about 0.25 M to about 1 M, about 0.25 M to about 2 M, about 0.25 M to about 3 M, about 0.25 M to about 4 M, about 0.25 M to about 5 M, about 0.5 M to about 0.75 M, about 0.5 M to about 1 M, about 0.5 M to about 2 M, about 0.5 M to about 3 M, about 0.5 M to about 4 M, about 0.5 M to about 5 M, about 0.75 M to about 1 M, about 0.75 M to about 2 M, about 0.75 M to about 3 M, about 0.75 M to about 4 M, about 0.75 M to about 5 M, about 1 M to about 2 M, about 1 M to about 3 M, about 1 M to about 4 M, about 1 M to about 5 M, about 2 M to about 3 M, about 2 M to about 4 M, about 2 M to about 5 M, about 3 M to about 4 M, about 3 M to about 5 M, or about 4 M to about 5 M. [0528] In some embodiments, a salt or electrolyte comprises sodium chloride, potassium chloride, guanidinium chloride, guanidine hydrochloride, potassium glutamate, an alkali metal salt, a halide salt, an ionic liquid, or an WSGR Docket Number: 64828-710.601 organic salt. Under a cis>trans salt concentration asymmetry both cations and anions may flow cis-to-trans at moderate to low applied voltages. Thus, salt applied electro-osmosis can be highly advantageous for creating or enhancing EOF under lower voltages where repulsive EPF effects on the polymer analyte may be reduced. High asymmetry salt conditions may be used in combination with pores that may be engineered with enhanced ion-selectivity. [0529] A difference in a concentration of a molecule between two sides of a membrane can modify an electro- osmotic flux by providing a competing or assisting osmotic flux. A difference in concentration across a membrane can create an osmotic gradient, wherein a solvent (e.g., water) may diffuse across a membrane in the direction of a higher concentration of the molecule so as to minimize the difference in concentration between the sides of the membrane. The osmotic gradient can be oriented so as to drive a water flow in the same direction as the electro-osmotic force, or in a different direction. For example, a high ion concentration on a first side (e.g., cis side) relative to a second side (e.g., trans side) can create an osmotic gradient that competes with a cis to trans electro-osmotic force, as the osmotic gradient can drive water flow in a trans to cis direction. The ion concentrations may support a cis to trans electro-osmotic flow even if they also provide an osmotic gradient. [0530] In some embodiments, the EOF can be generated by an asymmetric salt distribution between the first side (e.g., cis side) of the membrane and the second side (e.g., trans side) of the membrane. In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be different from the concentration of the one or more salts on the second side (e.g., trans side) of the membrane. In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be higher than the concentration of the one or more salts on the second side (e.g., trans side) of the membrane. In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be lower than the concentration of one or more salts on the second side (e.g., trans side) of the membrane. In some cases, the concentration of one or more salts on the second side (e.g., trans side) of the membrane can be higher than the concentration of the one or more salts on the first side (e.g., cis side) of the membrane. In some cases, the concentration of one or more salts on the second side (e.g., trans side) of the membrane can be lower than the concentration of the one or more salts on the first side (e.g., cis side) of the membrane. [0531] In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be between about 1 nanomolar (nM) to about 1,000 nM. In some instances, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be between about 1 nM to about 10 nM, between about 10 nM to about 100 nM, or between about 100 nM to about 1,000 nM. In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 WSGR Docket Number: 64828-710.601 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or more than 1,000 nM. In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can at most about 1,000 nM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 95 nM, at most about 90 nM, at most about 85 nM, at most about 80 nM, at most about 75 nM, at most about 70 nM, at most about 65 nM, at most about 60 nM, at most about 55 nM, at most about 45 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, at most about 5 nM, at most about 1 nM, or less than 1 nM. In some cases, the concentration of one or more salts on the first side (e.g., cis side) of the membrane can about 1 nM, about 5 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM, about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about 55 nM, about 60 nM, about 65 nM, about 70 nM, about 75 nM, about 80 nM, about 85 nM, about 90 nM, about 95 nM, about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 350 nM, about 400 nM, about 450 nM, about 500 nM, about 550 nM, about 600 nM, about 650 nM, about 700 nM, about 750 nM, about 800 nM, about 850 nM, about 900 nM, about 950 nM, or about 1,000 nM. [0532] In some embodiments, a salt, ion, osmolyte, or electrolyte concentration on the first side (e.g., cis side) can be at least about 0.01 M, at least about 0.05 M, at least about 0.10 M, at least about 0.20 M, at least about 0.30 M, at least about 0.40 M, at least about 0.50 M, at least about 0.60 M, at least about 0.70 M, at least about 0.80 M, at least about 0.90 M, at least about 1.00 M, at least about 1.10 M, at least about 1.25 M, at least about 1.50 M, at least about 1.75 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 3.5 M, at least about 4 M, at least about 4.5 M, at least about 5 M, or greater than about 5 M. In some embodiments, a salt, ion, osmolyte, or electrolyte concentration on the first side (e.g., cis side) can be at most about 5 M, at most about 4.5 M, at most about 4 M, at most about 3.5 M, at most about 3 M, at most about 2.5 M, at most about 2 M, at most about 1.75 M, at most about 1.50 M, at most about 1.25 M, at most about 1 M, at most about 0.90 M, at most about 0.80 M, at most about 0.70 M, at most about 0.60 M, at most about 0.50 M, at most about 0.40 M, at most about 0.30 M, at most about 0.20 M, at most about 0.10 M, at most about 0.05 M, at most about 0.01 M, or less than about 0.01 M. WSGR Docket Number: 64828-710.601 [0533] In some embodiments, a salt, ion, osmolyte, or electrolyte concentration on the first side (e.g., cis side) can be from about 0.01 M to about 5 M. In some embodiments, a salt, ion, osmolyte, or electrolyte concentration on the first side (e.g., cis side) can be from about 0.01 M to about 0.1 M, about 0.01 M to about 0.5 M, about 0.01 M to about 1 M, about 0.01 M to about 1.5 M, about 0.01 M to about 2 M, about 0.01 M to about 2.5 M, about 0.01 M to about 3 M, about 0.01 M to about 3.5 M, about 0.01 M to about 4 M, about 0.01 M to about 4.5 M, about 0.01 M to about 5 M, about 0.1 M to about 0.5 M, about 0.1 M to about 1 M, about 0.1 M to about 1.5 M, about 0.1 M to about 2 M, about 0.1 M to about 2.5 M, about 0.1 M to about 3 M, about 0.1 M to about 3.5 M, about 0.1 M to about 4 M, about 0.1 M to about 4.5 M, about 0.1 M to about 5 M, about 0.5 M to about 1 M, about 0.5 M to about 1.5 M, about 0.5 M to about 2 M, about 0.5 M to about 2.5 M, about 0.5 M to about 3 M, about 0.5 M to about 3.5 M, about 0.5 M to about 4 M, about 0.5 M to about 4.5 M, about 0.5 M to about 5 M, about 1 M to about 1.5 M, about 1 M to about 2 M, about 1 M to about 2.5 M, about 1 M to about 3 M, about 1 M to about 3.5 M, about 1 M to about 4 M, about 1 M to about 4.5 M, about 1 M to about 5 M, about 1.5 M to about 2 M, about 1.5 M to about 2.5 M, about 1.5 M to about 3 M, about 1.5 M to about 3.5 M, about 1.5 M to about 4 M, about 1.5 M to about 4.5 M, about 1.5 M to about 5 M, about 2 M to about 2.5 M, about 2 M to about 3 M, about 2 M to about 3.5 M, about 2 M to about 4 M, about 2 M to about 4.5 M, about 2 M to about 5 M, about 2.5 M to about 3 M, about 2.5 M to about 3.5 M, about 2.5 M to about 4 M, about 2.5 M to about 4.5 M, about 2.5 M to about 5 M, about 3 M to about 3.5 M, about 3 M to about 4 M, about 3 M to about 4.5 M, about 3 M to about 5 M, about 3.5 M to about 4 M, about 3.5 M to about 4.5 M, about 3.5 M to about 5 M, about 4 M to about 4.5 M, about 4 M to about 5 M, or about 4.5 M to about 5 M. [0534] In some embodiments, a salt, ion, osmolyte, or electrolyte concentration on the first side (e.g., cis side) can be about 0.01 M, about 0.05 M, about 0.10 M, about 0.20 M, about 0.30 M, about 0.40 M, about 0.50 M, about 0.60 M, about 0.70 M, about 0.80 M, about 0.90 M, about 1.00 M, about 1.10 M, about 1.25 M, about 1.50 M, about 1.75 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M. [0535] In some cases, the concentration of one or more ions on the first side (e.g., cis side) of the membrane can be between about 1 nanomolar (nM) to about 1,000 nM. In some instances, the concentration of one or more ions on the first side (e.g., cis side) of the membrane can be between about 1 nM to about 10 nM, between about 10 nM to about 100 nM, or between about 100 nM to about 1,000 nM. In some cases, the concentration of one or more ions on the first side (e.g., cis side) of the membrane can be at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 30 nM, at least about 35 nM, at least about 40 nM, at least about 45 nM, at least about 50 nM, at least about 55 nM, at least about 60 nM, at least about 65 nM, at least about 70 nM, at least about 75 nM, at least about 80 nM, at least about 85 nM, at least about 90 nM, at least about 95 nM, at least about 100 nM, at least about 150 nM, at least about 200 nM, at least about 250 nM, at least about 300 nM, at least about 350 nM, at least about WSGR Docket Number: 64828-710.601 400 nM, at least about 450 nM, at least about 500 nM, at least about 550 nM, at least about 600 nM, at least about 650 nM, at least about 700 nM, at least about 750 nM, at least about 800 nM, at least about 850 nM, at least about 900 nM, at least about 950 nM, at least about 1,000 nM, or more than 1,000 nM. In some cases, the concentration of one or more ions on the first side (e.g., cis side) of the membrane can at most about 1,000 nM, at most about 950 nM, at most about 900 nM, at most about 850 nM, at most about 800 nM, at most about 750 nM, at most about 700 nM, at most about 650 nM, at most about 600 nM, at most about 550 nM, at most about 500 nM, at most about 450 nM, at most about 400 nM, at most about 350 nM, at most about 300 nM, at most about 250 nM, at most about 200 nM, at most about 150 nM, at most about 100 nM, at most about 95 nM, at most about 90 nM, at most about 85 nM, at most about 80 nM, at most about 75 nM, at most about 70 nM, at most about 65 nM, at most about 60 nM, at most about 55 nM, at most about 45 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, at most about 5 nM, at most about 1 nM, or less than 1 nM. [0536] In some embodiments, the one or more ions can comprise chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, or oxide, or any combinations thereof. [0537] In some embodiments, the one or more ions on the first side (e.g., cis side) of the membrane can comprise chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, or oxide, or any combinations thereof. In some embodiments, the one or more ions on the second side (e.g., trans side) of the membrane can comprise chloride, carbonate, chlorite, chlorate, phosphate, bicarbonate, bromide, ammonium sulfate, ammonium, sulfate, sulfide, calcium, fluoride, hydroxide, aluminum, barium, bismuth, cadmium, cesium, chromium, cobalt, copper, hydrogen, iron, lead, lithium, magnesium, mercury, nickel, potassium, rubidium, silver, sodium, strontium, tin, zinc, iodide, nitride, or oxide, or any combinations thereof. [0538] In some embodiments, the one or more ions on the first side (e.g., cis side) of the membrane can be the same types of ions as the one or more ions on the second side (e.g., trans side) of the membrane. In some embodiments, one or more ions on the first side (e.g., cis side) of the membrane can be different types of ions from the one or more ions on the second side (e.g., trans side) of the membrane. [0539] Salt imbalances on two sides of a pore can create strong osmotic gradients, which can either enhance or compete with EOF depending on the relative direction of the fluid flow. For example, for a high-salt-cis WSGR Docket Number: 64828-710.601 low-salt-trans system that can be set up to create a net cis-to-trans EOF, the osmotic gradient can compete with the EOF. For systems where the osmotic gradient competes with EOF, the low salt compartment can contain an osmolyte to fully or partially balance the osmotic imbalance created by salt concentration asymmetry. In some embodiments, an osmolyte can comprise a non-ionic or a zwitterionic solute, e.g., glycine betaine, glucose, sucrose, glycerol, PEGs, or dextrans, or any combination thereof. For example, a salt imbalance of 0.5 M KCl can be balanced with about 1 M Glycine betaine. Specific osmolytes can be selected and balanced based on their osmolarity and their concentrations. In some embodiments, osmolytes can be added either to symmetrical salt concentration or asymmetric salt concentration systems to create an osmotic gradient that acts in the same direction as the EOF to enhance the capture and/or translocation of an analyte. For example, osmolyte (e.g.1 M glycine betaine) can be added to the trans compartment of an ion-selective pore system (e.g. 1 M K Glu cis and 1 M KGlu trans) to enhance the cis-to-trans EOF. In some embodiments, osmolytes can be added either to symmetrical salt concentration or asymmetric salt concentration systems to create an osmotic gradient that acts in a different direction as the EOF to enhance the capture and/or translocation of an analyte. [0540] In some embodiments, high mobility ions can be used on one side of a membrane and low mobility and/or sterically inhibited counterions on the other side of a membrane. For example, a salt with a high mobility ion can be used on the first side (e.g., cis side) of a membrane, and a salt with a low mobility (counter) ion used on the second side (e.g., trans side) of a membrane to create a stronger cis-to-trans ion-selectivity under an appropriate applied voltage. In some embodiments, low mobility ions comprises all or a part of a total ionic content in the system. In some embodiments, low mobility ions can comprise greater than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the low mobility ions flow across the pore. In some embodiments, low mobility ions can comprise less than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the low mobility ions flow across the pore. In some embodiments, high mobility ions can comprise greater than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the high mobility ions flow across the pore. In some embodiments, high mobility ions can comprise less than about 10, 20, 30, 40, 50, 60, 70, 80, or about 90 percent of the salt content on the side of a membrane from which the high mobility ions flow across the pore. For example, an analyte can be added to the first side (e.g., cis side) and translocated cis-to-trans via a strong cis-to-trans EOF – the system in this instance can be a pore system set up with a highly mobile cation salt on the cis (e.g. K+, Na+, NH4+) and a low mobility anion salt on the trans (e.g. glutamate, or acetate, or any combination thereof), wherein a negative voltage can be applied to the trans. The system can be set up with 1M KCl in the cis compartment and 1M KGlu in trans compartment, so that a greater EOF can be achieved cis-to-trans under negative applied voltage to trans than the EOF generated trans-to-cis WSGR Docket Number: 64828-710.601 when a positive voltage can be applied to the trans, due to relative lower mobility of glutamate anions versus chloride anions. [0541] In some embodiments, immobile and/or sterically hindered ions on one side of the membrane may be combined with ion selective pores to limit the flux of one or more of the ions. The ion permeability of large and/or immobile salts in ion-selective pore systems (e.g. based on mutated pores) can be determined experimentally using the GHK equation under asymmetric salt conditions, to create a system with a sufficiently large net ion flux to capture and translocate complex polymer analytes. In some embodiments, the flux of immobile or sterically hindered ions can be effectively zero under an applied voltage, either for an open state and/or the analyte-filled state of a pore, so that all ionic flux can be in one direction (e.g. cis to trans). In some embodiments, small and/or highly mobile ions on one side of the membrane can be combined with ion selective pores to further increase flux of given ions. For example, small and/or highly mobile cations (e.g. K+, Na+, or NH4+, or any combination thereof) can be combined with ion-selective pores with high internal net negative charge. The net negative charge inside a pore can interact favorably with cations, which can increase the absolute flux of the cations relative to the same pore with less negative charge. Thus, the increased cation flux can increase the relative proportion of net electro-osmotic flux in one direction (e.g. cis-to-trans), and it can increase the absolute net electro-osmotic flux at a given voltage, which can create a stronger EOF versus EPF. [0542] In some embodiments, the EOF can be generated by a cation biased flux through a pore. A cation- biased EOF (P(+) >> P(-)) can be created or enhanced by the choice of salts in either the cis or trans compartments. In some embodiments, EOF can be generated by high mobility monovalent cations such as K+ = NH4+ > Na+ > Li+ > or any combination thereof. High cation biased EOF can be further enhanced by exploiting salts with large anions that may be relatively immobile or otherwise restricted from translocating through a pore. In some embodiments, an anion can comprise high molecular mass inorganic anions (e.g., Br, Phosphate, sulphate, or FeCN6, and or any combination thereof), organic anions (e.g., Acetate, Glutamate, succinate, maleate, or butyrate dextrans, or any combination thereof), or ionic liquid anions (e.g., tetrafluoroborate (BF4), hexafluorophosphate (PF6), bis-trifluoromethanesulfonimide (NTf2), trifluoromethanesulfonate (OTf), dicyanamide (N(CN)2), hydrogen sulphate (HSO4), and ethyl sulphate (EtOSO3)). In some embodiments, where an analyte can be added to the cis and translocated cis-to-trans via a strong cis-to-trans cation biased EOF. A nanopore system described herein can be set up with an ionic salt on the first side (e.g., cis side) and an ionic salt on the second side (e.g., trans side). In some embodiments, a nanopore system described herein can be set up with a cation salt on the first side (e.g., cis side) and an anion salt on the second side (e.g., trans side). In some embodiments, a nanopore system described herein can be set up with a anion salt on the first side (e.g., cis side) and an cation salt on the second side (e.g., trans side). The cation may comprise any positively charged ion (e.g., K+, Ca2+, Na+, H+, H3O+, or NH4+, or any combination thereof). The anion may comprise any WSGR Docket Number: 64828-710.601 negatively charged ion (e.g., Cl-, I-, OH-, O2-, SO 2- 4 , or NO - 3, or any combination thereof). In some embodiments, a nanopore system described herein can be set up with a highly mobile cation salt (e.g. K+, Na+, or NH + 4 , or any combination thereof) on the first side and a low mobility anion salt (e.g. glutamate, acetate, or FeCN6, or any combination thereof) on the trans. A negative voltage may be applied to the first side (e.g., cis side), or the second side (e.g., trans side), or any combination thereof. For example, the cis and trans compartments can contain >0.3 M, >0.5 M, >1.0 M, >2.0 M, >3.0 M, or >4.0 M K Glu under an applied voltage of -40 mV to - 200 mV. In some embodiments, the trans compartment can contain >0.3 M, >0.5 M, >1.0 M, >2.0 M, >3.0 M, or >4.0 M potassium salts of butyrate, tetrafluoroborate, or ferri- or ferro-cyanide. In some embodiments, an analyte may comprise a polycationic tag so that EPF aids in the initial pore capture. [0543] In some embodiments, ion selectivity of an unoccupied pore can closely but not exactly match the state when the pore can be translocating an analyte (which can be variable). This can be in part due to the additional volume that an analyte occupies in the channel of a pore (which can thus create a further barrier to the passage of larger ions), and can also be in part due to any charges on the analyte (e.g. a charged polypeptide) that alters the ion-selectivity of the pore. In some embodiments, a pore can be provided in conditions suitable to create strong selective conditions. In some embodiments where larger ions that may be likely to experience a greater effect from an analyte-filled pore, the selectivity can be determined. [0544] In some embodiments, dominant net EOF can be created either cis-to-trans or trans-to-cis relative to the convention of stating the polarity of the applied voltage at the trans electrode. The EOF can be either cation biased or anion biased. In some embodiments, the direction of the net EOF can determine to which compartment an analyte can be added. In some embodiments, the absolute net electro-osmotic flow across the pore in any direction (which can be directly correlated to the magnitude of the force applied to a translocated analyte) can be dependent on the applied voltage (which, may be varying to a first approximation according to the GHK flux equations). Since EPF can be directly correlated to applied voltage (increasing in magnitude as the voltage can be increased), a pore system can be created with sufficiently large absolute net electro-osmotic flow at relatively low voltages to overcome repulsive EPF forces acting on an analyte . In some embodiments, the absolute relative net electro-osmotic flow over applied voltage (IrelV) can be given by:
Figure imgf000140_0001
[0546] which can be greater than 0.1 pA/mV, greater than 0.2 pA/mV, or greater than 0.3 pA/mV. Therefore, for example, at -80 mV a pore can have >8 pA, >16 pA, or >24 pA of net electro-osmotic flow in one direction. In some embodiments, IrelV can be greater than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or about 1.0 pA/mV. In some embodiments, IrelV can be less than about 0.01, 0.02, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or about 1.0 pA/mV. In some embodiments, a pore can have greater than about 2, 4, 6, 8, 16, 24, 32, or about 64 pA of net electro- WSGR Docket Number: 64828-710.601 osmotic flow in one direction. In some embodiments, a pore can have less than about 2, 4, 6, 8, 16, 24, 32, or about 64 pA of net electro-osmotic flow in one direction. [0547] In some embodiments, a pore can be comprised in a membrane, the membrane separating a fluidic chamber into a first side (e.g., cis side) and a second side (e.g., trans side). In some embodiments, a membrane can comprise a film-like structure. In some embodiments, a membrane than be thinner than a length of a channel of a pore. [0548] In some embodiments, a membrane can be prepared using surfactants. For example, pore monomers can be contacted with surfactant micelles to form pore-micelles which can then be contacted with a lipid bilayer to allow pore formation. Pore-micelles may be subsequently contacted with a second pore monomer to form a multi-component pore-micelle complex. The multi-component pore-micelle complex can then be contacted with a lipid bilayer to allow pore formation. [0549] An analyte can be added to the second side (e.g., trans side) or first side (e.g., cis side) of the pore. In some embodiments, a chamber comprising a membrane comprising a pore can be subjected to an electrical potential such that an analyte can be electrophoretically and/or electroosmotically translocated through the pore. The optimal value (absolute and direction) of the potential may depend on parameters such as the ion-selectivity of the pore, characteristics like size, shape and pI of the analyte, and whether analytes may be captured from the second side (e.g., trans side) or first side (e.g., cis side) of the pore. For instance, for an anion selective pore, at approximately neutral pH, a positive potential may be necessary to capture analytes from the first side (e.g., cis side), whereas for the second side (e.g., trans side) capture a negative potential may be used. The capture frequency and/or dwell time within the pore for a particular analyte may depend on the absolute value of the potential. [0550] In some embodiments, a membrane comprises an amphiphilic layer. In some embodiments, a membrane comprises lipids. In some embodiments, a membrane comprises a lipid bilayer. In some embodiments, a membrane comprises phospholipids. In some embodiments, a membrane comprises a monolayer or a bilayer. In some embodiments, a membrane comprises a bilayer of phospholipids. In some embodiments, a membrane comprises lipids or amphipathic molecules that may be chemically stable under low pH conditions, for example ether-linked phospholipids. In some embodiments, a membrane comprises phosphatidylcholine (PC), e.g., 1,2-diphytanoyl-sn-glycero-3phosphocholine (DPhPC). In some embodiments, a membrane comprises sphingomyelin (SM). In some embodiments, a membrane comprises synthetic or naturally occurring amphiphilic molecules. In some embodiments, a membrane comprises archaeal lipids. In some embodiments, a membrane comprises block copolymers. In some embodiments, a membrane comprises a solid state material (e.g. SiN). For example, a pore may comprise a solid state hole. For example, a pore can comprise a biological pore inserted in a solid state material. In some embodiments, a membrane may be a solid- WSGR Docket Number: 64828-710.601 state membrane. In some embodiments, a membrane may comprise silicon dioxide, silicon nitride, graphene, anodic alumina, polycarbonate, poly(ethylene terephthalate), polytetrafluoroethylene, polyurethane, thermoplastic polyurethane, boron nitride, molybdenum disulfide, transition metal carbide, gold, or platinum, or combinations thereof. In some embodiments, the membrane may comprise multiple layers. In some embodiments, the membrane may be functionalized. In some embodiments, the membrane may be functionalized with a thiol group, a peptide, a nucleic acid, or a biomolecule, or combinations thereof. [0551] In some embodiments, a membrane comprises a lipid. A lipid can comprise a head group, an interfacial moiety and two hydrophobic tail groups which may be the same or different. In some embodiments, a head group comprises a neutral head group, such as diacylglycerides (DG) and ceramides (CM); a zwitterionic head group, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM); a negatively charged head group, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); or a positively charged headgroup, such as trimethylammonium-Propane (TAP). In some embodiments, an interfacial moiety comprises a naturally- occurring interfacial moiety, such as glycerol-based or ceramide-based moiety. In some embodiments, a hydrophobic tail group comprises a saturated hydrocarbon chain, such as lauric acid (n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmitic acid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic (n-Eicosanoic); an unsaturated hydrocarbon chain, such as oleic acid (cis-9-Octadecanoic); or a branched hydrocarbon chain, such as phytanoyl. The length of the chain and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary. The length of the chains and the position and number of the branches, such as methyl groups, in the branched hydrocarbon chains can vary. The hydrophobic tail groups can be linked to the interfacial moiety as an ether or an ester. [0552] A lipid can also be chemically-modified. The bead group or the tail group of a lipid may be chemically- modified. In some embodiments, a lipid comprises a chemically-modified head group, e.g., PEG-modified lipids, 1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]; functionalized PEG Lipids, such as 1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N- [Biotinyl(Polyethylene Glycol)2000]; and lipids modified for conjugation, such as 1,2-Dioleoyl-sn-Glycero-3- Phosphoethanolamine-N-(succinyl) and 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). In some embodiments, a lipid comprises a chemically-modified tail group, e.g., polymerisable lipids, such as 1,2- bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinated lipids, such as l-Palmitoyl-2-(16- Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine; deuterated lipids, such as 1,2-Dipalmitoyl-D62-sn-Glycero-3- Phosphocholine; or ether linked lipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. [0553] In some embodiments, a membrane may be about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or about 150 nm thick. In some embodiments, a membrane WSGR Docket Number: 64828-710.601 may be between 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, or 9 and 10 nm thick. In some embodiments, a membrane may be at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or about 150 nm thick . In some embodiments, a membrane may be at less than about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or about 150 nm thick. [0554] In some embodiments, the system may further comprise methods for measuring a signal based on ionic current flowing through the nanopore during a period of time of translocation. These measuring mechanisms are set up to detect changes in the signal that reflect characteristics of the analyte as it can be translocated. In some embodiments, the analyte measured can be a protein. In some cases, the characteristic of the protein measured can comprise an amino acid sequence of the protein, one or more post-translational modifications of the protein, amino acid mutations in the sequence of the protein, domain structures of the protein, length of the protein, net charge of the protein, or conformation of the protein. In some embodiments, the analyte measured can be a nucleic acid molecule. In some cases, the characteristic of the nucleic acid molecule measured can comprise a nucleotide sequence of the nucleic acid molecule, nucleotide mutations in the sequence of the nucleic acid molecule, methylation of the nucleic acid molecule, acetylation of the nucleic acid molecule, length of the nucleic acid molecule, net charge of the nucleic acid molecule, or conformation of the nucleic acid molecule. In some embodiments, the analyte measured can be an oligosaccharide. In some cases, the characteristic of the oligosaccharide measured can comprise a sequence of the oligosaccharide, the length of the oligosaccharide, the net charge of the oligosaccharide, presence or absence of coupled lipids, presence or absence of coupled peptides, or structure of the oligosaccharide. In some embodiments, the analyte measured can be a lipid molecule. In some cases, the characteristic of the lipid measured can comprise length of the lipid, the net charge of the lipid, or a structure of a lipid. [0555] In some embodiments, methods are provided relating to the analysis of an analyte. In some embodiments, a change in ionic current can be measured while the analyte translocates through the nanopore. In some cases, the change in ionic current can be measured by a voltage based chip. In some cases, the voltage based chip can measure the voltage and/or change in current across the nanopore. In some cases, the voltage based chip can be a trans electrode (e.g. electrodes adjacent to the membrane/nanopore to measure the voltage across the nanopore). [0556] The characterization methods may involve measuring the ion current flow through the pore, typically by measurement of a current. Alternatively, the ion flow through the pore may be measured optically. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. The characterization methods may be carried out using a patch clamp or a voltage clamp. The characterization methods can involve the use of a voltage clamp. WSGR Docket Number: 64828-710.601 [0557] In some embodiments, an analyte comprises a polymer analyte. The analyte can comprise a nucleic acid based polymer analyte or a non-nucleic acid based polymer analyte. The analyte can be of synthetic, semi- synthetic, or biological origin. For example, a synthetic analyte may comprise an analyte constructed by a non- biological chemical process, such as polyethylene glycol (PEG), or a synthetically constructed DNA molecule. For example, a synthetic analyte may comprise an analyte constructed by a non-biological chemical process, such as polyethylene glycol (PEG), synthetically constructed peptides of proteins, or a synthetically constructed DNA molecule. A biological analyte can comprise an analyte produced by a biological process, such as a protein produced by a cell or by systems employing cellular (or cellular derived) components (e.g. enzymatic in vitro translation systems). A biological analyte can comprise an analyte produced by a biological process, such as a protein produced by a cell. A semi-synthetic analyte can comprise portions created by biological and non- biological origins, for example, a biologically-produced protein conjugated to a PEG molecule. Possible electrical measurements can include current measurements, impedance measurements, tunneling, electron tunneling measurements (Ivanov AP et al., Nano Lett. 201 1 Jan 12; 1 l(l):279-85), FET measurements (International Application WO2005/124888), or voltage FET measurements, or any combination thereof. In some embodiments, the signal may be electron tunneling across a solid state nanopore or a voltage FET measurement across a solid state nanopore. [0558] The characterization methods may involve measuring the ion current flow through the pore, by measurement of a current. Alternatively, the ion flow through the pore may be measured optically. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. The characterization methods may be carried out using a patch clamp or a voltage clamp. The characterization methods preferably involve the use of a voltage clamp. The characterization methods may be carried out on an array of nanopores or wells where each array comprises 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more nanopores or wells. The characterization methods may involve the measuring of a current flowing through the pore. The method can be typically carried out with a voltage applied across the membrane and pore. The voltage used can be typically from +2 V to -2 V, typically -400 mV to +400mV. The voltage used can be in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from +10 mV, 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used can be in the range 20 mV to 240mV, or the the voltage can be in the range of 120 mV to 220 mV. It can be possible to increase discrimination between different characteristics of the analyte by a pore by using an increased applied potential. [0559] In some embodiments, an analyte comprises a polymer analyte. The analyte can comprise a nucleic acid based polymer analyte or a non-nucleic acid based polymer analyte. The analyte can be of synthetic, semi- WSGR Docket Number: 64828-710.601 synthetic, or biological origin. For example, a synthetic analyte may comprise an analyte constructed by a non- biological chemical process, such as polyethylene glycol (PEG), or a synthetically constructed DNA molecule. A biological analyte can comprise an analyte produced by a biological process, such as a protein produced by a cell. A semi-synthetic analyte can comprise portions created by biological and non-biological origins, for example, a biologically-produced protein conjugated to a PEG molecule. [0560] In some embodiments, an analyte comprises a protein (e.g., a polypeptide) or peptide. A protein or peptide can comprise a folded state, an unfolded state, or intermediate states thereof (e.g., a partially unfolded state). A folded state comprises a state of a protein or peptide in which the polymer can be at a low-energy state such that the protein or peptide maintains a two or three dimensional structure. This low-energy state can be based on the interactions of the amino acids of the peptide or protein with each other. An unfolded state can comprise a state of a protein or peptide in which the polymer can be at a high-energy state such that the protein or peptide does not maintain a two or three dimensional structure. An intermediate state between a folded and unfolded state can be an energy state at which a portion or portions of the peptide or protein may maintain a two or three dimensional structure, and other portions of the peptide or protein may not maintain a two or three dimensional structure. In some embodiments, a protein (e.g., polypeptide) can comprise a folded protein structure. In some embodiments, a peptide can comprise a linear structure. In some cases, a peptide can comprise a portion of a protein. [0561] In some embodiments, methods described herein may comprise translocating a plurality of analytes. The plurality of analytes may be translocated through (i) a nanopore disposed in a membrane, (ii) a plurality of nanopores disposed in a membrane, or (iii) a plurality of nanopores disposed in a plurality of membranes, or (iv) any combination thereof. In some embodiments, each nanopore of the plurality of nanopores disposed in a plurality of membranes may be disposed in the same membrane. In some embodiments, each nanopore of the plurality of nanopores disposed in a plurality of membranes may be disposed in different membranes. In some embodiments, at least a portion of each analyte of a plurality of analytes may be translocated through the same pore. In some embodiments, at least a portion of each analyte of a plurality of analytes may be translocated through a different pore. The plurality of analytes can comprise a plurality of non-nucleic acid based polymer analytes (e.g., a plurality of proteins, a plurality of polypeptides, a plurality of peptides, or any combination thereof). [0562] In some embodiments, a plurality of signals or changes thereof (e.g., plurality of currents or changes thereof) may be detected from the plurality of analytes (e.g., the plurality of non-nucleic acid based polymer analytes). The plurality of signals or changes thereof (e.g., plurality of currents or changes thereof) may be used to generate a plurality of characteristics associated with the plurality of analytes. A characteristic of the plurality of characteristics can comprise a characteristic of an analyte described herein. In some embodiments, the WSGR Docket Number: 64828-710.601 plurality of characteristics comprises differences between at least a subset of analytes of a plurality of analytes. The plurality of characteristics can comprises one or more differences in sequence. The differences in sequence can be of at least about 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 10 amino acids, or greater than about 10 amino acids. The differences in sequence can be of at most about 10 amino acids, at most about 5 amino acids, at most about 4 amino acids, at most about 3 amino acids, at most about 2 amino acids, at most about 1 amino acid, or less than about 1 amino acid. The difference in sequence may be of at most 10 units between least the subset of analytes. [0563] In some embodiments, a relative concentration and/or an absolute concentration of one or more analytes may be determined. The relative concentration and/or absolute concentration of one or more analytes may be a concentration in a plurality of analytes. In some cases, a plurality of analytes may comprise a subset (e.g., percentage) of analytes that can be modified. In some embodiments, a percentage of modified analytes (e.g., different analytes) may be determined from a plurality of analytes. [0564] In some embodiments, one or more analytes may be captured by a nanopore with an EOF. The one or more analytes may be captured by a nanopore with an EOF and/or a motor protein (e.g., a translocase). The translocase may comprise a translocase as described herein. The translocase may unfold the one or more analytes and/or assist in translocating the analyte through the nanopore. [0565] FIGs. 138A-138E illustrate a system for translocating an analyte (e.g., a peptide, polypeptide, or protein, or fragment thereof, or combination thereof) through a nanopore. The analyte may be captured from a second side (e.g., a trans side). In some embodiments, an analyte may be captured on one side of a nanopore system and translocated by a motor protein (e.g., a translocase). The analyte may be translocated with a motor protein in a same direction as a net EOF. The analyte may be translocated with a motor protein in a different direction as a net EOF. For example, a target protein may be translocated as shown in FIGs. 138A-138E by operations of (i) adding the analyte (7) to a second side (e.g., a trans side) and one or more motor proteins (8) to a first side (e.g., a cis side) (FIG. 138A). A voltage may be applied and/or set up in the nanopore system to provide an EOF (e.g., a strong net trans-to-cis EOF). An analyte may be captured from a second side (e.g., a trans side) via EOF and/or electrophoresis acting with the analyte and/or a leader construct (1). The analyte may be able to translocate under the strong net trans-to-cis EOF until encountering a stopper domain (2). A stopper domain may be coupled to a C-terminus of an analyte. A stopper domain may be coupled to a N- terminus of an analyte. The stopper domain may prevent further translocation of the analyte through the nanopore (FIG.138B). In some embodiments, the translocase in the first side (e.g., cis side) of the system may bind to a leader construct and/or at least a portion of the analyte (FIG. 138C). An applied voltage may be reversed. The reversal of the applied voltage may switch the direction of a net EOF of the system. For example, WSGR Docket Number: 64828-710.601 reversing the voltage may switch a net EOF from a trans-to-cis direction to a cis-to-trans direction. An analyte may translocate back through to a side of the system (e.g., a trans side) after reversal of the voltage. A bound motor protein (e.g., translocase) may contact a top (e.g., opening) of a nanopore (FIG.138D). The translocase on the top of the nanopore may continue to translocate along an analyte. The analyte may be translocated in a direction opposite to a net EOF. For example, as the translocase rests on the top of a nanopore, the translocase may translocate an analyte in a trans-to-cis direction against the net cis-to-trans direction of the EOF. The net cis-to-trans direction of the EOF may maintain a stretch of the analyte and/or maintain the translocase against the nanopore (FIG. 138D). The analyte may be released on the first side (e.g., cis side) of the membrane if (i) the translocase can overcome the stopper domain and/or (ii) the voltage can be reversed to unbind the translocase and/or remove the analyte from the pore. [0566] Leader constructs can comprise one or more highly charged polymeric capture motifs as described herein. For example, a capture motif may comprise high polyanion (if negative voltage may be applied at a second side (e.g., trans side)) or polycation (if positive voltage may be applied at a second side (e.g., trans side)) content. For polycation capture motif, the motif may comprise (R)n, (K)n, or (H)n, or variations thereof, or any combination thereof. For polyanion capture motif, the motif may comprise (D)n, or (E)n, or variations thereof, or any combination thereof. Amino acid sequences of leader constructs are shown in Table 45. Table 45. Amino acid sequences of leader constructs. Leader SEQ ID number Leader description Leader sequence NO. *-linker-HTH-G12-R15- GSGSGSGDELAQLERELMKLKAQGVDSDELEALARKLAMLARGGGGGGG 187 1 ssrA GGGGGRRRRRRRRRRRRRRRAANDENYALAA *-linker-Alpha-G12- GSGSGSGDYMERWYRYYNEFGGGGGGGGGGGGRRRRRRRRRRRRRRRAA 188 2 R15-ssrA NDENYALAA *-linker-Beta-G12-R15- GSGSGSGRGKITVNGKTYEGRGGGGGGGGGGGGRRRRRRRRRRRRRRRA 189 3 ssrA ANDENYALAA 4 *-G18-R15-ssrA GGGGGGGGGGGGGGGGGGRRRRRRRRRRRRRRRAANDENYALAA 190 GGGGGGGGGGGGGGGGGGGGGGGGRRRRRRRRRRRRRRRAANDENYALA 191 5 *-G24-R15-ssrA A 6 *-G12-S5-R10-ssrA GGGGGGGGGGGGSSSSSRRRRRRRRRRAANDENYALAA 192 7 *-G12-S10-R5-ssrA GGGGGGGGGGGGSSSSSSSSSSRRRRRAANDENYALAA 193 8 *-G12-S15-R0-ssrA GGGGGGGGGGGGSSSSSSSSSSSSSSSAANDENYALAA 194 9 *-G12-K15-ssrA GGGGGGGGGGGGKKKKKKKKKKKKKKKAANDENYALAA 195 10 *-G12-R(KR)7-ssrA GGGGGGGGGGGGRKRKRKRKRKRKRKRAANDENYALAA 196 11 *-G12-R(GR)7-ssrA GGGGGGGGGGGGRGRGRGRGRGRGRGRAANDENYALAA 197 GGGGGGGGGGGGRGRGRGRGRGRGRGRGRGRGRGRGRGRGRAANDENYA 198 12 *-G12-R(GR)14-ssrA LAA 13 *-G12-K(GK)7-ssrA GGGGGGGGGGGGKGKGKGKGKGKGKGKAANDENYALAA 199 GGGGGGGGGGGGKGKGKGKGKGKGKGKGKGKGKGKGKGKGKAANDENYA 200 14 *-G12-K(GK)14-ssrA LAA 15 *-G12-R15-NYALAA GGGGGGGGGGGGRRRRRRRRRRRRRRRNYALAA 201 16 *-G12-R15-YALAA GGGGGGGGGGGGRRRRRRRRRRRRRRRYALAA 202 17 *-G12-R15-ALAA GGGGGGGGGGGGRRRRRRRRRRRRRRRALAA 203 WSGR Docket Number: 64828-710.601 18 *-G12-R15-LAA GGGGGGGGGGGGRRRRRRRRRRRRRRRLAA 204 19 *-G12-H15-ssrA GGGGGGGGGGGGHHHHHHHHHHHHHHHAANDENYALAA 205 20 *-G12-H(GH)7-ssrA GGGGGGGGGGGGHGHGHGHGHGHGHGHAANDENYALAA 206 GGGGGGGGGGGGHGHGHGHGHGHGHGHGHGHGHGHGHGHGHGHAANDEN 207 21 *-G12-H(GH)15-ssrA YALAA *G12-R15- 208 22 ALEQNRRKKAI GGGGGGGGGGGGRRRRRRRRRRRRRRRLEQNRRKKAI 23 *G12-R15-ANRRKKAI GGGGGGGGGGGGRRRRRRRRRRRRRRRNRRKKAI 209 24 *G24-ANRRKKAI GGGGGGGGGGGGGGGGGGGGGGGGNRRKKAI 210 [0567] A stopper domain may comprise an electrostatic domain that can prevent further translocation of an analyte or at least a portion of an analyte. The at least a portion of the analyte may be prevented from translocating after another portion of the analyte and/or a leader construct have translocated through the nanopore. A stopper domain may comprise one or more steric stopper motifs comprising GFP, titin, or maltose binding protein (MBP), or any combination thereof. A stopper domain may comprise a plurality of folded proteins that may fulfill two criteria: (i) the proteins may be too large to pass through a nanopore without being unfolded, and/or (ii) the proteins may be able to be unfolded by the force of a translocase pulling the analyte or at least a portion of an analyte into the nanopore after it binds on the opposite side of the membrane. A stopper domain may comprise one or more electrostatic stopper motifs comprising one or more regions of opposite charge to the capture motif. For example, if a capture motif may be polyanionic, then a stopper domain may be is polycationic. These “dipole” combination of one or more charges can prevent full translocation as the one or more charges between a capture motif and a stopper motif can balance under electrophoresis at the mid-point when both may be in the nanopore. At this point, an analyte may be left to reside in the port. A stopper domain can comprise one or more steric motifs, or one or more electrostatic motifs, or variants thereof, or any combination thereof. [0568] FIG.139 shows a schematic of the electrophysiology signal obtained from implementing a nanopore system described herein and as depicted in FIGs.138A-138E. State (i) can correspond to an unoccupied open- pore state; state (ii) can correspond to a capture of an analyte and/or at least a portion of an analyte. The analyte may be captured by a leader construct under electrophoretic forces (and against the EOF) into the second side (e.g., trans side) of the nanopore. This may lead to an block (e.g., an instantaneous block) measured in the current (e.g., state (iii)). The nanopore system may detect state (ii) and may hold a system static for the duration of state (iii) to enable one or more translocases to bind to at least a portion of a leader construct in a first side (e.g., cis side). In state (iv), a system may reverse a voltage and an EOF may be reversed. The analyte may then translocates in an opposite direction (e.g., a cis-to-trans direction) quickly until a bound translocase contacts the top of the nanopore. In state (v), the translocase may pull the analyte out of the nanopore in a direction (e.g., WSGR Docket Number: 64828-710.601 a trans-to-cis direction), which may lead to sequence of changing current levels representative of the composition of the analyte (state v) until the analyte is pulled out of the nanopore and exits (state vi). [0569] FIGs. 140A-140E illustrate a system for translocating an analyte (e.g., a peptide, polypeptide, or protein, or fragment thereof, or combination thereof) through a nanopore. The analyte may be captured from a side (e.g., a trans side). In some embodiments, an analyte may be captured on one side of a nanopore system and translocated by one or more motor proteins (e.g., one or more translocases). The analyte may be translocated with a motor protein in a same direction as a net EOF (e.g., a net cis-to-trans EOF). For example, in FIG.140A, an analyte may be added to a trans side of a system and a motor protein (e.g., a translocase) may be added to a cis side. A voltage may be applied to the system to establish a net trans-to-cis EOF. The analyte may be captured from a second side (e.g., a trans side) via EOF and/or an electrophoretic force (EPF) acting on the analyte and/or a leader construct. The analyte may be able to translocate under the strong net trans-to-cis EOF until encountering a stopper domain (2) (FIG. 140B). In some embodiments, one or more motor proteins (e.g., one or more translocases) may be present in a first side (e.g., a cis side). The one or more translocases may bind along at least one portion of an analyte (FIG.140C). An applied voltage may be reversed. The reversal of the applied voltage may switch the direction of a net EOF of the system. For example, reversing the voltage may switch a net EOF from a trans-to-cis direction to a cis-to-trans direction. An analyte may translocate back through to a side of the system (e.g., a trans side) after reversal of the voltage. A bound motor protein (e.g., translocase) may contact a top (e.g., opening) of a nanopore (FIG. 140D). The analyte may be released on the first side (e.g., cis side) of the membrane if (i) the translocase can overcome the stopper domain and/or (ii) the voltage can be reversed to unbind the translocase and/or remove the analyte from the pore (FIG.140E). [0570] FIG.141 shows a schematic of the electrophysiology signal obtained from implementing the system described herein and as depicted in FIGs. 140A-140E. State (i) can correspond to the unoccupied open-pore state, state (ii) can correspond to a capture of an analyte via its leader construct under electrophoretic forces and/or against an EOF into the nanopore. The system can detect state (ii) and may hold the system static for a duration of state (iii). Holding the system static may enable translocases to bind to the portion of the leader in a first side (e.g., a cis side). In state (iv), a system may then reverse a voltage and the EOF can be reversed. The reversal of EOF may translocate the analyte in an opposite direction (e.g., cis-to-trans) very quickly until one or more bound translocases contact the top of a nanopore. In state (v), the one or more translocases can control the translocation of the analyte into the nanopore cis-to-trans and may lead to sequence of changing current levels representative of the composition of the analyte (state v) until the analyte exits to the cis side (state vi). [0571] FIGs. 142A-142E illustrate a system for translocating an analyte (e.g., a peptide, polypeptide, or protein, or fragment thereof, or combination thereof) through a nanopore. The analyte (7) may be captured from a side (e.g., a trans side). In some embodiments, an analyte may be captured on one side of a nanopore system WSGR Docket Number: 64828-710.601 and translocated by one or more motor proteins (e.g., one or more translocases). The analyte may be translocated with a motor protein (8) in a same direction as a net EOF (e.g., a net cis-to-trans EOF). The analyte may be translocated with a motor protein in a different direction as a net EOF. An analyte may be added to a second side (e.g., a trans side) and a motor protein to a first side (e.g., a cis side) (FIG. 142A). A voltage may be applied or set up in the system so that a strong net cis-to-trans EOF can be applied to the system. An analyte can be captured from the second side (e.g., trans side) via electrophoresis acting on a charged leader construct (1) comprising a stopper domain (2). The stopper domain may allow at least a portion of an analyte to translocate across the nanopore so that a portion of the substrate may be exposed to the cis solution (FIG. 142B). The motor protein (e.g., translocase) in a first side (e.g., cis side) may bind to at least a portion of a leader. In some embodiments, after binding, the motor protein may translocate along the analyte until it contacts an opening of a nanopore (FIG. 142C). The translocase may overcome a stopper domain and pull an analyte through the nanopore in a direction that can be opposite to the EOF. For example, a translocase may overcome a stopper domain and pull an analyte through the nanopore in a trans-to-cis direction against a net cis-to-trans EOF (FIG. 142D). The analyte may be released on the first side (e.g., cis side) (FIG.142E). [0572] FIG.143 shows a schematic of the electrophysiology signal obtained from implementing the system described herein and in FIGs. 142A-142E. State (i) corresponds to the unoccupied open-pore state, state (ii) corresponds to the capture of an analyte via its leader under electrophoretic forces (and against the EOF) into the nanopore. This capture may lead to an instantaneous block measured in the current (state iii). During state (iii), one or more translocases can bind to at least a portion of a leader construct in the first side (e.g., cis side) compartment and begin to translocate along the polypeptide (in the cis-to-trans direction). When the translocase contacts the top of the nanopore it begin to start pulling the polypeptide through the nanopore trans-to-cis (state iv) leading to sequence of changing current levels representative of the composition of the polypeptide (state v) until the polypeptide is pulled out of the nanopore and exits to the cis side (state vi). [0573] FIGs. 144A-144D illustrate a system for translocating an analyte (e.g., a peptide, polypeptide, or protein, or fragment thereof, or combination thereof) through a nanopore. The analyte (7) may be captured from a side (e.g., a cis side). In some embodiments, an analyte may be captured on one side of a nanopore system and translocated by one or more motor proteins (e.g., one or more translocases). The analyte may be translocated with a motor protein (8) in a same direction as a net EOF (e.g., a net cis-to-trans EOF). For example, an analyte may be added to a first side (e.g., a cis side) and one or more motor proteins may be added to the first side (e.g., a cis side). One or more motor proteins (e.g., one or more translocases) may bind to at least a portion of an analyte. The bound one or more translocases may translocate along the analyte (e.g., forming a “train of enzymes” along the analyte) (FIG.144A). A voltage may be applied and/or a system may be set up to provide a net EOF (e.g., a net cis-to-trans EOF) to the system. An analyte and/or at least a portion of an analyte may be WSGR Docket Number: 64828-710.601 captured with one or more bound translocases. The analyte:translocase complex may be capture from a first side (e.g., a cis side) via an EOF and/or EPF which may act on the analyte. In some cases, the EOF and/or EPF can act on an analyte comprising a leader construct. The analyte may be translocated near a nanopore. One or more translocases may contact a nanopore (FIG.144B). The one or more translocases may continue to control translocation of the analyte and/or at least a portion of the analyte. The at least a portion of the analyte may translocate into the nanopore in a same direction as a net EOF (e.g., both in a cis-to-trans direction) (FIG. 144C). The analyte may be released on a first side (e.g., a cis side) (FIG. 144D) and/or an analyte may be released on a second side (e.g., a trans side). FIG.145 shows a schematic of the electrophysiology signal obtained from implementing the system described herein and in FIGs.144A-144D. State (i) corresponds to the unoccupied open-pore state, state (ii) corresponds to the capture of the analyte with one or more translocases bound, which may continue to control the translocation of the analyte cis-to-trans (state iii) until the analyte exits the nanopore on the trans side (state iv). DEVICES [0574] In some aspects, the present disclosure provides systems, apparatus, instruments, devices, components, and/or elements comprising a pore, a membrane, an analyte, a sample, a processor, and/or a computer disclosed herein. In some aspects, the present disclosure provides systems, apparatus, instruments, devices, components, and/or elements for implementing a method disclosed herein. [0575] In some embodiments, a device can provide an electric potential (e.g., from an electrical energy source such as a battery) across a membrane. For example, a pore can be embedded in a membrane, and the membrane can be provided in a device, wherein the membrane provides a partition between two solutions. FIGS. 71A- 71B illustrate a device, in accordance with some embodiments. One or both of the solutions can comprise an electrolyte, and an electric potential can be applied across the membrane such that a net ionic current can be generated from one side of the membrane to the other. The net ionic current can be a result of ions in the electrolyte passing through the pore, in response to the applied electric potential. In some embodiments, the ionic current can be faradaic current. [0576] The net ionic current can be expected to maintain a constant value when the electric potential and other environmental variables are held constant. However, when an analyte can be captured by a pore, the analyte can translocate through the pore from one side of the membrane to another. During the translocation, the analyte can create a perturbation in the net ionic current. The perturbation can arise from multiple mechanisms, including at least one of: (1) steric repulsion of ions from the channel – the analyte can physically obstruct a pore channel, thereby inhibiting or prohibiting ions from passing through the channel; (2) electrostatic attraction/repulsion of ions from/to the channel – the analyte can carry electrostatic charges which repel like- WSGR Docket Number: 64828-710.601 charged ions, and can attract ions to the channel – an ion may “piggy-back” with the analyte through a pore; (3) induction of conformational change of the pore – the presence of an analyte can change the conformation of a pore, which can promote or demote ion passage through the channel. Thus, an analyte passing through a pore can cause net ionic current to be perturbed. In some embodiments, a device can be configured to measure a perturbation of the net ionic current. [0577] The perturbation created by a translocation event can be measured in one or more ways. One method can be to measure the ionic current from one side of the membrane to the other side. Another method can be to measure electric potential from one side to the other side. In some embodiments, the impedance can be measured. In some embodiments, the conductivity can be measured. In some embodiments, current rectification can be measured. In some embodiments, an optical signal can be measured. In some embodiments, a tunneling current can be measured. In some embodiments, fluorescence probes for reporting ionic flux or field effect transistor systems can be used to measure properties of a translocation event. In some embodiments, surface enhanced Raman, plasmonics, or other spectroscopic techniques can be used to measure the property of the analyte, the pore, or both, directly. In some embodiments, perturbations can be measured without the application of an electric potential, e.g., a chemical gradient of ions and/or analytes can provide the driving force for translocation of analytes and create measurable signals. In some embodiments, the applied potential can be a chemical potential, electric potential, or another form of potential that can be sufficient to provide a gradient for analyte translocation and measurable perturbations. A device can comprise electrodes, spectroscopy tools, or microscopes, or any combination thereof to measure the signals. [0578] An applied electric potential can be maintained at a constant or fluctuating voltage for a fixed period (milliseconds, seconds, minutes, hours). In some embodiments, the voltage can be changed in discrete steps to alter the sensing conditions and/or obtain different information from the analytes. The voltage can be constantly changing, such as periodic waveforms (e.g. square wave, triangular wave, or sinusoidal, or any combination thereof). Waveforms of different amplitudes, frequencies, and shapes can be used to translocate analytes, which can produce different signals from the same analytes. The additional information provided by the different signals can provide additional dimensionality for distinguishing one analyte from another. In some embodiments, an applied electric potential can have a voltage from +50 mV to -50 mV, or +100 mV to -100 mV. In some embodiments, an applied electric potential can have a voltage of at least about -300 mV, -300 mV, -150 mV, -100 mV, -50 mV, -20 mV and about 0 mV. In some embodiments, an applied electric potential can have a voltage of at most about +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, about +300 mV. In some embodiments, an applied electric potential can have a voltage of +/-50 mV to +/-150 mV or +/- 50 mV to +/-100 mV. A device can comprise one or more active electronics to provide a constant or a modulating electric potential. WSGR Docket Number: 64828-710.601 [0579] In some embodiments, measurements can be made using a single channel recording equipment. In some embodiments, measurements can be made using multi-channel systems. In some embodiments, measurements can be made using multi-channel systems that may be capable of simultaneously acquiring signals from multiple independent pore systems (e.g. a plurality of membranes containing inserted pores). A recording equipment or multi-channel system can comprise integrated circuits, e.g., an ASIC, and/or transistor, e.g., CMOS. [0580] Thus, a device can comprise a pore embedded in a membrane, and a source for applying an electrical potential to the membrane, and a recording device. The device can be used to identify any one of the analytes disclosed herein. [0581] In some embodiments, a device can comprise a sensor. In some embodiments, a sensor can comprise a membrane and a pore. In some embodiments, a device can comprise an array of sensors. In some embodiments, a device can be configured to receive a sample. In some embodiments, a sensor can be configured to contact a sample. In some embodiments, a device can be configured to process the sample. In some embodiments, a sensor can comprise a membrane and a pore. In some embodiments, a sensor can comprise a membrane and an array of pores disposed in the membrane. In some embodiments, an array of pores may comprise more than about 1, 2, 4, 8, 16, 24, or 32 pores. In some embodiments, a sensor can comprise an array of membranes. In some embodiments, a sensor can comprise an array of different membranes. In some embodiments, a sensor can comprise an array of different pores. In some embodiments, a sensor can comprise an array of identical membranes. In some embodiments, a sensor can comprise an array of identical pores. In some embodiments, a sensor can comprise two or more electrodes. In some embodiments, an electrode comprises a sensing electrode. In some embodiments, an electrode comprises an anode or a cathode. In some embodiments, a sensor comprises a plurality of electrodes, comprising at least anodes and cathodes. In some embodiments, a sensor can comprise two sensing electrodes. In some embodiments, a sensor can comprise a voltage sensor configured to measure a voltage difference between the two sensing electrodes. In some embodiments, a sensor can comprise a current sensor configured to measure a current between the two sensing electrodes. In some embodiments, a sensor can comprise a current sensor configured to measure a difference in current between the two sensing electrodes. In some embodiments, a sensor can comprise a FET sensor. In some embodiments, the sensor electrodes can be located near the nanopore or membrane to sense the local voltage. In some embodiments, a sensor may be a part of a sensor array. In some embodiments, each sensor in a sensor array comprises its own set of one or more electrodes. The one or more electrodes can be electrically coupled to a channel in the sensor array. In some embodiments, a device comprises multiple different pores to generate different characteristic signals from analytes, which signal can be compared or combined to improve their discrimination and/or characterization. In some embodiments, a device can be configured to perform single molecule analysis. WSGR Docket Number: 64828-710.601 [0582] In some embodiments, a membrane may have a surface area from about 5 nm2 to about 1000 mm2. In some embodiments, a membrane may have a surface area less than about 5nm2. In some embodiments, a membrane may have a surface area greater than about 1000 mm2. In some embodiments, a membrane may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2. In some embodiments, a membrane may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 μm2. In some embodiments, a membrane may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2. [0583] In some embodiments, a combined surface area of a plurality of membranes may have a surface area from about 5 nm2 to about 1000 mm2. In some embodiments, a combined surface area of a plurality of membranes may have a surface area less than about 5nm2. In some embodiments, a combined surface area of a plurality of membranes may have a surface area greater than about 1000 mm2. In some embodiments, a combined surface area of a plurality of membranes may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2. In some embodiments, a combined surface area of a plurality of membranes may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 μm2. In some embodiments, a combined surface area of a plurality of membranes may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2. [0584] In some embodiments, a pore may have a surface area on a membrane of more than about 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 36, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450 or about 500 nm2. In some embodiments, a pore may have a surface area on a membrane of less than about 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 36, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450 or about 500 nm2. [0585] In some embodiments, each sensor in the sensor array can be fluidically coupled to the same fluidic system. In some embodiments, at least two sensors in the sensor array can be fluidically coupled to different fluidic systems. In some embodiments, the device can provide the same electrolyte solution to a side of the membrane of each sensor in the sensor array. In some embodiments, the device can provide a different WSGR Docket Number: 64828-710.601 electrolyte solution to a side of the membrane of each sensor in the sensor array. In some embodiments, the fluid environment of each sensor in the sensor array can be controlled individually. In some embodiments, each sensor in the sensor array can be fluidically isolated from one another. In some embodiments, at least two sensors in the sensor array can be in fluid communication on at least one side of the membrane of at least two sensors. [0586] In some embodiments, a sensor array comprises a plurality of chambers. In some embodiments, a sensor array comprises a plurality of chambers, wherein a plurality of membranes form a plurality of surfaces of the plurality of chambers. In some embodiments, a plurality of electrodes may be disposed on a second plurality of surfaces within the plurality of chambers. In some embodiments, the sensor array comprises an adhesive configured to adhere the plurality of membranes to the plurality of chambers. The terms “compartment” and “chamber” can be used interchangeably. [0587] In some embodiments, a plurality of chambers comprise at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 μL in volume. In some embodiments, a plurality of chambers comprise less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or about 300 μL in volume. In some embodiments, a plurality of chambers comprise a thickness of less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.50, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.25, 4.50, 4.75, or about 5 mm. In some embodiments, a plurality of chambers comprise a thickness of greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.25, 2.50, 2.75, 3.0, 3.25, 3.50, 3.75, 4.0, 4.25, 4.50, 4.75, or about 5 mm. [0588] In some embodiments, a chamber of the plurality of chambers comprise at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, 1000, 5000 or about 10000 nL in volume. In some embodiments, a chamber of the plurality of chambers comprise less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 750, 1000, 5000 or about 10000 nL in volume. [0589] In some embodiments, a plurality of surfaces may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2. In some embodiments, a plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 μm2. In some embodiments, a plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2. WSGR Docket Number: 64828-710.601 In some embodiments, a plurality of surfaces may have a surface area greater than about 1000 mm2. In some embodiments, a plurality of surfaces may have a surface area less than about 5 nm2. [0590] In some embodiments, a surface of the plurality of surfaces may have a surface area from about 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 nm2. In some embodiments, a surface of the plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 μm2. In some embodiments, a surface of the plurality of surfaces may have a surface area from about 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40 , 40 to 50 , 50 to 60, 60 to 70, 70 to 80, 80 to 90, 90 to 100, 100 to 125, 125 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 750, or 750 to about 1000 mm2. [0591] In some embodiments, the plurality of membranes comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or about 1000000 pores. In some embodiments, the plurality of membranes comprises less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, or about 1000000 pores. [0592] In some embodiments, a device can comprise a circuit that can both apply the voltage and measure the current. In some embodiments, the device comprises one circuit to apply the voltage difference and another to measure the current. In some embodiments, a voltage difference can be created with an asymmetric salt across the membrane. In some embodiments, a device comprises one or more electrodes directly associated with a pore at or near the pore channel or the opening. In some embodiments, one or more electrodes may be placed within the cis and/or trans chambers. In some embodiments, electrodes may be capable of detecting differences in ionic current around two chambers or tunneling electrical current around the pore channel or opening. [0593] In some embodiments, a device can comprise or be coupled to a computer or a processor. In some embodiments, a processor can be configured to receive a signal from a sensor. In some embodiments, a processor can be configured to receive an electrical signal from a sensor. In some embodiments, a processor can be configured to receive an optical signal from a sensor. In some embodiments, a processor can be configured to receive a mechanical signal from a sensor. In some embodiments, a processor can be configured to process a signal to generate an identification of an analyte. In some embodiments, a processor can be configured to process a signal to generate a probability of an identification of an analyte. In some embodiments, a processor can be configured to denoise a signal. In some embodiments, a processor can be configured to WSGR Docket Number: 64828-710.601 process a signal to generate a sequence of an analyte. In some embodiments, a processor can be configured to generate a file comprising a signal. In some embodiments, a processor can be configured to generate a file comprising an identification of an analyte. [0594] In some embodiments, a device can analyze multiple analytes simultaneously. In some embodiments, a device can analyze multiple analytes at a high throughput. [0595] In some embodiments, the device can sense an analyte at a sensing throughput of 128 simultaneous reads to 16,384 simultaneous reads. In some embodiments, the device can sense an analyte at a sensing throughput of 128 to 256, 128 to 512, 128 to 1,024, 128 to 2,048, 128 to 4,096, 128 to 8,192, 128 to 16,384 simultaneous reads. In some embodiments, the device can sense an analyte at a sensing throughput of about 128, 256, 512, 1,024, 2,048, 4,096, 8,192, or 16,384 simultaneous reads. In some embodiments, the device can sense an analyte at a sensing throughput of at least about 128, 256, 512, 1,024, 2,048, 4,096, 8,192, or 16,384 simultaneous reads. [0596] In some embodiments, the device can sense an analyte at a sensing throughput of 1,000 reads per run to 100,000,000 reads per run. In some embodiments, the device can sense an analyte at a sensing throughput of 1,000 to 5,000, 1,000 to 10,000, 1,000 to 50,000, 1,000 to 100,000, 1,000 to 500,000, 1,000 to 1,000,000, 1,000 to 5,000,000, 1,000 to 10,000,000, 1,000 to 50,000,000, 1,000 to 100,000,000 reads per run. In some embodiments, the device can sense an analyte at a sensing throughput of at least about 1,000, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, or 100,000,000 reads per run. [0597] In some embodiments, a device can operate with a turnaround time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. In some embodiments, a device can operate with a turnaround time of at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. In some embodiments, a device can operate with a turnaround time of more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. [0598] In some embodiments, a device can process a sample in about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. In some embodiments, a device can process a sample in at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. In some embodiments, a device can process a sample in more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. [0599] In some embodiments, a device can process a sample at a rate of about 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 7500, or about 10000 μL/minute. In some embodiments, a device can process a sample at a rate of at most about 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 7500, or about 10000 μL/minute. In some embodiments, a device can process a sample WSGR Docket Number: 64828-710.601 at a rate of greater than about 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 750, 1000, 1500, 2000, 5000, 7500, or about 10000 μL/minute. [0600] In some embodiments, a device can identify an analyte in a sample in about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. In some embodiments, a device can identify an analyte in a sample in at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. In some embodiments, a device can identify an analyte in a sample in more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, or about 240 minutes. [0601] In some embodiments, the analyte comprises a polypeptide. In some embodiments, the device can sequence a polypeptide with a read length of about 10 to about 500 amino acids. In some embodiments, the device can sequence a polypeptide with a read length of 10 to 21, 10 to 53, 10 to 99, 10 to 150, 10 to 200, 10 to 245, 10 to 250, 10 to 300, or 10 to 500 amino acids. In some embodiments, the device can sequence a polypeptide with a read length of about 10, 21, 53, 99, 150, 200, 245, 250, 300, or about 500 amino acids. In some embodiments, the device can sequence a polypeptide with a read length of at least about 10, 21, 53, 99, 150, 200, 245, 250, 300, or about 500 amino acids. In some embodiments, the device can sequence a polypeptide with a read length of at most about 10, 21, 53, 99, 150, 200, 245, 250, 300, about 500 amino acids. [0602] In some embodiments, the device can sequence a polypeptide with an average read length of about 2 to about 34000 amino acids. In some embodiments, the device can sequence a polypeptide with an average read length of about 2, 5, 10, 15, 21, 30, 40, 53, 60, 70, 80, 90, 99, 100, 150, 200, 225, 245, 250, 300, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000 or about 34000 amino acids. In some embodiments, the device can sequence a polypeptide with an average read length of at least about 2, 5, 10, 15, 21, 30, 40, 53, 60, 70, 80, 90, 99, 100, 150, 200, 225, 245, 250, 300, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000 or about 34000 amino acids. In some embodiments, the device can sequence a polypeptide with an average read length of at most about 2, 5, 10, 15, 21, 30, 40, 53, 60, 70, 80, 90, 99, 100, 150, 200, 225, 245, 250, 300, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000, 30000, or about 34000 amino acids. [0603] In some embodiments, a device can comprise an electromagnetic shield. In some embodiments, an electromagnetic shield can partially or fully enclose one or more membranes and/or measurement electronics and/or measurement electrodes. In some embodiments, the electromagnetic shield can comprise a Faraday cage, a Faraday bag, a Faraday cylinder, or a Faraday well. The electromagnetic shield can reduce noise in a measured signal, where noise can arise from environmental electromagnetic fields and/or active electronics of the device. [0604] In some embodiments, a device can comprise a support for a membrane. In some embodiments, the support can comprise a polymer. In some embodiments, the support can comprise Delrin® (polyoxymethylene WSGR Docket Number: 64828-710.601 or acetal homopolymer), a polyester, e.g. Mylar® (biaxially-oriented polyethylene terephthalate (boPET)polyester film), PC, PVC, PAN, PES, polysulphone, polyimide, polystyrene, polyethylene, PVF, PET, FIFE, PEEK, PTFE, or FEP. In some embodiments, the support can comprise a thickness of at least about 0.1, 0.5, 1, 5, 10, 50, 100, 500, or 1000 μm. In some embodiments, the support can comprise a thickness of at most about 0.1, 0.5, 1, 5, 10, 50, 100, 500, or 1000 μm. [0605] In some embodiments, a device comprises lipids. In some embodiments, the lipids can be configured to be formed into a membrane. In some embodiments, the lipids can be substantially dehydrated. In some embodiments, the lipids can comprise less than about 30, 25, 20, 15, 10, 5, 4, 3, 2, or about 1 wt% of water by mass. In some embodiments, the lipids can comprise less than about 0.1, 0.01, 0.001, or about 0.0001 wt% of water by mass. In some embodiments, the lipids can comprise one or more additives that affect the properties of a lipid bilayer membrane. In some embodiments, the one or more additives can comprise fatty acids, such as palmitic acid, myristic acid and oleic acid; fatty alcohols, such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol and stigmasterol; lysophospholipids, such as 1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; or ceramides. In some embodiments, the lipids can comprise cholesterol and/or ergosterol. [0606] In some embodiments, lipids can be configured to form a membrane. In some embodiments, an aqueous solution can be used to collect lipids from an internal surface of a device, and then form a lipid bilayer membrane. In some embodiments, an aqueous solution can comprise one or more properties that are close to physiological conditions. For example, a physiologically acceptable solution can be buffered to a pH of 3 to 9. The pH of the solution can be adapted to the lipids used, and the intended application of a lipid bilayer membrane. In some embodiments, a buffer can comprise phosphate buffered saline (PBS), N-2- Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid (HEPES) buffered saline, Piperazine-1,4-Bis-2- Ethanesulfonic Acid (PIPES) buffered saline, 3-(n-Morpholino)Propanesulfonic Acid (MOPS) buffered saline or Tris(Hydroxymethyl)aminomethane (TRIS) buffered saline. For example, an aqueous solution can be 10 mM PBS containing 1.0M sodium chloride (NaCl) and having a pH of about 6.9. [0607] In some embodiments, a device comprises a pore comprising a proteasome. In some embodiments, the proteasome comprises difference modes. In some embodiments, the proteasome comprises a (active) peptide- mode, wherein the proteasome can recognize a protein, cut it into pieces and can recognize the individual fragments. In some embodiments, the proteasome comprises a (inactive) strand-mode, wherein proteins can be recognized as they are linearized and transported across the pore at a controlled speed by an unfoldase, for example VAT, which threads intact analytes across the pore channel. In some embodiments, individual peptides can be directed by the electroosmotic flow through the proteasomal nanochannel to the pore where they can be recognized by specific current blockades. Accordingly, a device can comprise a multi-protein proteasome-pore WSGR Docket Number: 64828-710.601 assembly for real-time single-molecule protein sequencing applications. In some embodiments, the pore can degrade proteins, polypeptides, or peptides at physiological conditions and at more extreme conditions including high salt, high temperature, high pH, and/or low pH. In some embodiments, one or more proteasomes can be located on the first side (e.g., cis side) or the second side (e.g., trans side) of the membrane. In some embodiments, one or more unfoldases can be located on the first side (e.g., cis side) or the second side (e.g., trans side) of the membrane. In some embodiments, one or more translocases can be located on the first side (e.g., cis side) or the second side (e.g., trans side) of the membrane. In some embodiments, the pore can degrade polypeptides and translocate at least one of the degradation products. [0608] In some embodiments, a device can be configured to process a sample. In some embodiments, a sample comprises an analyte. In some embodiments, a sample comprises a polypeptide, a nucleic acid, a metabolite, or a small molecule, or combinations thereof. In some embodiments, a sample comprises a plurality of analytes. In some embodiments, a sample comprises a plurality of polypeptides, a plurality of nucleic acids, a plurality of metabolites, or a plurality of small molecules, or combinations thereof. In some embodiments, a sample can undergo at least a portion of an Edman degradation cycle prior to processing. In some embodiments, a sample comprises products of at least a portion of an Edman degradation cycle. In some embodiments, a sample can undergo at least a portion of an Edman degradation cycle as part of being processed. [0609] In some embodiments, a device may process a portion of a sample, or all of a sample. In some embodiments, a device may analyze measured signals as a sample can be processed. In some embodiments, a device may process an amount of sample based on analytes identified during processing. In some embodiments, a device may be configured to return a result once a particular analyte has been detected from a sample. In some embodiments, a device may be configured to stop sample processing once a particular criterion has been met. In some embodiments, a device may be configured to change operating parameters during the course of operation. In some embodiments, a device may be configured to change operating parameters once a particular criterion has been met. In some embodiments, a particular criterion comprises detecting an analyte, detecting a threshold number of analytes, detecting a combination of analytes, processing a volume of sample, running for a threshold amount of time, detecting a threshold ionic current, or detecting a threshold change in ionic current, or combinations thereof. In some embodiments, an operating parameter comprises an electrolyte concentration on a first side (e.g., cis side), an electrolyte concentration on a second side (e.g., trans side), an electrical potential, a flow rate, a sample preparation operation, a recording device operating condition, a sample source, determining a portion of the plurality of chambers to direct sample flow towards, determining a portion of the plurality of chambers to stop directing sample flow towards, or a selection of a portion of the sensor array to analyze, or combinations thereof. WSGR Docket Number: 64828-710.601 [0610] In some embodiments, a device can be configured to process at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or about 400 samples at once or in parallel. In some embodiments, a device can be configured to process at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or about 400 samples at once or in parallel. [0611] In some embodiments, a device can comprise a sample preparation portion. In some embodiments, the device can perform a sample preparation before identifying analytes. The sample preparation portion of the device can be integrated, in the device, via a fluidic connection to a sensor. In some embodiments, the fluidic connection can comprise a serpentine or otherwise a coiled channel. In some embodiments, the device comprises fluidic channels. In some embodiments, the fluidic channels comprise microfluidic length-scales. In some embodiments, the fluidic channels comprise laminar flow channels. In some embodiments, the fluidic channels can be in fluidic communication with a sample preparation portion and a sensor or a sensor array. In some embodiments, the fluidic channels can manipulate a fluid by capillary effects. In some embodiments, the fluidic channels can wick a fluid. In some embodiments, the fluidic channels comprise a coating that can enhance fluid adhesion to the fluidic channels. [0612] In some embodiments, a device can comprise a waste portion. The device can discard or store reagents, or byproducts, or any combination thereof, from processing a sample. [0613] In some embodiments, a device can comprise one or more ports. In some embodiments, a port can be configured to receive a sample. In some embodiments, a port can be configured to receive one or more reagents. [0614] In some embodiments, a device can comprise a temperature control device. A temperature control device can be a heater, a cooler, or both. A temperature control device can be used to adjust and/or maintain a temperature of at least a portion of a device. The portion can be a sample preparation portion, a sensor, or an array of sensors. In some embodiments, a device can comprise a temperature sensor. In some embodiments, the temperature can be at least about 0, 10, 20, 30, 40, 50, 60, 70, 80, or about 90 degrees Celsius. In some embodiments, the temperature can be at most about 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 degrees Celsius. [0615] In some embodiments, a device can comprise one or more magnets. In some embodiments, the magnets can provide a constant or a fluctuating magnetic field. The magnetic field can be used to separate, from a sample, magnetic or paramagnetic beads. [0616] In some embodiments, a device can comprise a recording device. A recording device can record an electrical signal at a sampling rate of at least about 1, 2 ,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 600, 7000, 8000, 9000, or about 10000 kHz. A recording device can record an electrical signal at a sampling rate of at most about 1, 2 ,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, WSGR Docket Number: 64828-710.601 600, 7000, 8000, 9000, or about 10000 kHz. In recording device can be configured to filter an electrical signal. For example, a recording device can perform Bessel filtering. The Bessel filtering can comprise a filter frequency of at least about 1, 2 ,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 600, 7000, 8000, 9000, or about 10000 kHz. The Bessel filtering can comprise a filter frequency of at most about 1, 2 ,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 600, 7000, 8000, 9000, or about 10000 kHz. In some embodiments, a recording device comprises a digital-to-analog converter. In some embodiments, a recording device comprises an amplifier. In some embodiments, a recording device comprises a patch clamp amplifier. In some embodiments, a device comprises multiple amplifiers. In some embodiments, a device comprises multiple digital-to-analog converters. [0617] In some embodiments, a device can comprise a display. In some embodiments, a display can display an electrical signal. In some embodiments, a display can display an electrical signal measured by an electrode. In some embodiments, the device can comprise a flow cell. In some embodiments, the flow cell can comprise or be fluidically coupled to the sensor or the sensor array. In some embodiments, the sensor or the sensor array can be integrated with the flow cell into a single piece, or they can be separate. In some embodiments, a flow cell can be in fluid communication with a plurality of sensor arrays. In some embodiments, a flow cell can be in fluid communication with at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 sensor arrays. In some embodiments, a flow cell can be in fluid communication with no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 sensor arrays. [0618] In some aspects, provided herein is a system (e.g., a nanopore system). The system may comprise a chamber (e.g., a fluidic chamber). The system may comprise one or more membranes. The one or more membranes can comprise one or more nanopores. One or more membranes may separate the fluidic chamber into at least a first side and a second side. The system (e.g., nanopore system) may comprise one or more controllers. The one or more controllers may be operatively coupled to the system. The one or more controllers may operate individually. In some embodiments, the one or more controllers may operate collectively. The one or more controllers may be configured to translocate at least a portion of an analyte. The analyte may comprise an analyte as described herein. In some embodiments, the one or more controllers may be configured to translocate at least a portion of an analyte through a nanopore. The one or more controllers of the system may be configured to detect (i) a current or change thereof, (ii) a voltage or change thereof, or (iii) a resistance or change thereof, or (iv) any combination thereof. The one or more controllers may detect (i) a current or change thereof, (ii) a voltage or change thereof, or (iii) a resistance or change thereof while at least a portion of the analyte is translocating. In some embodiments, the one or more controllers of the system may be configured to WSGR Docket Number: 64828-710.601 use a current or change thereof, (ii) a voltage or change thereof, or (iii) a resistance or change thereof, or (iv) any combination thereof to determine one or more characteristics of at least a portion of an analyte. Determining a characteristic can comprise measuring a characteristic of an analyte, or quantitating a characteristic of an analyte, or any combination thereof. The one or more characteristics may be determined with an accuracy as described herein. [0619] As an example, provided herein is a system comprising: a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side; one or more controllers operatively coupled to the nanopore system, wherein the one or more controllers are individually or collectively configured to: (a) translocate at least a portion of an analyte through the nanopore, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or fragments thereof, or a combination thereof, (b) detect (1) a current or change thereof; or (2) voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) use (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to deter-mine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. [0620] One or more controllers described herein may be configured to translocate at least a portion of an analyte. The analyte may translocate at an average rate of translocation. The rate can comprise an average rate of translocation through the pore. The average rate of translocation may comprise an average of an analyte (e.g., a non-nucleic acid based polymer analyte) translocating through a pore one or more times. The average rate of translocation may comprise an average of two or more analytes translocating through a pore one or more times. A rate of translocation of an analyte through a pore may be expressed as amino acids per second (amino acids/sec) and/or nanometers/sec (nm/s). In some embodiments, a rate of translocation (e.g., an average rate of translocation) may comprise translocation of an analyte with a motor protein (e.g., a translocase) or without a motor protein. [0621] As another example, provided herein is a system comprising: a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side; and one or more controllers operatively coupled to the nanopore system, wherein the one or more controllers are individually or collectively configured to: (a) translocate at least a portion of an analyte through the nanopore, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, at least a portion of a peptide, or fragments thereof, or a combination thereof, wherein (i) an average rate of translocation is be-tween about 0.1 amino acids per second to about 35000 amino acids per second or (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, (b) detect (1) a current or change thereof, or (2) voltage or change thereof WSGR Docket Number: 64828-710.601 while the at least the portion of the analyte is translocating through the nanopore; and (c) use (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte. [0622] The one or more controllers may be configured to repeat a translocation, detection, or use of (i) a current or change thereof, (ii) a voltage or change thereof, or (iii) a resistance or change thereof, or (iv) any combination thereof. The detection, translocation, or determination of one or more characteristics, or any combination thereof, may be repeated for an additional analyte by the one or more controllers. In some cases, the detection, translocation, or determination of one or more characteristics, or any combination thereof, may be repeated by one or more controllers for a plurality of analytes. The repetition for the plurality of analytes may generate a plurality of signals (e.g., electrical signals). The plurality of electrical signals may be used to determine one or more characteristics of the plurality of analytes. For example, the one or more controllers may repeat detection, translocation, or determination of one or more characteristics, or any combination thereof at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 25 times, at least about 50 times, at least about 100 times, at least about 500 times, or greater than about 500 times. In some embodiments, he one or more controllers may repeat detection, translocation, or determination of one or more characteristics, or any combination thereof at most about 500 times, at most about 100 times, at most about 50 times, at most about 25 times, at most about 10 times, at most about 5 times, at most about 4 times, at most about 3 times, at most about 2 times, or less than about 2 times. [0623] As another example, provided herein is a system comprising: a first nanopore system, wherein the first nanopore system comprises (1) a first fluidic chamber, and (2) a first membrane comprising a first nanopore, wherein the first mem-brane separates the first fluidic chamber into a first side and a second side; a second nanopore system, wherein the second nanopore system comprises (1) a second fluidic chamber, and (2) a second membrane comprising a second nanopore, wherein the second membrane separates the second fluidic chamber into a third side and a fourth side; and one or more controllers operatively coupled to the first nanopore system or the second nanopore system, wherein the one or more controllers are individually or collectively configured to: (a) translocate at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or first fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or second fragments thereof, or a combination thereof, wherein the first analyte and the second analyte is among a sample, (b) detect (i) (1) a first current or change thereof, or (2) a first voltage or change thereof while the at least the portion of the first analyte WSGR Docket Number: 64828-710.601 is translocating through the first nanopore, and (ii) (3) a second current or change thereof, or (4) a second voltage or change thereof while the at least the portion of the second analyte is translocating through the second nanopore; (c) use (i) (1) the first current or change thereof, or (2) the first voltage or change thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (3) the second current or change thereof, or (4) the second voltage or change thereof to determine a second characteristic of the at least the portion of the second analyte; and (d) characterize one or more properties of the sample using the first characteristic or the second characteristic determined in (c). [0624] In some embodiments, one or more samples can comprise a plurality of analytes (e.g., a plurality of non-nucleic acid based polymer analytes). At least a subset of the plurality of analytes may undergo any of the methods and/or system disclosed herein. In some embodiments, (i) a plurality of currents or changes thereof, (ii) a plurality of voltages or changes thereof, or (iii) a plurality of resistances or changes thereof, or (iv) any combinations thereof, may be used to determine a plurality of characteristics. The plurality of characteristics can comprise at least about 1 characteristics, at least about 2 characteristics, at least about 3 characteristics, at least about 4 characteristics, at least about 5 characteristics, at least about 10 characteristics, at least about 50 characteristics, at least about 100 characteristics, or greater than about 100 characteristics. At least a subset of a plurality of characteristics may be used to characterize the one or more properties of a sample. [0625] The first side, second side, third side, or fourth side, or any combination thereof may be different sizes. The first side, second side, third side, or fourth side, or any combination thereof may be the same size. One or more sides of a first nanopore system may be larger in size than one or more sides of a second nanopore system. One or more sides of a first nanopore system may be smaller in size than one or more sides of a second nanopore system. In some embodiments, a first side and a third side may be larger in size than a second side and a fourth side, respectively. In some embodiments, a first side and a third side may be a same size as a second side and a fourth side, respectively. The first side, second side, third side, or fourth side, or any combination thereof may comprise an enclosed compartment. The first side, second side, third side, or fourth side, or any combination thereof may comprise an open compartment. The first side, second side, third side, or fourth side, or any combination thereof may comprise a continuous phase (e.g., a continuous aqueous phase). [0626] In some embodiments, a device can comprise a computer, a software module, a non-transitory computer readable storage medium, a database, and/or a standalone application disclosed herein. [0627] FIGs.137A-137E provide schematic illustrations of a nanopore system as described herein. FIG.137A shows a small enclosed compartment (e.g., trans) connected to a larger compartment (e.g., cis). The cis can be open, wherein it may not be enclosed by a supporting structure. A membrane can be disposed between the small enclosed compartment (e.g., trans) connected to a larger compartment (e.g., cis). In some embodiments, the membrane comprises a nanopore. The trans compartment can be supported by a structure enclosing it from the WSGR Docket Number: 64828-710.601 open cis. The larger compartment (e.g., cis) can comprise a first electrode (e.g., Electrode 1). Electrode 1 (common) can refer to an electrode common in an array of connected nanopore systems. For example, Electrode 1 may be common for a number of nanopore systems fluidly connected in an array. The small enclosed compartment (e.g., trans) can comprise an Electrode 2. The Electrode 1 (common) and Electrode 2 can be connected via a circuit. The circuit may provide an applied voltage to the nanopore system. The applied voltage may translocate one or more analytes and/or at least a portion an analyte through a nanopore of the system. The trans can be connected (e.g., fluidically connected) to the cis. In some embodiments, the trans can be connected (e.g., fluidly connected) to one or more adjacent cis compartments. FIG.137B shows a first compartment (e.g., cis) directly connected to a second compartment (e.g., trans). For example, the first and second compartment can be directly connected in a solid structure (e.g., supporting substrate). The supporting structure can support a membrane between the cis and trans. In some embodiments, the solid structure may form chambers around the first compartment and second compartment. The membrane disposed between the two enclosed compartment (cis and trans) can comprise a nanopore. In some embodiments, the membrane comprises one or more nanopores. A circuit can connect an Electrode 1 disposed in cis and an Electrode 2 disposed in trans. FIG. 137C shows a nanopore system comprising one or more droplets. A droplet may be cis. The cis droplet may be an aqueous cis droplet. The first droplet may be connected to a second droplet. A second droplet can comprise a trans. The trans droplet may be an aqueous trans droplet. The cis droplet and trans droplet can be disposed in a hydrophobic medium. The hydrophobic medium may be a hydrophobic liquid and/or a semi-liquid. The one or more droplets (e.g., cis droplet and trans droplet) may be connected via a membrane. The membrane may comprise a nanopore as described herein. The one or more droplets (e.g., cis droplet and trans droplet) may be partially surround by the hydrophobic medium. In some cases, the one or more droplets (e.g., cis droplet and trans droplet) can be completely surrounded by the hydrophobic medium. In some embodiments, the one or more droplets (e.g., cis droplet and trans droplet) may comprise one or more electrodes. A cis droplet can comprise Electrode 1. Electrode 1 may provide an applied voltage to cis in the system. A trans droplet can comprise Electrode 2. Electrode 2 may provide an applied voltage to trans in the system. A circuit can connect Electrode 1 and Electrode 2 in the system. In some cases, the one or more electrodes may be connected to the one or more droplets. FIG.137D shows a nanopore system comprising a droplet. A droplet may be trans (e.g., a trans droplet). The trans droplet may be an aqueous trans droplet. The droplet can be connected to a phase. The phase may be a continuous phase (e.g., a continuous aqueous phase). In some embodiments, the continuous phase (e.g., a continuous aqueous phase) may be a liquid phase. In some embodiments, the continuous phase may be a semi-solid phase. The trans may connected to the continuous phase by a membrane. The membrane may comprise a nanopore as described herein. The trans droplet may be partially surround by a hydrophobic medium. In some cases, an Electrode 1 may be disposed in the continuous phase (e.g., the continuous aqueous WSGR Docket Number: 64828-710.601 phase). An Electrode 2 may be disposed in trans. The Electrode 1 and Electrode 2 can be connected via a circuit. In some embodiments, the circuit can provide an applied voltage to Electrode 1 and/or Electrode 2. FIG.137E shows a nanopore system comprising a droplet. A droplet may be cis (e.g., a cis droplet). The cis droplet may be an aqueous cis droplet. The droplet can be connected to a compartment (e.g., trans). The trans may be an open compartment. The trans may be an enclosed compartment. In some embodiments, a membrane can be disposed between the cis (e.g., aqueous droplet) and trans (e.g., enclosed compartment). In some embodiments, the membrane comprises a nanopore. The cis (e.g., aqueous droplet) and trans (e.g., enclosed compartment) may be fluidly connected. The cis (e.g., aqueous droplet) can be disposed in a hydrophobic medium. The hydrophobic medium may be a hydrophobic liquid and/or a semi-liquid. An Electrode 1 can be disposed in trans (e.g., enclosed compartment). An Electrode 2 can be disposed in cis. In some embodiments, the circuit can connect Electrode 1 and Electrode 2. [0628] In some embodiments, a first side of a system described herein may be larger in size than a second side. In some embodiments, a first side of a system described herein may be smaller in size than a second side. In some embodiments, a first side of a system described herein may be the same size as a second side. A first side and/or a second side of a system may comprise an enclosed compartment. The enclosed compartment may be supported by a solid structure described herein. The enclosed compartment may be connected (e.g., fluidly connected) to another side (e.g., another compartment) of the system. A first side and/or a second side of a system may comprise an open compartment. The open compartment may be a continuous open compartment. The continuous open compartment can comprise a continuous aqueous phase. The open compartment may be connected (e.g., fluidly connected) to another side (e.g., another compartment) of the system. In some embodiments, a first side can comprise an open compartment and the second side can comprise an enclosed compartment. [0629] A nanopore system can comprise one or more open chambers, or one or more closed chambers, or any combination thereof. In some embodiments, a nanopore system described herein may comprise one or more micropipettes. In some embodiments, two or more of the nanopore systems as represented in FIGs.137A-137E may be connected in any combination to provide one or more arrays of nanopores. For example, at least about 2 nanopore systems, at least about 3 nanopore systems, at least about 4 nanopore systems, at least about 5 nanopore systems, at least about 10 nanopore systems, or greater than about 10 nanopore systems as represented in FIGs.137A-137E may be connected to provide one or more arrays of nanopores. In some embodiments, an array can comprise at least about 2, at least about 5, at least about 10, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000, or greater than about 100,000 nanopore systems. In some embodiments, an array can comprise at most about 100,000, at most about 10,000, at most about 1,000, WSGR Docket Number: 64828-710.601 at most about 100, at most about 50, at most about 10, at most about 5, at most about 2, or less than about 2 nanopore systems. [0630] A compartment of a nanopore system described herein may be enclosed by a substrate (e.g., a solid substrate, see ”Supporting structure” in FIG.137B). The solid substrate may be composed of organic and/or inorganic materials. Inorganic materials can comprise silicon, glass, metals, ceramics, or plastics, or any combination thereof. The compartment of a nanopore system described herein may be enclosed by a semi-solid substrate, for example an organogel. In some embodiments, the compartment of a nanopore system described herein may be enclosed by a hydrophobic liquid (e.g., oil, silicone, or alkane, or any combination thereof). In some embodiments, a nanopore system described herein can comprise an amphipathic membrane (e.g., a lipid membrane). [0631] One or more compartments of a nanopore system described herein may be connected to another compartment of the nanopore system. For example, a first compartment (e.g., a cis compartment) can be connected to a second compartment (e.g., a trans compartment) by a channel. The channel can comprise a microfluidic pathway, in which a solution may be introduced, removed, or flow through the microfluidic pathway. In some embodiments, a nanopore system described herein can comprise one or more compartments connected by one or more nanopores. The one or more nanopores can be disposed in one or more membranes. For example, a nanopore system described herein can comprise a trans-cis-trans compartment arrangement and/or a cis-trans-cis arrangement. [0632] The one or more compartments of the nanopore system may each comprise a volume. The volume of the one or more compartments may be the same. The volume of the one or more compartments may be different. In some cases, a first compartment (e.g., a cis compartment) can be larger in volume than a second compartment (e.g., a trans compartment). In some cases, a first compartment (e.g., a cis compartment) can be smaller in volume than a second compartment (e.g., a trans compartment). In some cases, a first compartment (e.g., a cis compartment) can be the same volume as a second compartment (e.g., a trans compartment). A nanopore system described herein can comprise a ratio of a first compartment (e.g., a cis compartment) to a second compartment (e.g., a trans compartment). For example, a system described herein can comprise a 1:1 ratio, where there may be 1 first compartment (e.g., a cis compartment) to 1 second compartment (e.g., a trans compartment). In some embodiments, a nanopore system and/or system described herein can comprise a 1:1 ratio, a 1:2 ratio, a 1:3 ratio, a 1:4 ratio, a 1:5 ratio, a 2:1 ratio, a 3:1 ratio, a 4:1 ratio, or a 5:1 ratio of a first compartment (e.g., a cis compartment) to a second compartment (e.g., a trans compartment). A plurality of first compartments (e.g., cis compartments) may be connected to a plurality of second compartments (e.g., trans compartments). [0633] A membrane of a nanopore system can comprise any membrane as described herein. The membrane may be pre-formed (e.g., formed prior to insertion in the nanopore system). In some embodiments, the WSGR Docket Number: 64828-710.601 membrane may not be pre-formed. In some embodiments, each compartment and/or droplet of a nanopore system described herein may be separated by one or more membranes. The one or more membranes can comprise one or more nanopores described herein. [0634] In some embodiments, the compartments and/or droplets of a nanopore system may comprise one or more electrodes. In some embodiments, a first compartment (e.g., a cis compartment) can be addressed by one or more electrodes and a second compartment (e.g., a trans compartment) can be addressed by one or more electrodes. In some embodiments, a first droplet (e.g., a cis droplet) can be addressed by one or more electrodes and a second droplet (e.g., a trans droplet) can be addressed by one or more electrodes. For example, if one or more compartments are separate they can comprise separate (e.g., distinct) electrodes. If one or more compartments are connected (e.g., fluidly connected, electrically connected, or ionically connected, or any combination thereof), they can comprise a shared electrode (e.g., an electrode common to the one or more compartments). In some embodiments, a nanopore system described herein can comprise at least about 1 electrode, at least about 2 electrodes, at least about 3 electrodes, at least about 4 electrodes, at least about 5 electrodes, at least about 10 electrodes, or greater than about 10 electrodes. In some embodiments, a nanopore system described herein can comprise at most about 10 electrodes, at most about 5 electrodes, at most about 4 electrodes, at most about 3 electrodes, at most about 2 electrodes, at most about 1 electrode, or less than about 1 electrode. One or more electrodes may enable separate application of voltage across one or more membranes of the nanopore system. One or more electrodes may enable separate readout of a voltage and/or current difference across a nanopore, or membrane, or any combination thereof. [0635] In some embodiments, an array of one or more nanopore systems described herein may be addressed by circuitry. The circuitry may comprise control circuitry, or acquisition circuitry, or any combination thereof. A circuit can apply a voltage. In some embodiments, the circuit can apply the voltage across a membrane and/or a nanopore disposed in a membrane. A circuit can measure a current or change thereof (e.g., an ionic current or change thereof). In some embodiments, the circuit can measure the current or change thereof (e.g., an ionic current or change thereof) across a membrane and/or a nanopore disposed in a membrane. The nanopore and/or membrane of the nanopore system (or each nanopore and/or membrane in an array comprising two or more nanopore systems) can be addressed by an individual circuitry. Without wishing to be bound by theory, the nanopore and/or membrane in a nanopore system and/or array of connected nanopore systems can be individually addressed by control and/or acquisition circuitry to enable each nanopore and/or membrane to be controlled and/or measured separately. A measured current or change thereof and/or an applied voltage can be amplified, or digitized, or any combination thereof. For example, current or voltage signals across one or more membranes and/or one or more nanopores disposed in one or more membranes may be amplified and/or digitized in circuitry. WSGR Docket Number: 64828-710.601 [0636] A device described herein can comprise one or more electrochemical mediators. An electrochemical mediator can enable a flow (e.g., a continuous flow) of current through a system. The electrochemical mediator can comprise a solid mediator. The solid mediator can comprise a metal mediator. In some embodiments, the electrochemical mediator may comprise silver/silver chloride (Ag/AgCl). The electrochemical mediator can comprise ferrocene methanol (FcMeOH), ferricyanide (Fe(CN)6), Iodide (I), hexacyanoferrate (II), Meldola's Blue (MB), Dichlorophenolindophenol (DCPIP), p-Benzoquinone (p-BQ), or o-Phenylenediamine (o-PD), or any combination thereof. Readout of a device described herein may involve light. For example, a readout of the device can comprise using one or more fluorescent reporters to provide a quantification of the ionic current flowing through one or more nanopores of the one or more compartments of the device. In some embodiment, one or more controller circuits may be the same as one or more acquisition circuits of system described herein. In some embodiments, one or more controller circuits may be different from one or more acquisition circuits of system described herein. The controller circuit and acquisition circuit may use the same one or more electrodes. In some embodiments, the controller circuit and acquisition circuit may use different electrodes. In some embodiments, one or acquisition circuits and/or one or more controller circuits may not use at least one electrode. For example, one or acquisition circuits and/or one or more controller circuits may use one or more light sensors. The light sensor may be located adjacent to a pore and/or membrane. In some embodiments, an acquisition by one or more acquisition circuits may be performed by at least one optical reporter. The optical report can provide an indication of an ionic current. In some embodiments, the acquisition circuit can be a raman based readout. The raman based readout can comprise a readout of a composition of an analyte. The raman based readout may not provide a readout of the current. COMPUTERS [0637] In some aspects, the present disclosure describes a computer-implemented system comprising: a digital processing device comprising: at least one processor, an operating system configured to perform executable instructions, a memory, and a computer program. The computer program can include instructions executable by the digital processing device to control a manufacture a membrane in a device. The computer program can include instructions executable by the digital processing device to prepare a sample. The computer program can include instructions executable by the digital processing device to control a device to process a sample. The computer program can include instructions executable by the digital processing device to analyze a signal produced by an analyte in a sample. The computer program can include instructions executable by the digital processing device to store a signal produced by an analyte into a database. In some aspects, the present disclosure describes a computer-implemented method, implementing any one of the methods disclosed herein in a computer system. Referring to FIG.70, a block diagram is shown depicting an exemplary machine that WSGR Docket Number: 64828-710.601 includes a computer system 7700 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and/or methodologies for preparing a sample for analysis, processing a sample to generate a signal from an analyte in the sample, analyzing a signal to identify an analyte, storing a signal in a database, or any combination thereof. The components in FIG.70 are examples only and do not limit the scope of use or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments. [0638] Computer system 7000 may include one or more processors 7001, a memory 7003, and a storage 7008 that communicate with each other, and with other components, via a bus 7040. The bus 7040 may also link a display 7032, one or more input devices 7033 (which may, for example, include a keypad, a keyboard, a mouse, or a stylus, or any combination thereof), one or more output devices 7034, one or more storage devices 7035, and various tangible storage media 7036. All of these elements may interface directly or via one or more interfaces or adaptors to the bus 7040. For instance, the various tangible storage media 7036 can interface with the bus 7040 via storage medium interface 7026. Computer system 7000 may have any suitable physical form, including but not limited to one or more integrated circuits (ICs), printed circuit boards (PCBs), mobile handheld devices (such as mobile telephones or PDAs), laptop or notebook computers, distributed computer systems, computing grids, or servers. [0639] Computer system 7000 includes one or more processor(s) 7001 (e.g., central processing units (CPUs), general purpose graphics processing units (GPGPUs), or quantum processing units (QPUs)) that carry out functions. Computer system 7000 may be one of various high performance computing platforms. For instance, the one or more processor(s) 7001 may form a high performance computing cluster. In some embodiments, the one or more processors 7001 may form a distributed computing system connected by wired and/or wireless networks. In some embodiments, arrays of CPUs, GPUs, or QPUs, or any combination thereof may be operably linked to implement any one of the methods disclosed herein. Processor(s) 7001 optionally contains a cache memory unit 7002 for temporary local storage of instructions, data, or computer addresses. Processor(s) 7001 are configured to assist in execution of computer readable instructions. Computer system 7000 may provide functionality for the components depicted in FIG. 70 as a result of the processor(s) 7001 executing non- transitory, processor-executable instructions embodied in one or more tangible computer-readable storage media, such as memory 7003, storage 7008, storage devices 7035, and/or storage medium 7036. The computer- readable media may store software that implements particular embodiments, and processor(s) 7001 may execute the software. Memory 7003 may read the software from one or more other computer-readable media (such as mass storage device(s) 7035, 7036) or from one or more other sources through a suitable interface, such as network interface 7020. The software may cause processor(s) 7001 to carry out one or more processes or one WSGR Docket Number: 64828-710.601 or more steps of one or more processes described or illustrated herein. Carrying out such processes or steps may include defining data structures stored in memory 7003 and modifying the data structures as directed by the software. [0640] The memory 7003 may include various components (e.g., machine readable media) including, but not limited to, a random access memory component (e.g., RAM 7004) (e.g., static RAM (SRAM), dynamic RAM (DRAM), ferroelectric random access memory (FRAM), or phase-change random access memory (PRAM), or any combination thereof), a read-only memory component (e.g., ROM 7005), and any combinations thereof. ROM 7005 may act to communicate data and instructions unidirectionally to processor(s) 7001, and RAM 7004 may act to communicate data and instructions bidirectionally with processor(s) 7001. ROM 7005 and RAM 7004 may include any suitable tangible computer-readable media described below. In one example, a basic input/output system 7006 (BIOS), including basic routines that help to transfer information between elements within computer system 7000, such as during start-up, may be stored in the memory 7003. [0641] Fixed storage 7008 can be connected bidirectionally to processor(s) 7001, optionally through storage control unit 7007. Fixed storage 7008 provides additional data storage capacity and may also include any suitable tangible computer-readable media described herein. Storage 7008 may be used to store operating system 7009, executable(s) 7010, data 7011, applications 7012 (application programs), and the like. Storage 7008 can also include an optical disk drive, a solid-state memory device (e.g., flash-based systems), or a combination of any of the above. Information in storage 7008 may, in appropriate cases, be incorporated as virtual memory in memory 7003. [0642] In one example, storage device(s) 7035 may be removably interfaced with computer system 7000 (e.g., via an external port connector (not shown)) via a storage device interface 7025. Particularly, storage device(s) 7035 and an associated machine-readable medium may provide non-volatile and/or volatile storage of machine- readable instructions, data structures, program modules, and/or other data for the computer system 7000. In one example, software may reside, completely or partially, within a machine-readable medium on storage device(s) 7035. In another example, software may reside, completely or partially, within processor(s) 7001. [0643] Bus 7040 connects a wide variety of subsystems. Herein, reference to a bus may encompass one or more digital signal lines serving a common function, where appropriate. Bus 7040 may be any of several types of bus structures including, but not limited to, a memory bus, a memory controller, a peripheral bus, or a local bus, and any combinations thereof, using any of a variety of bus architectures. As an example, and not by way of limitation, such architectures include an Industry Standard Architecture (ISA) bus, an Enhanced ISA (EISA) bus, a Micro Channel Architecture (MCA) bus, a Video Electronics Standards Association local bus (VLB), a Peripheral Component Interconnect (PCI) bus, a PCI-Express (PCI-X) bus, an Accelerated Graphics Port (AGP) WSGR Docket Number: 64828-710.601 bus, HyperTransport (HTX) bus, or serial advanced technology attachment (SATA) bus, and any combinations thereof. [0644] Computer system 7000 may also include an input device 7033. In one example, a user of computer system 7000 may enter commands and/or other information into computer system 7000 via input device(s) 7033. Examples of an input device(s) 7033 include, but are not limited to, an alpha-numeric input device (e.g., a keyboard), a pointing device (e.g., a mouse or touchpad), a touchpad, a touch screen, a multi-touch screen, a joystick, a stylus, a gamepad, an audio input device (e.g., a microphone, or a voice response system, or any combination thereof), an optical scanner, a video or still image capture device (e.g., a camera), and any combinations thereof. In some embodiments, the input device can be a Kinect, Leap Motion, or the like. Input device(s) 7033 may be interfaced to bus 7040 via any of a variety of input interfaces 7023 (e.g., input interface 7023) including, but not limited to, serial, parallel, game port, USB, FIREWIRE, THUNDERBOLT, or any combination of the above. In some embodiments, an input device 7033 may be used to prepare a sample for analysis, process a sample to generate a signal from an analyte in the sample, analyze a signal to identify an analyte, store a signal in a database, or any combination thereof. In some embodiments, the input device can be used using human inputs through the input device 7033. [0645] In particular embodiments, when computer system 7000 can be connected to network 7030, computer system 7000 may communicate with other devices, specifically mobile devices and enterprise systems, distributed computing systems, cloud storage systems, cloud computing systems, and the like, connected to network 7030. Communications to and from computer system 7000 may be sent through network interface 7020. For example, network interface 7020 may receive incoming communications (such as requests or responses from other devices) in the form of one or more packets (such as Internet Protocol (IP) packets) from network 7030, and computer system 7000 may store the incoming communications in memory 7003 for processing. Computer system 7000 may similarly store outgoing communications (such as requests or responses to other devices) in the form of one or more packets in memory 7003 and communicated to network 7030 from network interface 7020. Processor(s) 7001 may access these communication packets stored in memory 7003 for processing. [0646] Examples of the network interface 7020 include, but are not limited to, a network interface card, or a modem, and any combination thereof. Examples of a network 7030 or network segment 7030 include, but are not limited to, a distributed computing system, a cloud computing system, a wide area network (WAN) (e.g., the Internet, an enterprise network), a local area network (LAN) (e.g., a network associated with an office, a building, a campus or other relatively small geographic space), a telephone network, a direct connection between two computing devices, or a peer-to-peer network, and any combinations thereof. A network, such as WSGR Docket Number: 64828-710.601 network 7030, may employ a wired and/or a wireless mode of communication. In general, any network topology may be used. [0647] Information and data can be displayed through a display 7032. Examples of a display 7032 include, but are not limited to, a cathode ray tube (CRT), a liquid crystal display (LCD), a thin film transistor liquid crystal display (TFT-LCD), an organic liquid crystal display (OLED) such as a passive-matrix OLED (PMOLED) or active-matrix OLED (AMOLED) display, or a plasma display, and any combinations thereof. The display 7032 can interface to the processor(s) 7001, memory 7003, and fixed storage 7008, as well as other devices, such as input device(s) 7033, via the bus 7040. The display 7032 can be linked to the bus 7040 via a video interface 7022, and transport of data between the display 7032 and the bus 7040 can be controlled via the graphics control 7021. In some embodiments, the display can be a video projector. In some embodiments, the display can be a head-mounted display (HMD) such as a VR headset. In further embodiments, suitable VR headsets include, by way of non-limiting examples, HTC Vive, Oculus Rift, Samsung Gear VR, Microsoft HoloLens, Razer OSVR, FOVE VR, Zeiss VR One, Avegant Glyph, Freefly VR headset, and the like. In still further embodiments, the display can be a combination of devices such as those disclosed herein. [0648] In addition to a display 7032, computer system 7000 may include one or more other peripheral output devices 7034 including, but not limited to, an audio speaker, a printer, a storage device, and any combinations thereof. Such peripheral output devices may be connected to the bus 7040 via an output interface 7024. Examples of an output interface 7024 include, but are not limited to, a serial port, a parallel connection, a USB port, a FIREWIRE port, a THUNDERBOLT port, and any combinations thereof. [0649] In addition, or as an alternative, computer system 7000 may provide functionality as a result of logic hardwired or otherwise embodied in a circuit, which may operate in place of or together with software to execute one or more processes or one or more steps of one or more processes described or illustrated herein. Reference to software in this disclosure may encompass logic, and reference to logic may encompass software. Moreover, reference to a computer-readable medium may encompass a circuit (such as an IC) storing software for execution, a circuit embodying logic for execution, or both, where appropriate. The present disclosure encompasses any suitable combination of hardware, software, or both. [0650] Various illustrative logical blocks, modules, circuits, and algorithm steps described in connection with the embodiments disclosed herein may be implemented as electronic hardware, computer software, or combinations of both. To clearly illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules, circuits, and steps have been described above generally in terms of their functionality. [0651] The various illustrative logical blocks, modules, and circuits described in connection with the embodiments disclosed herein may be implemented or performed with a general purpose processor, a digital WSGR Docket Number: 64828-710.601 signal processor (DSP), an application specific integrated circuit (ASIC), a field programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic, or discrete hardware components, or any combination thereof designed to perform the functions described herein. A general purpose processor may be a microprocessor, but in the alternative, the processor may be any conventional processor, controller, microcontroller, or state machine. A processor may also be implemented as a combination of computing devices, e.g., a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors in conjunction with a DSP core, or any other such configuration. [0652] The steps of a method or algorithm described in connection with the embodiments disclosed herein may be embodied directly in hardware, in a software module executed by one or more processor(s), or in a combination of the two. A software module may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, a removable disk, a CD-ROM, or any other form of storage medium. An exemplary storage medium can be coupled to the processor such the processor can read information from, and write information to, the storage medium. In the alternative, the storage medium may be integral to the processor. The processor and the storage medium may reside in an ASIC. The ASIC may reside in a user terminal. In the alternative, the processor and the storage medium may reside as discrete components in a user terminal. [0653] In accordance with the description herein, suitable computing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, and tablet computers. [0654] In some embodiments, the computing device includes an operating system configured to perform executable instructions. The operating system is, for example, software, including programs and data, which manages the device’s hardware and provides services for execution of applications. Suitable server operating systems include, by way of non-limiting examples, FreeBSD, OpenBSD, NetBSD®, Linux, Apple® Mac OS X Server®, Oracle® Solaris®, Windows Server®, and Novell® NetWare®. Suitable personal computer operating systems include, by way of non-limiting examples, Microsoft® Windows®, Apple® Mac OS X®, UNIX®, and UNIX-like operating systems such as GNU/Linux®. In some embodiments, the operating system can be provided by cloud computing. Suitable mobile smartphone operating systems include, by way of non-limiting examples, Nokia® Symbian® OS, Apple® Ios®, Research In Motion® BlackBerry OS®, Google® Android®, Microsoft® Windows Phone® OS, Microsoft® Windows Mobile® OS, Linux®, and Palm® WebOS®. [0655] In some embodiments, a computer system 7000 may be accessible through a user terminal to receive user commands. The user commands may include line commands, scripts, programs, or any combination thereof, and various instructions executable by the computer system 7000. A computer system 7000 may receive WSGR Docket Number: 64828-710.601 instructions to prepare a sample for analysis, process a sample to generate a signal from an analyte in the sample, analyze a signal to identify an analyte, store a signal in a database, schedule a computing job for the computer system 7000 to carry out any instructions, or any combination thereof. ANALYTES [0656] Provided herein are various analytes that can be detected and/or identified using a pore, membrane, device, or a method disclosed herein. The terms “analyte” and “substrate” may be used interchangeably. [0657] In some embodiments, an analyte can be an antibiotic. In some embodiments, an analyte can be a drug. In some embodiments, an analyte can be a metabolite. In some embodiments, an analyte can be a lipid. In some embodiments, an analyte can be a small biological molecule. In some embodiments, an analyte can be a steroid, a carbohydrate, an amino acid, a nucleotide, a hormone, a fatty acid, a vitamin, a flavin, a protein-cofactor, a lipid, or a phenolic compound. In some embodiments, analytes can generate different signals based on volume, shape, charge, structure, cross-linking, post-translational modifications (e.g., phosphorylation, glycosylation, or rhamnosylation, or any combination thereof), damage, oxidation, reduction, (D/L-) chirality, and/or sequence of the analyte. [0658] In some embodiments, an analyte comprises a polymer. In some embodiments, an analyte can be a protein-protein complex. In some embodiments, an analyte can be a DNA-protein complex. In some embodiments, an analyte can be an RNA-protein complex. [0659] In some embodiments, the analyte can be a non-nucleic acid based polymer analyte. In some embodiments, an analyte comprises a polymer. In some embodiments, a polymer comprises a protein. In some embodiments, a polymer comprises a polypeptide. The polypeptide (e.g., one or more polypeptide) may comprise one or more expressible polypeptides. In some embodiments, a polymer comprises a peptide. In some embodiments, a polymer comprises a polyamino acid. In some embodiments, a polymer comprises a polysaccharide. In some embodiments, a polymer comprises a glycoprotein. In some embodiments, a polymer comprises a nucleic acid. In some embodiments, a polymer comprises DNA. In some embodiments, a polymer comprises RNA. In some embodiments, a polymer comprises mRNA. In some embodiments, a polymer comprises a sequence. In some embodiments, a polymer can be a label-free analyte. In some embodiments, a polymer can be labeled. In some embodiments, the termini of a polymer may be unstructured, e.g., when the polymer can be denatured or partially denatured. In some embodiments, a contour length of a polymer can be longer than a length of a channel of a pore. In some embodiments, a contour length of a polymer can be shorter than a length of a channel of a pore. [0660] In some embodiments, a polymer comprises heterogeneous charge. In some embodiments, a heterogeneously charged polymer comprises positively and negatively charged repeating units. In some WSGR Docket Number: 64828-710.601 embodiments, charged repeating units may be evenly distributed along the sequence of a polymer. In some embodiments, charged repeating units may be unevenly distributed along the sequence of a polymer. In some embodiments, a polymer may be labeled. In some embodiments, a polymer may be synthetic, semi-synthetic or biological in origin. [0661] In some embodiments, a polymer comprises a protein. In some embodiments, a protein comprises two or more amino acids. In some embodiments, a protein comprises two or more peptides. In some embodiments, a protein comprises a polypeptide. In some embodiments, a protein comprises a negative charge. In some embodiments, a protein comprises a positive charge. In some embodiments, a protein can be negatively charged. In some embodiments, a protein can be positively charged. In some embodiments, a protein can be zwitterionic. [0662] In some embodiments, an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or any combination thereof can comprise one or more characteristics. The one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or any combination thereof can comprise a shape of the analyte, a volume of the analyte, a mass of the analyte, a structure of the analyte (e.g., a secondary structure, a tertiary structure, or a quaternary structure, or any combination thereof), a length of an analyte, a truncation of an analyte, an orientation of an analyte, one or more mutations of the analyte, a sequence of an analyte (e.g., a non-nucleic acid polymer analyte), a hydrophobicity of the analyte, a polarity of the analyte, an isoform of the analyte, a surface charge of the analyte, one or more post-translation modifications of the analyte, or one or more ligands coupled to the analyte, or any combination thereof. [0663] In some embodiments, the sequence information of the analyte may be captured in layers. The sequence information may be one or more layers of data (e.g., metadata) that may each comprise the sequence information and/or at least a portion of sequence information. The one or more layers of sequence information may be annotated to a reference sequence (e.g., a reference sequence of a database) with a probabilistic metric. The sequence information may comprise properties of the analyte (e.g., charge of the analyte, hydrophobicity of the analyte, aromaticity of the analytes, acidity of the analyte, chirality of the analyte, polarity of the analyte, or side chain composition of the analyte, or any combination thereof). The sequence information may comprise a partial set of natural amino acids, or a partial set of unnatural amino acids, or any combination thereof. In some embodiments, the sequence information may comprise an amino acid composition of the analyte. The amino acid composition can comprise the natural amino acid residues of the analyte, the unnatural amino acid residues of the analyte, or any combination thereof. In some embodiments, the sequence information can comprise one or more natural post-translational modifications, one or more unnatural post-translational modifications, one or more conjugations (e.g., drugs and/or small molecules), or any combination thereof. WSGR Docket Number: 64828-710.601 [0664] In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more translation errors. The translation errors may be associated with a portion of the analyte (e.g., an N- terminal portion, a C-terminal portion, or any combination thereof). In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more degradations. The degradations may comprise degradations to at least a portion of the analyte, or a molecular entity coupled to the analyte, or any combination thereof. In some embodiments, an analyte may comprise an antibody. The one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more variable regions and/or one or more constant regions. In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise buried and/or exposed amino acid residues. At least a portion of an analyte may be associated with buried and/or exposed amino acid residues. The buried and/or exposed amino acid residues can refer to the folded and/or unfolded state of the analyte (e.g., the peptide, polypeptide, or protein, or fragments thereof, or any combination thereof). The exposed and/or buried state of the one or more amino acid residues of the analyte may be present in the sample analyzed with the nanopore and/or nanopore system described herein. The exposed and/or buried state of the one or more amino acid residues of the analyte may be present in the original sample (e.g., the original biological sample). In some embodiments, identification and/or determination of buried and/or exposed amino acid residues may occur by secondary analysis. For example, identification and/or determination of a modified amino acid residue (e.g., an amino acid residue comprising a PTM and/or conjugation) may indicate an exposed and/or buried state of the amino acid residue. [0665] In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise an average speed of translocation (e.g., a rate of translocation) through a nanopore. In some cases, the average speed of translocation is the representative time taken for a population of polypeptides to move by translocation across a nanopore from a cis to trans, or trans to cis compartment, typically expressed in units such as nm/second. In other cases, the average speed of translocation is the representative time taken under specified conditions for an individual amino acid or other feature of a given polypeptide, once engaged with a nanopore, to translocate through the nanopore from an entry aperture of the nanopore to an exit aperture of the nanopore (e.g., from a first side to a second side, or a second side to a first side), typically expressed in units such as nm/second. A first side can comprise a cis side or a trans side, and a second side may comprise a cis side or a WSGR Docket Number: 64828-710.601 trans side. In some embodiments, translocation of at least a portion of an analyte through a nanopore comprises translocation from a a cis side to a trans side. [0666] In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more translocation kinetics. The translocation kinetics can be expressed as a function along a sequence of the analyte. The kinetics may comprise a function of speed and/or position along an analyte as the analyte (e.g., the at least a portion of the analyte) translocates through a pore. The kinetics of the analyte translocation can refer to different speeds of translocation that may be seen as an analyte (e.g., a polypeptide, peptide, or protein, or fragments thereof, or any combination thereof) translocates across a nanopore. The different speeds may be due to different amino acid and/or peptide sub-sequences. The different speeds may be due to modifications and/or moieties (e.g., PTMs, or conjugations, or any combination thereof) along structure of the analyte (e.g., along a polypeptide chain and/or peptide chain). Without wishing to be bound by theory, the modifications and/or moieties may interact with a nanopore (e.g., a channel, or constriction region, or any combination thereof) differently and may influence a translocation speed accordingly. The modifications and/or moieties interacting with the nanopore (e.g., a channel, or constriction region, or any combination thereof) may speed up or slow down a translocation speed of an analyte through a nanopore. In some embodiments, kinetics of translocation speed may refer to how a secondary structure, a tertiary structure, or a quaternary structure, or any combination thereof of an analyte influence translocation. For example, , kinetics of translocation speed may refer to how a secondary structure, a tertiary structure, or a quaternary structure, or any combination thereof slows an analyte’s translocation through a constriction region of a nanopore. This relates to the kinetics of translocation within/along a particular polypeptide. Changes in the kinetics of translocation can provide information on the secondary structure, tertiary structure, and/or quaternary structure of the domains of the polypeptide, amino acid composition of the polypeptide, molecular entities bound to the polypeptide (e.g., small molecules, drugs, co-factors, peptides, proteins, or nanoparticles, or any combination thereof), internal cross- links (e.g., cysteine-cysteine disulphide), conjugations (e.g., cysteine-cysteine disulphide crosslinks to other molecules or proteins, conjugate drugs, large PTMs such as ubiquitin and/or sumo), or any combination thereof. [0667] In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise determining one or more molecular entities of the analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof. Molecular entities may comprise variants and/or conjugations to an analyte as described herein. A molecular entity of the one or more molecular entities may comprise a compound. The compound may comprise a drug, or a small molecule, or any combination thereof. A molecular entity of the one or more molecular entities may comprise a particle, nucleic WSGR Docket Number: 64828-710.601 acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof. The one or more molecular entities may be coupled to an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof. For example, one or more molecular entities may be covalently coupled to an analyte. One or more molecular entities may be coupled to an analyte by a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, a non-helical linker, or any combination thereof. One or more molecular entities may be non-covalently coupled to an analyte. As another example, one or more molecular entities may be coupled via electrostatic interactions to analyte. [0668] In some embodiments, a characteristic of an analyte can comprise a property of one or more molecular entities. A property of one or more molecular entities can comprise a mass of the molecular entity, one or more charges of the molecular entity, one or more classes of the molecular entity, or identity of the molecular entity, or any combination thereof. The one or more charges can comprise a positive charge, a neutral charge, or a negative charge, or any combination thereof. In some embodiments, an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to at least about 1 molecular entity, at least about 2 molecular entities, at least about 3 molecular entities, at least about 4 molecular entities, at least about 5 molecular entities, at least about 8 molecular entities, at least about 10 molecular entities, or greater than about 10 molecular entities. In some embodiments, an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to at most about 10 molecular entities, at most about 8 molecular entities, at most about 5 molecular entities, at most about 4 molecular entities, at most about 3 molecular entities, at most about 2 molecular entities, at most about 1 molecular entity, or less than about 1 molecular entity. In some embodiments, an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to between about 1 molecular entity to about 10 molecular entities. In some embodiments, an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof may be coupled to between about 1 molecular entity to about 2 molecular entities, about 1 molecular entity to about 3 molecular entities, about 1 molecular entity to about 4 molecular entities, about 1 molecular entity to about 5 molecular entities, about 1 molecular entity to about 6 molecular entities, about 1 molecular entity to about 7 molecular entities, about 1 molecular entity to about 8 molecular entities, about 1 molecular entity to about 9 molecular entities, about 1 molecular entity to about 10 molecular entities, about 2 molecular entities to about 3 molecular entities, about 2 molecular entities to about 4 molecular entities, about 2 molecular entities to about 5 molecular entities, about 2 molecular entities to about 6 molecular entities, about 2 molecular entities to about 7 molecular entities, about 2 molecular entities to about 8 molecular entities, about 2 molecular entities to about 9 molecular entities, about WSGR Docket Number: 64828-710.601 2 molecular entities to about 10 molecular entities, about 3 molecular entities to about 4 molecular entities, about 3 molecular entities to about 5 molecular entities, about 3 molecular entities to about 6 molecular entities, about 3 molecular entities to about 7 molecular entities, about 3 molecular entities to about 8 molecular entities, about 3 molecular entities to about 9 molecular entities, about 3 molecular entities to about 10 molecular entities, about 4 molecular entities to about 5 molecular entities, about 4 molecular entities to about 6 molecular entities, about 4 molecular entities to about 7 molecular entities, about 4 molecular entities to about 8 molecular entities, about 4 molecular entities to about 9 molecular entities, about 4 molecular entities to about 10 molecular entities, about 5 molecular entities to about 6 molecular entities, about 5 molecular entities to about 7 molecular entities, about 5 molecular entities to about 8 molecular entities, about 5 molecular entities to about 9 molecular entities, about 5 molecular entities to about 10 molecular entities, about 6 molecular entities to about 7 molecular entities, about 6 molecular entities to about 8 molecular entities, about 6 molecular entities to about 9 molecular entities, about 6 molecular entities to about 10 molecular entities, about 7 molecular entities to about 8 molecular entities, about 7 molecular entities to about 9 molecular entities, about 7 molecular entities to about 10 molecular entities, about 8 molecular entities to about 9 molecular entities, about 8 molecular entities to about 10 molecular entities, or about 9 molecular entities to about 10 molecular entities. In some embodiments, a characteristic of an analyte and/or at least a portion of an analyte may comprise a quantity of two or more molecular entities (e.g., 2 molecular entities, 3 molecular entities, 4 molecular entities, 5 molecular entities, or greater than about 5 molecular entities). [0669] In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise at least one property of one or more intra cross-linkages. The intra cross-linkages may be disposed within an analyte, at least a portion of analyte, or an analyte of a plurality of analytes, or fragments thereof, or any combination thereof. In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise at least one property of one or more inter cross-linkages. The one or more inter cross-linkages can comprise a linkage formed between an analyte, at least a portion of analyte, and/or an analyte of a plurality of analytes with another an analyte, a portion of an analyte, or analyte of the plurality of analytes. Intra cross- linkages and/or inter cross-linkages may comprise one or more covalent or non-covalent interactions. Intra cross-linkages and/or inter cross-linkages may comprise one or more linkers. The one or more linkers may comprise any linker described herein. In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise one or more couplings/linkages (e.g., one or more disulfide bonds) with a molecular entity described herein. In some embodiments, a property can comprise a positioning and/or quantity WSGR Docket Number: 64828-710.601 of the one or more intra cross-linkages, the one or more inter cross-linkages, or the one or more covalent linkages with a molecular entity, or any combination thereof. In some embodiments, a property can comprise a presence and or absence of the one or more intra cross-linkages, the one or more inter cross-linkages, the one or more covalent linkages with a molecular entity, or any combination thereof. For example, a signal or change thereof of an analyte translocating through a nanopore described herein may be used to identify a presence and or absence of the one or more intra cross-linkages, the one or more inter cross-linkages, the one or more covalent linkages with a molecular entity, or any combination thereof. [0670] In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, one or more analytes (e.g., a plurality of analytes), or any combination thereof can comprise a category and/or identity associated with the analyte, at least a portion of analyte, one or more analytes (e.g., a plurality of analytes), or any combination thereof. A category of an analyte, at least a portion of analyte, one or more analytes (e.g., a plurality of analytes), or any combination thereof may comprise one or more of a type, class, gene ontology, sub-domains, functional domains, secondary structure elements, tertiary structural elements, quaternary structures, protein binding cavities, or any combination thereof. [0671] In some embodiments, one or more characteristics of an analyte, at least a portion of analyte, or one or more analytes (e.g., a plurality of analytes), or fragments thereof, or any combination thereof can comprise a force, energy, or time, or any combination thereof. A force, an energy, or a kinetic constant, or any combination thereof may be related to (i) structural domains, (ii) coupled molecular entities, or (iii) any combination thereof. For example, a structural domain and a coupled molecular entity may be detected by changes in the speed/kinetics during a portion of the translocation. These changes may be measured in the current or change thereof. In some embodiments, a force, energy, and/or time may comprise a folding energy, an unfolding force, denaturation dwell time kinetics, a binding energy, an unbinding force, or binding/unbinding dwell time kinetics, or any combination thereof. In some embodiments, one or more characteristics of one or more non- covalently bound entities may comprise binding force, binding energy, or unbinding dwell time, or any combination thereof. In some embodiments, one or more characteristics of one or more non-covalently bound entities may comprise unfolding force, folding energy, or denaturation dwell time, or any combination thereof. [0672] In some embodiments, the analyte may comprise a mass. In some embodiments, the analyte may comprise a mass of at least about 1 kDa, at least about 2 kDa, at least about 3 kDa, at least about 4 kDa, at least about 5 kDa, at least about 6 kDa, at least about 7 kDa, at least about 8 kDa, at least about 9 kDa, at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, at least about 25 kDa, at least about 30 kDa, at least about 35 kDa, at least about 40 kDa, at least about 45 kDa, at least about 50 kDa, at least about 55 kDa, at least about 60 kDa, at least about 65 kDa, at least about 70 kDa, at least about 75 kDa, at least about 80 kDa, at least about 85 kDa, at least about 90 kDa, at least about 95 kDa, at least about 100 kDa, at least about 125 kDa, at least WSGR Docket Number: 64828-710.601 about 150 kDa, at least about 175 kDa, at least about 200 kDa, at least about 250 kDa, at least about 300 kDa, at least about 350 kDa, at least about 400 kDa, at least about 450 kDa, at least about 500 kDa, at least about 550 kDa, at least about 600 kDa, at least about 650 kDa, at least about 700 kDa, at least about 750 kDa, at least about 800 kDa, at least about 850 kDa, at least about 900 kDa, at least about 950 kDa, at least about 1000 kDa, at least about 1500 kDa, at least about 2000 kDa, at least about 2500 kDa, at least about 3000 kDa, at least about 3500 kDa, at least about 4000 kDa, or greater than about 4000 kDa. In some embodiments, the analyte may comprise a mass of at most about 4000 kDa, at most about 3500 kDa, at most about 3000 kDa, at most about 2500 kDa, at most about 2000 kDa, at most about 1500 kDa, at most about 1000 kDa, at most about 950 kDa, at most about 900 kDa, at most about 850 kDa, at most about 800 kDa, at most about 750 kDa, at most about 700 kDa, at most about 650 kDa, at most about 600 kDa, at most about 550 kDa, at most about 500 kDa, at most about 450 kDa, at most about 400 kDa, at most about 350 kDa, at most about 300 kDa, at most about 250 kDa, at most about 200 kDa, at most about 175 kDa, at most about 150 kDa, at most about 125 kDa, at most about 100 kDa, at most about 95 kDa, at most about 90 kDa, at most about 85 kDa, at most about 80 kDa, at most about 75 kDa, at most about 70 kDa, at most about 65 kDa, at most about 60 kDa, at most about 55 kDa, at most about 50 kDa, at most about 45 kDa, at most about 40 kDa, at most about 35 kDa, at most about 30 kDa, at most about 25 kDa, at most about 20 kDa, at most about 15 kDa, at most about 10 kDa, at most about 9 kDa, at most about 8 kDa, at most about 7 kDa, at most about 6 kDa, at most about 5 kDa, at most about 4 kDa, at most about 3 kDa, at most about 2 kDa, at most about 1 kDa, or less than about 1 kDa. [0673] In some embodiments, the analyte may comprise a mass from about 1 kDa to about 100 kDa. In some embodiments, the analyte may comprise a mass from about 1 kDa to about 5 kDa, about 1 kDa to about 10 kDa, about 1 kDa to about 20 kDa, about 1 kDa to about 30 kDa, about 1 kDa to about 40 kDa, about 1 kDa to about 50 kDa, about 1 kDa to about 60 kDa, about 1 kDa to about 70 kDa, about 1 kDa to about 80 kDa, about 1 kDa to about 90 kDa, about 1 kDa to about 100 kDa, about 5 kDa to about 10 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 50 kDa, about 5 kDa to about 60 kDa, about 5 kDa to about 70 kDa, about 5 kDa to about 80 kDa, about 5 kDa to about 90 kDa, about 5 kDa to about 100 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 60 kDa, about 10 kDa to about 70 kDa, about 10 kDa to about 80 kDa, about 10 kDa to about 90 kDa, about 10 kDa to about 100 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 60 kDa, about 20 kDa to about 70 kDa, about 20 kDa to about 80 kDa, about 20 kDa to about 90 kDa, about 20 kDa to about 100 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 50 kDa, about 30 kDa to about 60 kDa, about 30 kDa to about 70 kDa, about 30 kDa to about 80 kDa, about 30 kDa to about 90 kDa, about 30 kDa to about 100 kDa, about 40 kDa to about 50 kDa, about 40 kDa to about 60 kDa, about 40 kDa to about 70 kDa, about 40 kDa to WSGR Docket Number: 64828-710.601 about 80 kDa, about 40 kDa to about 90 kDa, about 40 kDa to about 100 kDa, about 50 kDa to about 60 kDa, about 50 kDa to about 70 kDa, about 50 kDa to about 80 kDa, about 50 kDa to about 90 kDa, about 50 kDa to about 100 kDa, about 60 kDa to about 70 kDa, about 60 kDa to about 80 kDa, about 60 kDa to about 90 kDa, about 60 kDa to about 100 kDa, about 70 kDa to about 80 kDa, about 70 kDa to about 90 kDa, about 70 kDa to about 100 kDa, about 80 kDa to about 90 kDa, about 80 kDa to about 100 kDa, or about 90 kDa to about 100 kDa. [0674] In some embodiments, the analyte may comprise a mass from about 100 kDa to about 4,000 kDa. In some embodiments, the analyte can be from about 100 kDa to about 250 kDa, about 100 kDa to about 500 kDa, about 100 kDa to about 1,000 kDa, about 100 kDa to about 1,500 kDa, about 100 kDa to about 2,000 kDa, about 100 kDa to about 2,500 kDa, about 100 kDa to about 3,000 kDa, about 100 kDa to about 3,500 kDa, about 100 kDa to about 4,000 kDa, about 250 kDa to about 500 kDa, about 250 kDa to about 1,000 kDa, about 250 kDa to about 1,500 kDa, about 250 kDa to about 2,000 kDa, about 250 kDa to about 2,500 kDa, about 250 kDa to about 3,000 kDa, about 250 kDa to about 3,500 kDa, about 250 kDa to about 4,000 kDa, about 500 kDa to about 1,000 kDa, about 500 kDa to about 1,500 kDa, about 500 kDa to about 2,000 kDa, about 500 kDa to about 2,500 kDa, about 500 kDa to about 3,000 kDa, about 500 kDa to about 3,500 kDa, about 500 kDa to about 4,000 kDa, about 1,000 kDa to about 1,500 kDa, about 1,000 kDa to about 2,000 kDa, about 1,000 kDa to about 2,500 kDa, about 1,000 kDa to about 3,000 kDa, about 1,000 kDa to about 3,500 kDa, about 1,000 kDa to about 4,000 kDa, about 1,500 kDa to about 2,000 kDa, about 1,500 kDa to about 2,500 kDa, about 1,500 kDa to about 3,000 kDa, about 1,500 kDa to about 3,500 kDa, about 1,500 kDa to about 4,000 kDa, about 2,000 kDa to about 2,500 kDa, about 2,000 kDa to about 3,000 kDa, about 2,000 kDa to about 3,500 kDa, about 2,000 kDa to about 4,000 kDa, about 2,500 kDa to about 3,000 kDa, about 2,500 kDa to about 3,500 kDa, about 2,500 kDa to about 4,000 kDa, about 3,000 kDa to about 3,500 kDa, about 3,000 kDa to about 4,000 kDa, or about 3,500 kDa to about 4,000 kDa. [0675] In some embodiments, the analyte can be about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, about 55 kDa, about 60 kDa, about 65 kDa, about 70 kDa, about 75 kDa, about 80 kDa, about 85 kDa, about 90 kDa, about 95 kDa, about 100 kDa, about 125 kDa, about 150 kDa, about 175 kDa, about 200 kDa, about 250 kDa, about 300 kDa, about 350 kDa, about 400 kDa, about 450 kDa, about 500 kDa, about 550 kDa, about 600 kDa, about 650 kDa, about 700 kDa, about 750 kDa, about 800 kDa, about 850 kDa, about 900 kDa, about 950 kDa, about 1000 kDa, about 1500 kDa, about 2000 kDa, about 2500 kDa, about 3000 kDa, about 3500 kDa, or about 4000 kDa. [0676] In some embodiments, a protein comprises a pI of at least about 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or about 13. In some embodiments, a protein comprises a pI of at WSGR Docket Number: 64828-710.601 most about 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or about 13. In some embodiments, a protein can be denatured prior to translocating through a pore. In some embodiments, a protein may not be denatured prior to translocating through a pore. In some embodiments, a protein can be reduced prior to translocating through a pore. In some embodiments, a protein may not be reduced prior to translocating through a pore. In some embodiments, a protein can be alkylated prior to translocating through a pore. In some embodiments, a protein may not be alkylated prior to translocating through a pore. In some embodiments, a protein can be isotope labeled prior to translocating through a pore. In some embodiments, a protein may not be isotope labeled prior to translocating through a pore. In some embodiments, a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophobic amino acids. In some embodiments, a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophilic amino acids. In some embodiments, a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of charged amino acids. In some embodiments, a protein comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of neutral amino acids. [0677] In some embodiments, a non-nucleic acid based polymer analyte (e.g., polymer) comprises a peptide. In some embodiments, a non-nucleic acid based polymer analyte comprises two or more amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises a negative charge. In some embodiments, a non-nucleic acid based polymer analyte comprises a positive charge. In some embodiments, a non-nucleic acid based polymer analyte can be negatively charged. In some embodiments, a non-nucleic acid based polymer analyte can be positively charged. In some embodiments, a non-nucleic acid based polymer analyte can be zwitterionic. In some embodiments, a non-nucleic acid based polymer analyte comprises at least about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises at most about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises between 5 and 20 amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises proteolytically cleaved fragments of a protein. In some embodiments, a non-nucleic acid based polymer analyte comprises trypsinized fragments of a protein. A protease can be used to cleave a protein into peptides. In some embodiments, a protease comprises trypsin, chymotrypsin, pepsin, elastase, or any combination thereof. In some embodiments, a non-nucleic acid based polymer analyte can be denatured prior to translocating through a pore. In some embodiments, a non- nucleic acid based polymer analyte may not be denatured prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte can be reduced prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte may not be reduced prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte can be alkylated prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte may not be WSGR Docket Number: 64828-710.601 alkylated prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte can be isotope labeled prior to translocating through a pore. In some embodiments, a non-nucleic acid based polymer analyte may not be isotope labeled prior to translocating through a pore. In some embodiments, a non- nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophobic amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophilic amino acids. In some embodiments, a non- nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of charged amino acids. In some embodiments, a non-nucleic acid based polymer analyte comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of neutral amino acids. [0678] In some embodiments, a polymer comprises a polypeptide. In some embodiments, a polypeptide comprises two or more peptides. In some embodiments, a polypeptide may be a protein that can be unfolded during or before translocation through a pore. In some embodiments, a polypeptide comprises a molecular weight above 20 kDa. In some embodiments, a polypeptide comprises a molecular weight greater than about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or about 5000 kDa. In some embodiments, a polypeptide comprises a molecular weight less than about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or about 5000 kDa. In some embodiments, a polypeptide comprises a pI of at least about 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or about 13. In some embodiments, a polypeptide comprises a pI of at most about 2, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, or about 13. In some embodiments, a polypeptide can be denatured prior to translocating through a pore. In some embodiments, a polypeptide can be not denatured prior to translocating through a pore. In some embodiments, a polypeptide can be reduced prior to translocating through a pore. In some embodiments, a polypeptide may not be reduced prior to translocating through a pore. In some embodiments, a polypeptide can be alkylated prior to translocating through a pore. In some embodiments, a polypeptide may not be alkylated prior to translocating through a pore. In some embodiments, a polypeptide can be isotope labeled prior to translocating through a pore. In some embodiments, a polypeptide may not be isotope labeled prior to translocating through a pore. In some embodiments, a polypeptide comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophobic amino acids. In some embodiments, a polypeptide comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of hydrophilic amino acids. In some embodiments, a polypeptide comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of charged amino acids. In some embodiments, a polypeptide comprises at least about 10, 20, 30, 40, 50, 60, 70, 80, or about 90% of neutral amino acids. WSGR Docket Number: 64828-710.601 [0679] In some embodiments, a polysaccharide comprises a negative charge. In some embodiments, a polysaccharide comprises two or more monosaccharides. In some embodiments, a polysaccharide comprises a positive charge. In some embodiments, a polysaccharide can be negatively charged. In some embodiments, a polysaccharide can be positively charged. In some embodiments, a polysaccharide can be zwitterionic. In some embodiments, a polysaccharide can be linear. In some embodiments, a polysaccharide can be non-linear. In some embodiments, a polysaccharide can be branched. In some embodiments, a polysaccharide comprises a molecular topology comprising at least about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 branches. In some embodiments, a polysaccharide comprises a molecular topology comprising at most about 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 branches. In some embodiments, a polysaccharide comprises a sulfate pattern. In some embodiments, a polysaccharide comprises osidic bonds. In some embodiments, a polysaccharide comprises epimers of uronic acid residues. A polysaccharide can comprise various monosaccharides. A monosaccharide can be glucose, fructose, galactose, ribose, deoxyribose, or any other monosaccharide, or any stereoisomer thereof. In some embodiments, a polysaccharide comprises glycosidic linkages between monosaccharides. In some embodiments, a polysaccharide comprises starch, glycogen, galactogen, structural polysaccharides such as cellulose or chitin, or any combination thereof. In some embodiments, a polysaccharide can be a homopolysaccharide (or homoglycan) comprising monosaccharides of the same type. In some embodiments, a polysaccharide can be a heteropolysaccharide (or heteroglycan) comprising more than one type of monosaccharide repeating unit. [0680] In some embodiments, a polymer analyte can be a polypeptide of at least 30 peptide units and comprising positively and negatively charged residues, preferably wherein the polypeptide can be in a denatured/unfolded state, more preferably wherein the polypeptide can be added in a pre-denatured state. In some embodiments the polymer analyte can be an unmodified protein or a portion thereof, or a naturally occurring polypeptide or a portion thereof. In some embodiments, the polymer analyte can be a full length protein or naturally occurring polypeptide. In some embodiments polypeptides or polypeptide fragments can be conjugated to form a longer polypeptide. [0681] In some embodiments, one or more characteristics of an analyte and/or at least a portion of an analyte may comprise a fold portion of an analyte and/or at least a portion of an analyte. In some embodiments, one or more characteristics of an analyte and/or at least a portion of an analyte may comprise an unfolded portion of the analyte and/or at least a portion of an analyte. In some embodiments, one or more characteristics of an analyte and/or at least a portion of an analyte may comprise a partially folded portion of the analyte and/or at least a portion of an analyte. In some embodiments, one or more characteristics may comprise a percentage of unfolded portions associated with an analyte, at least a portion of an analyte, or any combination thereof. In some embodiments, one or more characteristics may comprise a percentage of folded portions associated with WSGR Docket Number: 64828-710.601 an analyte and/or at least a portion of an analyte. In some embodiments, an analyte and/or at least a portion of an analyte can comprise a structure comprising at least about 1% unfolded portions, at least about 5% unfolded portions, at least about 10% unfolded portions, at least about 20% unfolded portions, at least about 30% unfolded portions, at least about 40% unfolded portions, at least about 50% unfolded portions, or greater than about 50% unfolded portions. In some embodiments, an analyte and/or at least a portion of an analyte can comprise a structure comprising at most about 50% unfolded portions, at most about 40% unfolded portions, at most about 30% unfolded portions, at most about 20% unfolded portions, at most about 10% unfolded portions, at most about 5% unfolded portions, at most about 1% unfolded portions, or less than about 1% unfolded portions. In some embodiments, an analyte and/or at least a portion of an analyte can comprise a structure comprising between about 1% unfolded portions to about 50% unfolded portions. In some embodiments, an analyte and/or at least a portion of an analyte can comprise a structure comprising between about 1% unfolded portions to about 5% unfolded portions, about 1% unfolded portions to about 10% unfolded portions, about 1% unfolded portions to about 20% unfolded portions, about 1% unfolded portions to about 30% unfolded portions, about 1% unfolded portions to about 40% unfolded portions, about 1% unfolded portions to about 50% unfolded portions, about 5% unfolded portions to about 10% unfolded portions, about 5% unfolded portions to about 20% unfolded portions, about 5% unfolded portions to about 30% unfolded portions, about 5% unfolded portions to about 40% unfolded portions, about 5% unfolded portions to about 50% unfolded portions, about 10% unfolded portions to about 20% unfolded portions, about 10% unfolded portions to about 30% unfolded portions, about 10% unfolded portions to about 40% unfolded portions, about 10% unfolded portions to about 50% unfolded portions, about 20% unfolded portions to about 30% unfolded portions, about 20% unfolded portions to about 40% unfolded portions, about 20% unfolded portions to about 50% unfolded portions, about 30% unfolded portions to about 40% unfolded portions, about 30% unfolded portions to about 50% unfolded portions, or about 40% unfolded portions to about 50% unfolded portions. [0682] Any number of analytes can be translocated, detected, or identified using a method, sensor, or device disclosed herein. In some embodiments about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more unique analytes can be translocated, detected, or identified. In some embodiments, about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000 or more unique analytes can be translocated, detected, or identified. In some embodiments, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or less unique analytes can be translocated, detected, or identified. In some embodiments, about 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000 or less unique analytes can be translocated, detected, or identified. [0683] In some embodiments, a protein, a polypeptide, or a peptide can comprise a post-translational modification. A post-translational modification can be non-natural, for instance, chemical modifications WSGR Docket Number: 64828-710.601 introduced in a laboratory for biotechnological or biomedical purposes. In some embodiments, a post- translational modification can be natural, where a living organism or a cell performed the post-translational modification if an analyte. A post-translation modification can comprise modification with a hydrophobic group, modification with a cofactor, addition of a chemical group, glycation (the non-enzymatic attachment of a sugar), biotinylation, PEGylation, oxidation, or reduction, damage, or any combination thereof. In some embodiments, post-translational modification with a hydrophobic group comprises myristoylation, palmitoylation, isoprenylation or prenylation, the attachment of an isoprenoid group; farnesylation, the attachment of a farnesol group; geranylgeranylation, the attachment of a geranylgeraniol group; and glypiation, or glycosylphosphatidylinositol (GPI) anchor formation via an amide bond. In some embodiments, post- translational modification with a cofactor comprises lipoylation, attachment of a lipoate (Cs) functional group; flavination, attachment of a flavin moiety (e.g. flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)); attachment of heme C, for instance via a thioether bond with cysteine; phosphopantetheinylation, the attachment of a 4’-phosphopantetheinyl group; or retinylidene Schiff base formation. In some embodiments, post-translational modification by addition of a chemical group comprises acylation, e.g. O-acylation (esters), N-acylation (amides) or S-acylation (thioesters); acetylation, the attachment of an acetyl group for instance to the N-terminus or to lysine; formylation; alkylation, the addition of an alkyl group, such as methyl or ethyl; methylation, the addition of a methyl group for instance to lysine or arginine; amidation; butyrylation; gamma- carboxylation; glycosylation, the enzymatic attachment of a glycosyl group for instance to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine or tryptophan; polysialylation, the attachment of polysialic acid; malonylation; hydroxylation; iodination; bromination; citrulination; nucleotide addition, the attachment of any nucleotide such as any of those discussed above, ADP ribosylation; oxidation; phosphorylation, the attachment of a phosphate group for instance to serine, threonine or tyrosine (O-linked) or histidine (N-linked); adenylylation, the attachment of an adenylyl moiety for instance to tyrosine (O-linked) or to histidine or lysine (N-linked); propionylation; pyroglutamate formation; S-glutathionylation; Sumoylation; S- nitrosylation; succinylation, the attachment of a succinyl group for instance to lysine; selenoylation, the incorporation of selenium; or ubiquitinilation, the addition of ubiquitin subunits (N-linked). In some embodiments, one or more post-translational modifications can comprise phosphorylation, acetylation, amidation, deamidation, glycosylation, oxidation, ubiquitination, sumolation, lipidation, carbonylation, or any combination thereof. [0684] One or more analytes described herein can be associated with one or more characteristics. The characteristics may be clustered (e.g., grouped together). Grouping of characteristics can provide one or more properties of a sample comprising one or more analytes. For example, clustering characteristics of one or more analytes of a sample can provide one or more properties of the sample, comprising populations of analytes, quantifications of analytes, presence and/or absence of impurities, differences in analyte structure (e.g., WSGR Docket Number: 64828-710.601 secondary, tertiary, and quaternary structure), or purity of different molecules, or any combination thereof. In some cases, one or more characteristics (e.g., identity, mutations, sequences) may be associated with an analyte. In other cases, a sample may comprise a plurality of analytes. The plurality of analytes can comprise a plurality of characteristics, which can then be used to determine (e.g., by clustering) one or more properties (e.g., quantification, presence or absence of impurities, differences in secondary, tertiary, quaternary structure, purity tests of different molecules) associated with the sample. In some cases, the one or more characteristics may be the primary determination (e.g., associated with an analyte) and the one or more properties may be the secondary determination (e.g., associated with a sample that comprises the analyte). The secondary determination may be a result of the primary determination. In some cases, the secondary determination of the sample may be obtained from a plurality of characteristics associated with a plurality of analytes in the sample. SAMPLE PREPARATION [0685] Provided herein are various samples that can be assayed using a pore, membrane, device, or a method disclosed herein, and methods of preparing samples for assays. [0686] A sample can be a sample that may be known to contain or is suspected of containing one or more predetermined analytes. For instance, a biological sample from a patient may be suspected of containing a protein biomarker for cancer. The sample can be assayed to detect the presence of the one or more predetermined analytes. In some embodiments, a sample (e.g., a biological sample) described herein may be from a subject with a disease, or condition, or any combination thereof. In some embodiments, the disease or condition may comprise cancer, heart disease, neurodegenerative disease, diabetes, autoimmune disease, or infectious disease, or any combination thereof. The sample may be from an aged subject (e.g., a subject who is at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or greater than about 90 years of age). [0687] The sample may comprise an analyte described herein. The sample may comprise one or more analytes. The sample may comprise at least about 1 analyte, at least about 2 analytes, at least about 3 analytes, at least about 4 analytes, at least about 5 analytes, at least about 10 analytes, at least about 15 analytes, at least about 20 analytes, or greater than about 20 analytes. The sample may comprise at most about 20 analytes, at most about 15 analytes, at most about 10 analytes, at most about 5 analytes, at most about 4 analytes, at most about 3 analytes, at most about 2 analytes, at most about 1 analyte, or less than about 1 analyte. The sample may comprise between about 1 analyte to about 50 analytes. The sample may comprise between about 1 analyte to about 2 analytes, about 1 analyte to about 3 analytes, about 1 analyte to about 4 analytes, about 1 analyte to about 5 analytes, about 1 analyte to about 10 analytes, about 1 analyte to about 15 analytes, about 1 analyte to about 20 analytes, about 1 analyte to about 25 analytes, about 1 analyte to about 30 analytes, about 1 analyte to about 40 analytes, about 1 analyte to about 50 analytes, about 2 analytes to about 3 analytes, about 2 analytes WSGR Docket Number: 64828-710.601 to about 4 analytes, about 2 analytes to about 5 analytes, about 2 analytes to about 10 analytes, about 2 analytes to about 15 analytes, about 2 analytes to about 20 analytes, about 2 analytes to about 25 analytes, about 2 analytes to about 30 analytes, about 2 analytes to about 40 analytes, about 2 analytes to about 50 analytes, about 3 analytes to about 4 analytes, about 3 analytes to about 5 analytes, about 3 analytes to about 10 analytes, about 3 analytes to about 15 analytes, about 3 analytes to about 20 analytes, about 3 analytes to about 25 analytes, about 3 analytes to about 30 analytes, about 3 analytes to about 40 analytes, about 3 analytes to about 50 analytes, about 4 analytes to about 5 analytes, about 4 analytes to about 10 analytes, about 4 analytes to about 15 analytes, about 4 analytes to about 20 analytes, about 4 analytes to about 25 analytes, about 4 analytes to about 30 analytes, about 4 analytes to about 40 analytes, about 4 analytes to about 50 analytes, about 5 analytes to about 10 analytes, about 5 analytes to about 15 analytes, about 5 analytes to about 20 analytes, about 5 analytes to about 25 analytes, about 5 analytes to about 30 analytes, about 5 analytes to about 40 analytes, about 5 analytes to about 50 analytes, about 10 analytes to about 15 analytes, about 10 analytes to about 20 analytes, about 10 analytes to about 25 analytes, about 10 analytes to about 30 analytes, about 10 analytes to about 40 analytes, about 10 analytes to about 50 analytes, about 15 analytes to about 20 analytes, about 15 analytes to about 25 analytes, about 15 analytes to about 30 analytes, about 15 analytes to about 40 analytes, about 15 analytes to about 50 analytes, about 20 analytes to about 25 analytes, about 20 analytes to about 30 analytes, about 20 analytes to about 40 analytes, about 20 analytes to about 50 analytes, about 25 analytes to about 30 analytes, about 25 analytes to about 40 analytes, about 25 analytes to about 50 analytes, about 30 analytes to about 40 analytes, about 30 analytes to about 50 analytes, or about 40 analytes to about 50 analytes. [0688] One or more analytes of a sample may of the same species. In some embodiments, the one or more analytes of the sample may be of different species. In some embodiments, the sample comprises at least about 2 analytes of the same species, at least about 3 analytes of the same species, at least about 4 analytes of the same species, at least about 5 analytes of the same species, at least about 10 analytes of the same species, or greater than about 10 analytes of the same species. [0689] One or more analytes of the sample may be the same type of analyte. One or more analytes of the sample may be different types of analytes. For example, a sample may comprise a first type of analyte and a second type of analyte. In the first type of analyte, characteristics of the analyte may be determined. For example, in the first type of analyte, one or more of a number of analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or any combination thereof may be determined. In the second type of analyte, characteristics of the analyte may be determined. For example, in the second type of analyte, one or more of a number of analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or any combination thereof may be determined. In some WSGR Docket Number: 64828-710.601 cases, a plurality of analytes may comprise a plurality of different type of analytes. The difference in type between two or more analytes can comprise a difference in identity, sequence, structure, type of protein of proteins (e.g., antibody vs. antigen), or class (e.g., human protein vs plant protein), or a combination thereof. In other cases, the plurality of analytes may comprise a two or more analytes with different characteristics. The characteristics may be any disclosed herein. In some cases, less than all units (e.g., amino acids) may be determined to determine the identity of the analyte. [0690] In some embodiments, a presence of one or more analytes may be determined. In some embodiments, an absence of one or more analytes may be determined. A presence and/or absence of one or more analytes may be determined using a current or change thereof described herein. For example, an absence of a signal may be indicative of an absence of an analyte, an analyte type, a modification of an analyte, or any combination thereof, in a sample. [0691] A sample can be a sample that may be being interrogated for its composition. For instance, a biological sample from a patient can be human plasma. The biological sample can be analyzed to detect the presence and/or the concentration levels of various proteins in the human plasma. [0692] In some embodiments, a sample can be a biological sample. In some embodiments, a biological sample comprises blood, serum, plasma, urine, sweat, saliva, tears, mucus, phlegm, cerebrospinal fluid, cell, cell lysate, tissue, organ, organelle, bone marrow, or semen. In some embodiments, a biological sample comprises cell- free DNA, protein, or peptides, or any combination thereof. [0693] In some embodiments, a sample can be an inorganic sample. In some embodiments, a sample can comprise a water sample comprising one or more impurities, such as fluorinated substances (e.g., perfluoroalkyls; perfluorooctanoic acid; perfluorosulfonic acid), or heavy metals. [0694] A sample can be obtained, isolated or extracted from any organism or microorganism. For example, it can be obtained from a human or animal, e.g. from a bodily fluid, such as urine, lymph, saliva, mucus, seminal fluid or amniotic fluid, or from whole blood, plasma or serum. The sample may be obtained from a plant e.g. a cereal, legume, ornamental plant, fruit or vegetable, or part thereof including tubers, roots and bulbs. A sample can be produced inside (animal) cells such that it can be extracted from cells for characterization. A sample may comprise the products of cellular expression of a plasmid in a (microbial) host cell. In some embodiments, a sample can be secreted from cells. [0695] In some embodiments, a sample can comprise a protein, a polypeptide, a peptide, a polysaccharide, a DNA, a RNA, an antibiotic, a drug, a metabolite, a lipid, a steroid, a carbohydrate, an amino acid, a nucleotide, a hormone, a fatty acid, a vitamin, a flavin, a protein-cofactor, a lipid, or a phenolic compound, or any combination thereof. In some embodiments, a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, WSGR Docket Number: 64828-710.601 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique analytes. In some embodiments, a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique analytes. [0696] In some embodiments, a sample comprises proteins. In some embodiments, a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or about 6000 unique proteins. In some embodiments, a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or about 6000 unique proteins. [0697] In some embodiments, proteins or peptides in a sample can be cleaved. In some embodiments, proteins or peptides in a sample can be cleaved using an enzyme. In some embodiments, the enzyme can be a protease. In some embodiments, the protease can be trypsin, chymotrypsin, Lys-C protease, elastase, or peptidase. In some embodiments, cleaved proteins comprise peptides. [0698] In some embodiments, a peptide can be reacted with phenyl isothiocyanate. The peptide can form a cyclical phenylthiocarbamoyl derivative in response to reaction with phenyl isothiocyanate. The derivative can be cleaved as a thiazolinone derivative. In some embodiments, a peptide can undergo at least a portion of an Edman degradation cycle. In some embodiments, products of at least a portion of an Edman degradation cycle can be translocated through a pore. In some embodiments, a sample can undergo trypsinization followed by at least a portion of an Edman degradation cycle. [0699] In some embodiments, a sample comprises peptides. In some embodiments, a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique peptides. In some embodiments, a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, or about 50000 unique peptides. [0700] In some embodiments, a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 μL in volume. In some embodiments, a sample comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 mL in volume. In some embodiments, a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 μL in volume. In some embodiments, a sample comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 mL in volume. WSGR Docket Number: 64828-710.601 [0701] In some embodiments, a sample can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ng of analytes. In some embodiments, a sample can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 μg of analytes. In some embodiments, a sample can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 ng of analytes. In some embodiments, a sample can comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, or about 900 μg of analytes. [0702] A sample can be provided as an impure mixture of one or more analytes and one or more impurities. The impurities can be proteinaceous or non-proteinaceous (e.g., carbohydrates, or lipids, or any combination thereof). For example, an analyte can be an uncleaved polypeptide, and impurities can be cleaved products of a polypeptide. Impurities may comprise truncated forms of the analyte. Impurities may also comprise proteins other than the protein analyte e.g. which may be co-purified from a cell culture or obtained from a sample. [0703] A protein may comprise one or any combination of any amino acids, amino acid analogs, and naturally or artificial modified amino acids (i.e. amino acid derivatives). Amino acids (and derivatives, analogs or any combination thereof) in a polypeptide can be distinguished by their physical size and charge. An amino acids/derivatives/analogs can be naturally occurring or artificial. [0704] In some embodiments, a sample can be assayed in vitro. In some embodiments, a sample can be assayed in vivo, e.g., to sense insulin levels in diabetic patients. In some embodiments, a sample can be obtained from or extracted from an organism of various kinds (e.g. archaeal, prokaryotic, or eukaryotic). In some embodiments, a sample can comprise a body fluid of a patient (e.g. urine, lymph, mucus or amniotic fluid, sweat, saliva, blood, plasma, or serum). In some embodiments, the sample can be from a human, a mouse, a rat, a macaque, a pig, a cow, a chicken, or a plant. In some embodiments, a sample can be a non-biological sample, e.g., surgical fluids, water such as drinking water, sea water, or river water, which can provide environmental testing results, as well as reagents for laboratory tests. [0705] In some embodiments, a sample can be preprocessed prior to being assayed. In some embodiments, a sample can be purified, e.g., to isolate certain class of analytes such as proteins or peptides. In some embodiments, purification can be performed with affinity binding, such as by antibodies. In some embodiments, purification can be performed with chromatographic methods. In some embodiments, purification can be performed to isolate and/or purify specific components of the sample and/or remove unwanted background impurities. [0706] In some embodiments, a sample can be preprocessed using a denaturing agent. In some embodiments, a sample can be preprocessed using applied heat. In some embodiments, a sample can be preprocessed using chaotropic agents or detergents. In some embodiments, a sample can be preprocessed to provide a WSGR Docket Number: 64828-710.601 predetermined pH or salt condition. In some embodiments, a sample can be preprocessed using a reducing agent. In some embodiments, a sample can be preprocessed to break cross-links such as disulphide bridges, e.g., to disrupt certain secondary structures. In some embodiments, a sample can be preprocessed to label certain moieties, e.g., labeling cysteines or lysines with tags to modulate a signal arising from translocation of a labeled analyte through a pore. In some embodiments, a moiety can comprise an amine, a carbonyl, a thiol, an alkyne, or an azide. In some embodiments, a label can target a particular amino acid. In some embodiments, a moiety can be labeled or tagged with a fluorescent molecule, a radioisotopic molecule, a dye, a quantum dot, or a molecular label, or combinations thereof. In some embodiments, a label or tag can cause a greater change in measured signal when a sample can be translocated through a pore. In some embodiments, a label or tag can decrease a change in measured signal when a sample can be translocated through a pore. In some embodiments, a label or tag can increase a sensitivity of analysis for an analyte. In some embodiments, N- and C- termini of amino acid-based molecules can be modified in a sample. In some embodiments, N- or C- termini of amino acid-based molecules can be chemically modified. In some embodiments, amino acid-based molecules can be modified by adding a molecular label or tag (e.g. adding a barcode to register a precursor sample, or to facilitate capture and/or detection in a pore system). In some embodiments, a sample label or tag can comprise a peptide, a protein, or a nucleic acid, or any combination thereof. In some embodiments, a sample label or tag can comprise an amine-reactive dye, a thiol reactive dye, a carbonyl-reactive dye, a click chemistry reactive group, or a copper-free click chemistry group. In some embodiments, a sample label or tag comprises an active ester, succinimidyl ester, tetrafluorophenyl ester, sulfodichlorophenol ester, isothiocyanate, sulfonyl chloride, hydrazine, hydroxylamine, an alkoxyamine, maleimide, an iodoacetyl group, or a pyridyl disulfide. [0707] In some embodiments, an analyte can be modified. In some embodiments, the polypeptide can be modified by a leader according to embodiments involving a translocase as disclosed herein below. In some embodiments, the disclosed methods are for characterizing modifications in an analyte. In some embodiments, one or more of the amino acids/derivatives/analogs in an analyte can be post-translationally modified. [0708] In some embodiments, an analyte can be coupled to a leader. In some embodiments, a leader or leader- coupled analyte can preload and/or stall translocases. In some embodiments, an analyte may not be coupled to a leader. In some embodiments, a leader can comprise an exogenous sequence, a recognition motif, a capture motif, a stall motif, a block motif, or a coupling motif, or any combination thereof, and any permutation thereof. The motifs in a leader can be arranged in various orders. For example, the leader can comprise motifs in the order of recognition-capture-stall-block-coupling, or capture-stall-block-coupling, or recognition-stall-block- coupling. In some embodiments, the leader can comprise an exogenous sequence. In some embodiments, an exogenous sequence can be greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, or about 600 amino acids long. In some embodiments, an WSGR Docket Number: 64828-710.601 exogenous sequence can be less than about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 500, or about 600amino acids long. In some embodiments, an exogenous sequence can be from about 2 to about 600 amino acids long. In some embodiments, an exogenous sequence can be from about 5 to about 50 amino acids long. An exogenous element and/or an exogenous sequence can be one or more leader constructs, barcodes, adaptors, recognition motifs, or any combination thereof. [0709] In some embodiments, a translocase fully or partially comprises amino acids. In some embodiments, a translocase does not comprise amino acids. In some embodiments, the amino acids can be natural, non-natural, or a combination thereof. In some embodiments, translocases can move along proteins either in the C to N direction or the N to C direction. In some embodiments, the components of the leader may be arranged in either the C to N or N to C orientation, or different combinations thereof in a single construct. In some embodiments, the components of the leader may be arranged to promote translocation in either the C to N or N to C orientation. In some embodiments, a leader can comprise stretches of synthetic polymer molecules, such as PEG, PHEMA, polyacrylamide, polynucleotide, or peptide nucleic acids, or combinations thereof. The overall composition of the leader may be adapted for good water solubility. The composition may be adapted for low structural propensity in regions of capture to provide more efficient pore capture. [0710] In some embodiments, a leader can be added to the N- or C- terminus of a protein, or to both ends. In some embodiments, a leader (e.g. of the same design) can be added to both ends of an analyte for capturing and threading of the protein in both the C to N and the N to C directions, which can provide different information that can be combined informatically to improve accuracy of the analysis. In some embodiments, both termini of an analyte can be coupled to different leader designs, creating separate “leader” and “tail” ends. In some embodiments, leaders and tails can be used to control which behaviors occur at which end, for example to control the orientation of the capture of the analyte, or the loading and relative direction of the translocase, or any combination thereof. For example, in combination with a leader that directs binding of translocase and capture in a pore (e.g. leader contains translocase recognition motif/capture motif/stall motif/block motif), a tail sequence (e.g. simple unstructured amino acid sequence) can be added to the opposite end to add additional sequence for translocase to travel along during pore translocation so that the entirety of an analyte sequence passes through the pore before the translocase encounters the end of the molecule and unbinds. [0711] In some embodiments, a recognition motif promotes binding of the leader-conjugated analyte to the translocase/unfoldase. A recognition motif can include a peptide tag to enhance binding of the tagged protein. For example, a recognition motif can include ssrA (AANDENYALAA; SEQ ID NO: 38) to enhance binding of the tagged protein to ClpX, ClpA, ClpC, ClpE, PAN, FtsH, or VAT or any combination thereof. The recognition motif can comprise any of the motif sequences as shown in Table 25. Table 25. Sequences of recognition motifs. WSGR Docket Number: 64828-710.601 Description Sequence SEQ ID NO. ssrA AANDENYALAA 38 Pup tag (Prokaryotic Ubiquitin-like MAQEQTKRGGGGGDDDDIAGSTAAGQERREKLTEETDDLLDEID 39 Protein) DVLEENAEDFVRAYVQKGGQ C-terminal from SulA SASSHATRQLSGLKIHSNLYH 40 Pex15 254-309 (portion of Pex15) AKSKGKQRGVKQKIHHFHEPMLHNSSEEQVKVEDAFNQRTSTDS 41 RLQSTGTAPRKK Pex15 43-309 (portion of Pex15) SEVFQECVNLFIKRDIKDCLEKMSEVGFIDITVFKSNPMILDLF 42 VSACDIMPSFTKLGLTLQSEILNIFTLDTPQCIETRKIILGDLS KLLVINKFFRCCIKVIQFNLTDHTEQEEKTLELESIMSDFIFVY ITKMRTTIDVVGLQELIEIFIFQVKVKLHHKKPSPNMYWALCKT LPKLSPTLKGLYLSKDVSIEDAILNSIDNKIQKDKAKSKGKQRG VKQKIHHFHEPMLHNSSEEQVKVEDAFNQRTSTDSRLQSTGTAP RKK Pex15 1-309 (portion of Pex15) MAASEIMNNLPMHSLDSSLRDLLNDDLFIESDESTKSVNDQRSE 43 VFQECVNLFIKRDIKDCLEKMSEVGFIDITVFKSNPMILDLFVS ACDIMPSFTKLGLTLQSEILNIFTLDTPQCIETRKIILGDLSKL LVINKFFRCCIKVIQFNLTDHTEQEEKTLELESIMSDFIFVYIT KMRTTIDVVGLQELIEIFIFQVKVKLHHKKPSPNMYWALCKTLP KLSPTLKGLYLSKDVSIEDAILNSIDNKIQKDKAKSKGKQRGVK QKIHHFHEPMLHNSSEEQVKVEDAFNQRTSTDSRLQSTGTAPRK K [0712] A recognition motif can comprise pup tag (Prokaryotic Ubiquitin-like Protein, MAQEQTKRGGGGGDDDDIAGSTAAGQERREKLTEETDDLLDEIDDVLEENAEDFVRAYVQKGGQ ) to enhance binding to Mpa. A recognition motif can comprise C-terminal residues from SulA (SASSHATRQLSGLKIHSNLYH) to enhance binding to HslU, Lon, or any combination thereof. A recognition motif can comprise portions of Pex15 (Pex15254-309 AKSKGKQRGVKQKIHHFHEPMLHNSSEEQVKVEDAFNQRTSTDSRLQSTGTAPRKK, Pex1543-309 SEVFQECVNLFIKRDIKDCLEKMSEVGFIDITVFKSNPMILDLFVSACDIMPSFTKLGLTLQSEILNIFTL DTPQCIETRKIILGDLSKLLVINKFFRCCIKVIQFNLTDHTEQEEKTLELESIMSDFIFVYITKMRTTIDV VGLQELIEIFIFQVKVKLHHKKPSPNMYWALCKTLPKLSPTLKGLYLSKDVSIEDAILNSIDNKIQKDK AKSKGKQRGVKQKIHHFHEPMLHNSSEEQVKVEDAFNQRTSTDSRLQSTGTAPRKK, or Pex151-309 MAASEIMNNLPMHSLDSSLRDLLNDDLFIESDESTKSVNDQRSEVFQECVNLFIKRDIKDCLEKMSEV GFIDITVFKSNPMILDLFVSACDIMPSFTKLGLTLQSEILNIFTLDTPQCIETRKIILGDLSKLLVINKFFR CCIKVIQFNLTDHTEQEEKTLELESIMSDFIFVYITKMRTTIDVVGLQELIEIFIFQVKVKLHHKKPSPN MYWALCKTLPKLSPTLKGLYLSKDVSIEDAILNSIDNKIQKDKAKSKGKQRGVKQKIHHFHEPMLHN SSEEQVKVEDAFNQRTSTDSRLQSTGTAPRKK) to enhance binding to Msp1. Also provided herein are genetically engineered (mutated) variants of recognition elements. [0713] In some embodiments, the recognition motif can comprise between about one repeating unit to about 30 repeating units. In some cases, the recognition motif can comprise between about on repeating unit to about WSGR Docket Number: 64828-710.601 5 repeating units, between about 5 repeating units to about 10 repeating units, between about 10 repeating units to about 15 repeating units, between about 15 repeating units to about 20 repeating units, between about 20 repeating units to about 25 repeating units, or between about 25 repeating units. In some cases, the recognition motif can comprise at least about one repeating unit, at least about 2 repeating units, at least about 3 repeating units, at least about 4 repeating units, at least about 5 repeating units, at least about 6 repeating units, at least about 7 repeating units, at least about 8 repeating units, at least about 9 repeating units, at least about 10 repeating units, at least about 11 repeating units, at least about 12 repeating units, at least about 13 repeating units, at least about 14 repeating units, at least about 15 repeating units, at least about 16 repeating units, at least about 17 repeating units, at least about 18 repeating units, at least about 19 repeating units, at least about 20 repeating units, at least about 21 repeating units, at least about 22 repeating units, at least about 23 repeating units, at least about 24 repeating units, at least about 25 repeating units, at least about 26 repeating units, at least about 27 repeating units, at least about 28 repeating units, at least about 29 repeating units, at least about 30 repeating units, or more than 30 repeating units. In some cases, the recognition motif can comprise at most about 30 repeating units, at most about 29 repeating units, at most about 28 repeating units, at most about 27 repeating units, at most about 26 repeating units, at most about 25 repeating units, at most about 24 repeating units, at most about 23 repeating units, at most about 22 repeating units, at most about 21 repeating units, at most about 20 repeating units, at most about 19 repeating units, at most about 18 repeating units, at most about 17 repeating units, at most about 16 repeating units, at most about 15 repeating units, at most about 14 repeating units, at most about 13 repeating units, at most about 12 repeating units, at most about 11 repeating units, at most about 10 repeating units, at most about 9 repeating units, at most about 8 repeating units, at most about 7 repeating units, at most about 6 repeating units, at most about 5 repeating units, at most about 4 repeating units, at most about 3 repeating units, at most about 2 repeating units, at most about one repeating unit, or less than one repeating unit. In some cases, the recognition motif can comprise about one repeating unit, about two repeating units, about 3 repeating units, about 4 repeating units, about 5 repeating units, about 6 repeating units, about 7 repeating units, about 8 repeating units, about 9 repeating units, about 10 repeating units, about 11 repeating units, about 12 repeating units, about 13 repeating units, about 14 repeating units, about 15 repeating units, about 16 repeating units, about 17 repeating units, about 18 repeating units, about 19 repeating units, about 20 repeating units, about 21 repeating units, about 22 repeating units, about 23 repeating units, about 24 repeating units, about 25 repeating units, about 26 repeating units, about 27 repeating units, about 28 repeating units, about 29 repeating units, or about 30 repeating units. [0714] In some embodiments, a capture motif promotes capture in a pore. The recognition motif and pore- capture motif may be combined in one, bifunctional motif. In some embodiments, the capture motif comprises amino acids with a net charge to facilitate capture in a pore under the appropriate applied voltage by using WSGR Docket Number: 64828-710.601 electrophoretic attraction. In some embodiments, a motif can be comprised of some or all positively charged amino acids. In some embodiments, capture can be improved in conditions where a negative applied voltage is applied to the opposite side of a membrane in which an analyte can be contained so that the analyte can be attracted into the pore (or vice versa for a negatively charged capture motif). [0715] In some embodiments, a capture motif can be unstructured, which can promote efficient capture in a pore. In some embodiments, a capture motif can be long enough to aid efficient capture in a pore and, in combination with other motifs, can be configured so that when the capture motif can be captured in the pore from the first side (e.g., cis side) it reaches to the trans exit of the pore (or vice versa) when the bound translocase contacts the top of the pore and prevents further uncontrolled translocation. [0716] In some embodiments, a capture motif can comprise a polycationic tag. A polycationic tag can comprise one or more repeats of one or more amino acid sequences comprising one or more positively charged amino acids. For example, a polycation tag can be wholly or partially created from n repeats of (SGR)n, (SR)n, or (R)n or various combinations thereof. A polycationic tag can be used in a device with a pore with net negative internal charge where a negative voltage can be applied to the second compartment (e.g., the trans compartment) to create a net EOF cis-to-trans, wherein the tagged analyte can be added to the first side (e.g., cis side). [0717] In some embodiments, a capture motif can comprise a polyanion tag. A polyanionic tag can comprise one or more repeats of one or more amino acid sequences comprising one or more negatively charged amino acids. For example, a polyanionic tag can be wholly or partially created from n repeats of (SGD)n, (SD)n, or (D)n or various combinations thereof. A polyanionic capture tag can be used in combination with a pore with a net positive internal charge in a device with a positive voltage applied to the second compartment (e.g., the trans compartment) to create a net EOF cis-to-trans, wherein the tagged analyte can be added to the first side (e.g., cis side). [0718] In some embodiments, a capture motif can comprise a polyzwitterionic tag. A polyzwitterionic tag can comprise one or more repeats of one or more amino acid sequences comprising one or more positively charged amino acids. [0719] In some embodiments, the capture motif can comprise between about one repeating unit to about 30 repeating units. In some cases, the capture motif can comprise between about on repeating unit to about 5 repeating units, between about 5 repeating units to about 10 repeating units, between about 10 repeating units to about 15 repeating units, between about 15 repeating units to about 20 repeating units, between about 20 repeating units to about 25 repeating units, or between about 25 repeating units. In some cases, the capture motif can comprise at least about one repeating unit, at least about 2 repeating units, at least about 3 repeating units, at least about 4 repeating units, at least about 5 repeating units, at least about 6 repeating units, at least about 7 repeating units, at least about 8 repeating units, at least about 9 repeating units, at least about 10 repeating units, WSGR Docket Number: 64828-710.601 at least about 11 repeating units, at least about 12 repeating units, at least about 13 repeating units, at least about 14 repeating units, at least about 15 repeating units, at least about 16 repeating units, at least about 17 repeating units, at least about 18 repeating units, at least about 19 repeating units, at least about 20 repeating units, at least about 21 repeating units, at least about 22 repeating units, at least about 23 repeating units, at least about 24 repeating units, at least about 25 repeating units, at least about 26 repeating units, at least about 27 repeating units, at least about 28 repeating units, at least about 29 repeating units, at least about 30 repeating units, or more than 30 repeating units. In some cases, the capture motif can comprise at most about 30 repeating units, at most about 29 repeating units, at most about 28 repeating units, at most about 27 repeating units, at most about 26 repeating units, at most about 25 repeating units, at most about 24 repeating units, at most about 23 repeating units, at most about 22 repeating units, at most about 21 repeating units, at most about 20 repeating units, at most about 19 repeating units, at most about 18 repeating units, at most about 17 repeating units, at most about 16 repeating units, at most about 15 repeating units, at most about 14 repeating units, at most about 13 repeating units, at most about 12 repeating units, at most about 11 repeating units, at most about 10 repeating units, at most about 9 repeating units, at most about 8 repeating units, at most about 7 repeating units, at most about 6 repeating units, at most about 5 repeating units, at most about 4 repeating units, at most about 3 repeating units, at most about 2 repeating units, at most about one repeating unit, or less than one repeating unit. In some cases, the capture motif can comprise about one repeating unit, about two repeating units, about 3 repeating units, about 4 repeating units, about 5 repeating units, about 6 repeating units, about 7 repeating units, about 8 repeating units, about 9 repeating units, about 10 repeating units, about 11 repeating units, about 12 repeating units, about 13 repeating units, about 14 repeating units, about 15 repeating units, about 16 repeating units, about 17 repeating units, about 18 repeating units, about 19 repeating units, about 20 repeating units, about 21 repeating units, about 22 repeating units, about 23 repeating units, about 24 repeating units, about 25 repeating units, about 26 repeating units, about 27 repeating units, about 28 repeating units, about 29 repeating units, or about 30 repeating units. [0720] In some embodiments, a stall motif provides a region of low traction for a translocase. In some embodiments, a low traction region can be a region upon which a translocase cannot easily gain traction, e.g. due to a lack of side chains or small side chains, or side chains that do not interact favorably with the certain translocase structures (e.g., “paddles”) through which the translocase can exert its NTP driven power stroke. In some embodiments, when encountering the stall region, the translocase can struggle to proceed and can undergo futile NTP turnover, which can manifest into periods of moving, slipping, back sliding, rebinding, moving, and so on. In some embodiments, the stall motif can comprise a suitably long stretch amino acid residues such as glycines, or alanines or any combination thereof and other sequences. In some embodiments, the glycines can be interspersed with other residues, such as serine, to improve polypeptide properties such as structure or WSGR Docket Number: 64828-710.601 solubility. In some embodiments, the stall motif comprises a region comprising a non-amino acid chemistry. In some cases, the stall motif can comprise a region of one or more nucleotides. In some cases, the stall motif can comprise a region of one or more polymeric molecules. In some cases, the one or more polymeric molecules can comprise polyethylene glycol, ethylene, polystyrene, vinyl chloride, polyethylene, polypropylene, polycarbonates, polytetrafluoroethylene, polyamide, silicone based polymers, PMOXA polymers, polyglycans, polyacrylamide polymers, polyacrylic acid polymers, polyamines, polyethyleneimines, quaternaray ammonium polymers, polyvinyl alcohol polymers, pluronic polymers, ethylene oxide polymers, propylene oxide polymers, polyvinylpyrrolidone polymers, or carboxypolymethylene polymers, or any combination thereof. In some embodiments, the stall motif can comprise a region of one or more nucleic acid molecules. In some cases, the one or more nucleic acid comprises can comprise DNA, RNA, or any combination thereof. In some cases, the stall motif can comprise a region of one or more nucleic acid molecule analogues. In some cases, the one or more nucleic acid molecule analogues can comprise PNA, LNA, BNA, GNA, TNA, or HNA, or any combination thereof. [0721] In some embodiments, at least a portion of a stall motif comprises a region that does not interact favorably with a paddle of a translocase. In some embodiments, the paddles of the translocase may be unable to grasp the analyte-leader construct complex at the stall motif. In some cases, the translocase may not be able to move the analyte through the translocase and the nanopore when the translocase is unable to grasp the analyte-leader construct complex. [0722] In some embodiments, amino acids with no side chains can include glycine. In some embodiments, amino acids with small side chains can include alanine, serine, cysteine, valine, threonine, leucine, or isoleucine, or any combination thereof. [0723] In some embodiments, the stall motif can comprise any combination of amino acids with no side chains or amino acids with small side chains. In some embodiments, the stall motif can comprise any combination of amino acids with no side chains or amino acids with small side chains. In some cases, the stall motif can comprise glycine. In some embodiments, the stall motif can comprise alanine. In some embodiments, the stall motif can comprise serine. In some embodiments, the stall motif can comprise cysteine. In some embodiments, the stall motif can comprise valine. In some embodiments, the stall motif can comprise threonine. In some embodiments, the stall motif can comprise leucine. In some embodiments, the stall motif can comprise isoleucine. In Some embodiments, the stall motif can comprise glycine, alanine, serine, cysteine, valine, threonine, leucine, or isoleucine, or any combination thereof. [0724] A stall motif can comprise a region of amino acid residues including at least one of glycine, alanine, valine, or serine. In some embodiments, the stall motif comprises a region comprising n repeats of glycine ((G)n), alanine, ((A)n), or valine, ((V)n). In some embodiments, the stall motif can comprise n repeats of WSGR Docket Number: 64828-710.601 glycine. In some embodiments, the stall motif can comprise n repeats of alanine. In some embodiments, the stall motif can comprise n repeats of valine. In some cases, the stall motif can comprise n repeats of glycine and n repeats of alanine. In some cases, the stall motif can comprise n repeats of alanine and n repeats of valine. In some cases, the stall motif can comprise n repeats of glycine and n repeats of valine. In some cases, the stall motif can comprise n repeats of glycine, n repeats of alanine, and n repeats of valine. [0725] In some embodiments, the stall motif comprises a region comprising n repeats of glycine-serine ((GS)n), serine-glycine ((SG)n), alanine-serine ((AS)n), serine-alanine ((SA)n), valine-serine ((VS)n), or serine-valine ((SV)n). [0726] In some embodiments, a the stall motif comprises a region comprising n repeats of glycine-serine ((GS)n), serine-glycine ((SG)n), alanine-serine ((AS)n), serine-alanine ((SA)n), valine-serine ((VS)n), or serine-valine ((SV)n) and n can be between about 1 to about 50. In some cases, n can be between about 1 to about 5, between about 5 to about 10, between about 10 to about 15, between about 15 to about 20, between about 20 to about 25, between about 25 to about 30, between about 30 to about 35, between about 35 to about 40, between about 40 to about 45, or between about 45 to about 50. [0727] In some embodiments, the stall motif comprises a region comprising n repeats of glycine-serine ((GS)n), serine-glycine ((SG)n), alanine-serine ((AS)n), serine-alanine ((SA)n), valine-serine ((VS)n), or serine-valine ((SV)n) and n can be greater than about 1, greater than about 2, greater than about 3, greater than about 4, greater than about 5, greater than about 6, greater than about 7, greater than about 8, greater than about 9, greater than about 10, greater than about 11, greater than about 12, greater than about 13, greater than about 14, greater than about 15, greater than about 16, greater than about 17, greater than about 18 , greater than about 19, greater than about 20, greater than about 21, greater than about 22, greater than about 23, greater than about 24, greater than about 25, greater than about 26, greater than about 27, greater than about 28, greater than about 29, greater than about 30, greater than about 31, greater than about 32, greater than about 33, greater than about 34, greater than about 35, greater than about 36, greater than about 37, greater than about 38, greater than about 39, greater than about 40, greater than about 41, greater than about 42, greater than about 43, greater than about 44, greater than about 45, greater than about 50, or more. [0728] In some embodiments, the stall motif comprises a region comprising n repeats of glycine-serine ((GS)n), serine-glycine ((SG)n), alanine-serine ((AS)n), serine-alanine ((SA)n), valine-serine ((VS)n), or serine-valine ((SV)n) and n can be less than about 50, less than about 49, less than about 48, less than about 47, less than about 46, less than about 45, less than about 44, less than about 43, less than about 42, less than about 41, less than about 40, less than about 39, less than about 38, less than about 37, less than about 36, less than about 35, less than about 34, less than about 33, less than about 32, less than about 31, less than about 30, less than about 29, less than about 28, less than about 27, less than about 26, less than about 25, less than about 24, WSGR Docket Number: 64828-710.601 less than about 23, less than about 22, less than about 21, less than about 20, less than about 19, less than about 18, less than about 17, less than about 16, less than about 15, less than about 14, less than about 13, less than about 12, less than about 11, less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, less than about 1, or less. In some embodiments, n can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50. [0729] In some embodiments, the stall motif can be coupled to the leader construct. In some cases, the stall motif can be coupled to the leader construct via a covalent bond. In some cases, the stall motif can be coupled to the leader construct via a noncovalent bond. In some cases, the stall motif can be coupled to the leader construct via a linker. In some cases, the stall motif can be coupled to the leader construct via Click chemistry. [0730] In some embodiments, the stall motif can comprise between about one repeating unit to about 30 repeating units. In some cases, the stall motif can comprise between about on repeating unit to about 5 repeating units, between about 5 repeating units to about 10 repeating units, between about 10 repeating units to about 15 repeating units, between about 15 repeating units to about 20 repeating units, between about 20 repeating units to about 25 repeating units, or between about 25 repeating units. In some cases, the stall motif can comprise at least about one repeating unit, at least about 2 repeating units, at least about 3 repeating units, at least about 4 repeating units, at least about 5 repeating units, at least about 6 repeating units, at least about 7 repeating units, at least about 8 repeating units, at least about 9 repeating units, at least about 10 repeating units, at least about 11 repeating units, at least about 12 repeating units, at least about 13 repeating units, at least about 14 repeating units, at least about 15 repeating units, at least about 16 repeating units, at least about 17 repeating units, at least about 18 repeating units, at least about 19 repeating units, at least about 20 repeating units, at least about 21 repeating units, at least about 22 repeating units, at least about 23 repeating units, at least about 24 repeating units, at least about 25 repeating units, at least about 26 repeating units, at least about 27 repeating units, at least about 28 repeating units, at least about 29 repeating units, at least about 30 repeating units, or more than 30 repeating units. In some cases, the stall motif can comprise at most about 30 repeating units, at most about 29 repeating units, at most about 28 repeating units, at most about 27 repeating units, at most about 26 repeating units, at most about 25 repeating units, at most about 24 repeating units, at most about 23 repeating units, at most about 22 repeating units, at most about 21 repeating units, at most about 20 repeating units, at most about 19 repeating units, at most about 18 repeating units, at most about 17 repeating units, at most about 16 repeating units, at most about 15 repeating units, at most about 14 repeating units, at most about 13 repeating units, at WSGR Docket Number: 64828-710.601 most about 12 repeating units, at most about 11 repeating units, at most about 10 repeating units, at most about 9 repeating units, at most about 8 repeating units, at most about 7 repeating units, at most about 6 repeating units, at most about 5 repeating units, at most about 4 repeating units, at most about 3 repeating units, at most about 2 repeating units, at most about one repeating unit, or less than one repeating unit. In some cases, the stall motif can comprise about one repeating unit, about two repeating units, about 3 repeating units, about 4 repeating units, about 5 repeating units, about 6 repeating units, about 7 repeating units, about 8 repeating units, about 9 repeating units, about 10 repeating units, about 11 repeating units, about 12 repeating units, about 13 repeating units, about 14 repeating units, about 15 repeating units, about 16 repeating units, about 17 repeating units, about 18 repeating units, about 19 repeating units, about 20 repeating units, about 21 repeating units, about 22 repeating units, about 23 repeating units, about 24 repeating units, about 25 repeating units, about 26 repeating units, about 27 repeating units, about 28 repeating units, about 29 repeating units, or about 30 repeating units. [0731] In some embodiments, a block motif comprises a region positioned after a stall region through which a translocase cannot progress. In some embodiments, a block region after a stall region can prevent a translocase from diffusing past the stall region and regaining traction on the analyte beyond the stall region. In some embodiments, the block motif can be used without a stall region. Alternatively, the block motif can replace the stall region if it can be sufficiently robust in which the translocase is unable to overcome on its own. [0732] In some embodiments, a block motif provides a steric blockade. A steric blockade (e.g., steric obstruction) can prevent a translocase from pulling the analyte through the translocase by providing a physical barrier to the translocase In some embodiments, the block can comprise a folded protein structure that can be part of the polypeptide backbone of the translocated analyte that the protein cannot easily unfold. In some embodiments, a block motif can comprise a protein such as Maltose Binding Protein (MBP), or Titin or any combination thereof. In some embodiments, a block motif can comprise proteins (enzymes) that may be resistant to unfolding by a pore (e.g., ClpX), for example dihydrofolate reductase or barnase in presence of stabilizing ligands. [0733] In some embodiments, a block motif comprises a large bulky side chain. In some embodiments, a large bulky side-chain can be a covalently bound large molecule, such as a carbohydrate, a multi-ring molecule, or a branched dextran. In some embodiments, a side chain comprises a relatively small binder to which a larger species can be bound non-covalently (and which can be displaced when the motor proceeds through the block), e.g., biotin to which streptavidin can be bound, or a small antigen element to which a nanobody or antibody can be bound. [0734] In some cases, the block motif can be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about WSGR Docket Number: 64828-710.601 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% larger than the opening of channel of the nanopore. [0735] In some embodiments, the block motif may not be larger than the opening of the channel of the nanopore. In some embodiments, the block motif can enter into the channel of the nanopore in the absence of a translocase. In some embodiments, the block motif may not enter into the channel of the nanopore in the absence of a translocase. [0736] A steric obstruction or blockade can be formed by a folded protein or a bulky side chain. A folded protein may be difficult to unfold, or unfolding-resistant. In some embodiments, the steric obstruction can be one or more bulky amino acids. In some cases, the one or more bulky amino acids can comprise histidine, phenylalanine, tyrosine, or tryptophan, or any combination thereof. In some embodiments, the steric obstruction can have between about 1 to about 20 bulky amino acids. In some embodiments, the steric obstruction can have between about 1 to about 5 bulky amino acids, between about 5 to about 10 bulky amino acids, between about 10 to about 15 bulky amino acids, or between about 15 to about 20 bulky amino acids. In some cases, the steric obstruction can have at least about 1 bulky amino acid, at least about 2 bulky amino acids, at least about 3 bulky amino acids, at least about 4 bulky amino acids, at least about 5 bulky amino acids, at least about 6 bulky amino acids, at least about 7 bulky amino acids, at least about 8 bulky amino acids, at least about 9 bulky amino acids, at least about 10 bulky amino acids, at least about 11 bulky amino acids, at least about 12 bulky amino acids, at least about 13 bulky amino acids, at least about 14 bulky amino acids, at least about 15 bulky amino acids, at least about 16 bulky amino acids, at least about 17 bulky amino acids, at least about 18 bulky amino acids, at least about 19 bulky amino acids, at least about 20 bulky amino acids, or more than 20 bulky amino acids. In some cases, the steric obstruction can have at most about 20 bulky amino acids, at most about 19 bulky amino acids, at most about 18 bulky amino acids, at most about 17 bulky amino acids, at most about 16 bulky amino acids, at most about 15 bulky amino acids, at most about 14 bulky amino acids, at most about 13 bulky amino acids, at most about 12 bulky amino acids, at most about 11 bulky amino acids, at most about 10 bulky amino acids, at most about 9 bulky amino acids, at most about 8 bulky amino acids, at most about 7 bulky amino acids, at most about 6 bulky amino acids, at most about 5 bulky amino acids, at most about 4 bulky amino acids, at most about 3 bulky amino acids, at most about 2 bulky amino acids, at most about 1 bulky amino acid, or less than 1 bulky amino acid. In some cases, the steric obstruction can have about 1 bulky amino acid, about 2 bulky WSGR Docket Number: 64828-710.601 amino acids, about 3 bulky amino acids, about 4 bulky amino acids, about 5 bulky amino acids, about 6 bulky amino acids, about 7 bulky amino acids, about 8 bulky amino acids, about 9 bulky amino acids, about 10 bulky amino acids, about 11 bulky amino acids, about 12 bulky amino acids, about 13 bulky amino acids, about 14 bulky amino acids, about 15 bulky amino acids, about 16 bulky amino acids, about 17 bulky amino acids, about 18 bulky amino acids, about 19 bulky amino acids, or about 20 bulky amino acids. In some cases, the steric obstruction can have any combination of bulky amino acids disclosed herein. [0737] In some embodiments, a translocase may unfold an analyte with a three-dimensional structure by pulling at the analyte’s backbone with such a force to overcome the energetic bonds that maintain the three- dimensional structure of the analyte and thereby disassemble (e.g., unfold) a steric obstruction in the analyte. In some embodiments, the analyte can be a protein with a three-dimensional structure. In some cases, a translocase may unfold the protein by pulling at the peptide backbone with such force as to overcome the energetic bonds that maintain the three dimensional structure, and thereby disassemble a steric obstruction. [0738] To prevent this from occurring, a block motif can comprise a protein, peptide, polypeptide, or any combination thereof that may be unfolding-resistant so as to prevent the translocase from disassembling the steric obstruction provided by the protein. In some embodiments, an unfolding-resistant protein, peptide, or polypeptide, or fragments thereof, or any combination thereof can comprise a protein, peptide, or polypeptide, or fragments thereof, or any combination thereof that does not naturally unfold. In some cases, the unfolding- resistant protein, peptide, or polypeptide, or fragments thereof, or any combination thereof may require additional energy in order to be unfolded. In some cases, the additional energy can comprise the movement of the unfolding resistant protein, peptide, or polypeptide, or fragments thereof, or any combination thereof by a translocase. In some cases, the additional energy can comprise the movement of the analyte through the nanopore. In some cases, the additional energy can comprise the movement of the unfolding resistant protein, or peptide, polypeptide, or fragments thereof, or any combination thereof by a translocase and the movement of the analyte through the nanopore. [0739] In some embodiments, the unfolding resistant protein, peptide, or polypeptide can be a protein, peptide, or polypeptide that can be coupled to a cognate partner. In some cases, the protein, peptide, or polypeptide coupled to a cognate partner can have a higher melting temperature than a protein, peptide, or polypeptide not coupled to a cognate partner. In some cases, the protein, peptide, or polypeptide coupled to a cognate partner can have a melting temperature that can be from 0.1% to about 500% higher than a protein, peptide, or polypeptide not coupled to a cognate partner. In some cases, the protein, peptide, or polypeptide coupled to a cognate partner can have a melting temperature that can be about 0.1%, about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about WSGR Docket Number: 64828-710.601 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% higher than a protein, peptide, or polypeptide not coupled to a cognate partner. [0740] In some cases, the cognate partner can be a small molecule. In some instances, the small molecule can comprise sugars, lipids, fatty acids, phenolic compounds, or alkaloids, or any combination thereof. In some cases, the cognate partner can be one or more metal salts. In some instances, the one or more metal salts can comprise aluminum sulfate, sodium aluminate, ferric chloride, ferric sulfate, ferrous sulfate, ferrous chloride, or any combinations thereof. In some cases, the cognate partner can be a peptide. In some cases, the peptide can comprise amino acid residues. In some instances, the peptide cognate residue can comprise from about one amino acid residue to about 20 amino acid residues. In some instances, the peptide can comprise at least about one amino acid residue, at least about 5 amino acid residues, at least about 10 amino acid residues, at least about 15 amino acid residues, at least about 20 amino acid residues, or more than 20 amino acid residues, In some instances, the peptide can comprise at most about 20 amino acid residues, at most about 15 amino acid residues, at most about 10 amino acid residues, at most about 5 amino acid residues, at most about one amino acid residues, or less than one amino acid residue. In some instances, the peptide can comprise about one amino acid residue, about 5 amino acid residues, about 10 amino acid residues, about 15 amino acid residues, or about 20 amino acid residues. In some cases, the peptide cognate partner can comprise a fluorophore. In some cases, the cognate partner can be a whole protein, peptide, or polypeptide. In some cases, the cognate partner can be cross- linked peptides. In some cases, the cognate partner can be an aptamer. In some cases, the cognate partner can be an antibody. In some instances, the antibody can comprise anti-fluorophore antibody. In some cases, the cognate partner can be a nanobody. In some cases, the cognate partner can be biotin. In some cases, the cognate partner can be a monobody. In some cases, the cognate partner can be an affimer. In some cases, the cognate partner can be an avidin molecule. In some cases, the cognate partner can be a darpin. [0741] A protein, peptide, or polypeptide can be unfolding-resistant by comprising stabilizing bonds such that a translocase may not be able to exert sufficient energy to disrupt the bonds. In some cases, a stabilizing bond can be a disulfide bridge between cysteine residues, wherein the energy of the bond can be greater than the energy exerted by the translocase. An unfolding-resistant protein, peptide, or polypeptide may be a protein, peptide, or polypeptide that has been cross-linked to form multiple stabilizing bonds. A protein, peptide, or polypeptide may be cross-linked multiple stabilizing bonds. In some cases, the stabilizing bond can be a WSGR Docket Number: 64828-710.601 disulfide bridge, a desmosine bond, an ionic bond, a hydrogen bond, hydrophobic interactions, or van der Waals forces, or combinations thereof. [0742] A protein, peptide, or polypeptide may be unfolding-resistant due to a stabilizing ligand. A stabilizing ligand can comprise a molecule that binds to the protein, peptide, or polypeptide. The stabilizing ligand may bind exclusively in the folded state. The binding of the stabilizing ligand may provide an additional energetic barrier to protein, peptide, or polypeptide unfolding by lowering the energy state of the protein, peptide, or polypeptide, which provides an additional energetic barrier for the translocase to overcome in order to remove the steric obstruction provided by the protein, peptide, or polypeptide in a block motif. The binding of the stabilizing ligand may provide an additional kinetic barrier to protein, peptide, or polypeptide unfolding by adding an additional kinetic operation to overcome to reach an unfolded state and remove the steric blockade provided by the protein, peptide, or polypeptide. In some embodiments, the stabilizing ligand can comprise one or more internal bonds within the unfolding-resistant protein, peptide, or polypeptide. In some cases, the one or more internal bonds can comprise disulfide bonds, ionic bonds, hydrogen bonds, metallic bonds, or a polar covalent bond, a non-polar covalent bond, or any combination thereof. In some embodiments, the stabilizing ligand can comprise one or more ligands. In some cases, the one or more ligands can connect two or more locations on the unfolding resistant protein, peptide, or polypeptide. In some cases, the binding of the one or more ligands to the two or more locations on the unfolding resistant protein, peptide, or polypeptide can increase the stabilization of the unfolding resistant protein, peptide, or polypeptide. In some cases, increasing the stabilization of unfolding resistant protein, peptide, or polypeptide may require additional energy to unfold the unfolding resistant protein, peptide, or polypeptide. In some embodiments, the one or more ligands can comprise one or more peptide ligands. In some embodiments, the one or more ligands can comprise one or more nucleic acid ligands. In some cases, the one or more nucleic acid ligands can comprise DNA molecules, RNA molecules, nucleic acid analog molecules, or any combination thereof. In some embodiments, the one or more ligands can comprise one or more oligosaccharide ligands. In some cases, the one or more oligosaccharide ligands can comprise mannose, galactose, glucose, or lactose, or any combination thereof. In some embodiments, the one or more ligands can comprise one or more lipid ligands. In some cases, the one or more lipid ligands can comprise tricylglycerols, phospholipids, sterols, or any combination thereof. In some embodiments, the one or more ligands can comprise one or more chemical ligands. In some cases, the one or more chemical ligands can comprise anilinonapththalene sulfonate derivatives, iodide, bromide, sulfide, thiocyanate, chloride, nitrate, azide, fluoride, hydroxide, oxalate, water, nitrite, isothiocyanate, acetonitrile, pyridine, ammonia, ethylenediamine, 2,2’-bipyridine, 1,10-phenanthroline, nitrite, triphenylphosphine, cyanide, or carbon monoxide, or any combination thereof. In some cases, the one or more ligands can comprise WSGR Docket Number: 64828-710.601 one or more stabilizing molecules. In some cases, the one or more stabilizing ligands can comprise Barstar, methotrexate, biotin, or streptavidin, or any comination thereof. [0743] In some embodiments, the stabilizing ligand can comprise at least one portion of an unfolding-resistant protein, peptide, or polypeptide. In some cases, an unfolding-resistant protein, peptide, or polypeptide can comprise a protein, peptide, or polypeptide that requires additional energy in order for the protein, peptide, or polypeptide to be unfolded. In some cases, the at least one portion of the unfolding-resistant protein, peptide, or polypeptide can be a region of the unfolding-resistant protein, peptide, or polypeptide. [0744] An unfolding-resistant protein, peptide, or polypeptide may comprise Maltose Binding Protein (MBP), Titin, dihydrofolate reductase, barnase, mNeonGreen, dihydrofolate reductase, or streptavidin, or any combination thereof. [0745] In some embodiments, a coupling motif conjugates a motif to an analyte. In some embodiments, the coupling motif provides a reactive chemical species for chemically attaching a leader or a tail to an analyte. In some embodiments, the coupling motif may bind to a cysteine or a lysine residue in a protein. In some embodiments, the coupling motif comprises a tag for specifically coupling to either the N-termini, C-termini, or both of a protein. [0746] In some embodiments, the coupling motif may be a comprised of sequence for enzymatically coupling the leader to the analyte. For example, a coupling motif can comprise a motif for binding/loading of an enzyme having peptide ligase activity, such as a broad spectrum peptiligase, omniligase, or a similar enzyme. In some embodiments, the coupling motif may employ recognition sequences that couple enzymes such as Sortases either to the N- or C- terminus. [0747] In some cases, the coupling motif can couple to the analyte via a covalent bond. In some cases, the coupling motif can couple to the analyte via a non-covalent bond. In some cases, the coupling motif can couple to the analyte via a linker. In some cases, the coupling motif can couple to the analyte via click chemistry reactions. In some cases, the click chemistry reactions can comprise cycloaddition reactions, hetero Diels-Alder reactions, nucleophilic ring-opening reaction, carbonyl chemistry, epoxidation, dihydroxylation, or azide- phosphine coupling, or any combination thereof. In some cases, the click chemistry reactions can involve one or more click reagents. In some cases, the one or more click reagents can include 1,3-dipolar families, epoxides, aziridines, cyclic sulfates, epoxides, aziridines, cyclic sulfates, oxine ethers, hydrazones, aromatic heterocycles, or any combination thereof. In some cases, the coupling motif can couple to the analyte via a cysteine amino acid residue of the analyte. In some examples, the coupling motif can couple to a cysteine amino acid residue via a sulfide based conjugative reaction. In some cases, the coupling motif can couple to the analyte via an ester reaction. In some cases, the coupling motif can couple to the analyte via a thioester reaction. In some cases, the coupling motif can couple to the analyte via an amide reaction. In some cases, the coupling motif can couple to WSGR Docket Number: 64828-710.601 the analyte via a native chemical ligation reaction. In some cases, the native chemical ligation reaction can comprise reacting a peptide thioester with a cysteinyl peptide. In some cases, the coupling motif can couple to the analyte via a bioconjugation reaction. In some instances, the bioconjugation reaction can comprise reacting lysine with a N-hydroxysuccinimidyl (NHS) ester, a lysine acylation reaction, reacting lysine with isocyanates, reacting lysine with isothiocyanates, reacting lysine with benzoyl fluorides, reacting cysteine with maleimides, reacting cysteine with iodoacetamides, reacting cysteine with 2-thiopyridine, reacting cysteine with 3- arylpropiolonitrile, an electrophilic aromatic substitution reactions, reacting tyrosine with diazonium salts, reacting tyrosine with 4-phenyl-1,2,4-triazole-3,5-dione (PTAD), a mannich reaction, reacting a N-terminal serine or threonine with NaIO4, reacting N-terminal cysteine with iodoacetamides, reacting N-terminal of analyte with pyridoxal phosphate, staudinger ligation with azides, huisgen cyclization of azides, strain promoted huisgen cyclization of azides, cysteine or Tryptophan RH-catalyzed alkylation, lysine or N-terminal Ir- catalyzed alkylation, tyrosine Pd-catalyzed O-alkylation. cysteine Au-catalyzed alkylation, tryptophan arylation, cysteine arylation, or lysine arylation, or any combination thereof. [0748] In some embodiments, the coupling motif can selectively couple to the N-terminal of the analyte. In some cases, the coupling motif can selectively couple to the N-terminal of the analyte via a N-terminal specific reaction. In some cases, the N-terminal specific reaction can comprise transamination reaction, an azolation reaction, a condensation reaction, an oxidation reaction, an acylation reaction, or an alkylation imine formation reaction, or any combination thereof. In some embodiments, the coupling motif can selectively couple to the C-terminal of the analyte. In some cases, the coupling motif can selectively couple to the N-terminal of the analyte via a C-terminal specific reaction. In some cases, the C-terminal specific reaction can comprise decarboxylative photoredox alkylation, thioacid/azide amidation, C-terminal bioconjugation with asparaginyl endopeptides, or any combination thereof. [0749] A coupling motif can comprise a chemical group. The chemical group can react with a portion of an analyte to covalently couple the leader construct to the analyte. The chemical group can react with proteins. The chemical group can react with amino acid groups, including cysteines, lysines, tyrosine, tryptophan, arginine, methionine, N-terminal residues, or C-terminal residues, or any combination thereof. In some cases, a maleimide chemical group can react with cysteine. In some cases, an iodoacetamide chemical group can react with cysteine. In some cases, a 2-thiopyridine group can react with cysteine. In some cases, a 3- arylpropiolonitrile can react with cysteine. In some cases, an NHS-ester group can react with lysine. In some cases, an isocyanate group can react with lysine. In some cases, an isothiocyanate group can react with lysine. In some cases, a benzoyl fluoride group can react with lysine. In some cases, a diazonium salt can react with tyrosine. In some cases, PTAD can react with tyrosine. In some cases, tyrosine can overgo a Mannich reaction. In some cases, a N-terminal serine or a N-terminal threonine can react with a NaIO4 group. In some cases, a N- WSGR Docket Number: 64828-710.601 terminal cysteine can react with an iodoacetamide group. In some cases, an N-terminal residue can react with PLP. [0750] A coupling motif can couple a leader construct to an analyte via enzymatic activity. In some embodiments, the coupling motif can comprise an enzyme coupling region. In some cases, an enzyme can couple to the enzyme coupling region of the coupling motif. A coupling motif can comprises a portion for binding or loading of an enzyme. In some cases, the enzyme can have peptide ligase activity. In some embodiments, the enzyme can comprise peptiligase, omniligase, sortase, butelase, trypsiligase, peptide amidase, or asparaginyl endopeptidase, or any combination thereof. [0751] A coupling motif may comprise a recognition sequence. The recognition sequence may help the enzyme target an N-terminus or a C-terminus of the analyte. The recognition sequence may help the enzyme target a 3’ end or a 5’ end of the analyte. In some embodiments, a leader construct couples to an N- terminus, a C-terminus or both a N-terminus and C-terminus of an analyte. [0752] In some cases, the leader construct can comprise a stall motif and a block motif. In some cases, the leader construct can comprise a stall motif and a coupling motif. In some cases, the leader construct can comprise a stall motif and a recognition motif. In some cases, the leader construct can comprise a stall motif and a capture motif. In some cases, the leader construct can comprise a block motif and a coupling motif. In some cases, the leader construct can comprise a block motif and a recognition motif. In some cases, the leader construct can comprise a block motif and a capture motif. In some cases, the leader construct can comprise a coupling motif and a recognition motif. In some cases, the leader construct can comprise a coupling motif and a capture motif. In some cases, the leader construct can comprise a recognition motif and a capture motif. In some instances, the leader construct can comprise a block motif, a stall motif, and a coupling motif. In some instances, the leader construct can comprise a block motif, a stall motif, and a recognition motif. In some instances, the leader construct can comprise a block motif, a stall motif, and a capture motif. In some instances, the leader construct can comprise a block motif, a coupling motif, and a recognition motif. In some instances, the leader construct can comprise a block motif, a coupling motif, and a capture motif. In some instances, the leader construct can comprise a block motif, a recognition motif, and a capture motif. In some instances, the leader construct can comprise a stall motif, a coupling motif, and a recognition motif. In some instances, the leader construct can comprise a stall motif, a coupling motif, and a capture motif. In some instances, the leader construct can comprise a stall motif, a recognition motif, and a capture motif. In some instances, the leader construct can comprise a coupling motif, a recognition motif, and a capture motif. In some cases, the leader construct can comprise a block motif, a stall motif, a coupling motif, and a recognition motif. In some cases, the leader construct can comprise a block motif, a stall motif, a coupling motif, and a capture motif. In some cases, the leader construct can comprise a stall motif, a coupling motif, a recognition motif, and a capture motif. WSGR Docket Number: 64828-710.601 In some cases, the leader construct can comprise a block motif, a coupling motif, a recognition motif, and a capture motif. In some cases, the leader construct can comprise a block motif, a stall motif, a recognition motif, and a capture motif. In some cases ,the leader construct can comprise a block motif, a stall motif, a coupling motif, and a capture motif. In some embodiments, the capture motif can comprise a block motif, a stall motif, a coupling motif, a recognition motif, and a capture motif. [0753] In some embodiments, the leader construct can be present in a 5’ to a 3’ orientation. In some embodiment, the leader construct can be present in a N-terminal to a C-terminal orientation. [0754] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a stall motif, a block motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a stall motif, a block motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a stall motif, a recognition motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a stall motif, a recognition motif, a capture motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a stall motif, a block motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a stall motif, a capture motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a stall motif, a capture motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a stall motif, a capture motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a stall motif, a capture motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a block motif, a stall motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a coupling motif, a block motif, a stall motif, a recognition motif, and a capture motif. [0755] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a block motif, a stall motif, a capture motif, and a recognition motif. In some embodiments, the leader WSGR Docket Number: 64828-710.601 construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a block motif, a stall motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a block motif, a recognition motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a block motif, a recognition motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a block motif, a recognition motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a block motif, a recognition motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a block motif, a capture motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a block motif, a capture motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a block motif, a capture motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a block motif, a capture motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a recognition motif, a capture motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a recognition motif, a capture motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a recognition motif, a capture motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a recognition motif, a capture motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a recognition motif, a stall motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a recognition motif, a stall motif, a block motif, and a capture motif. [0756] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a recognition motif, a stall motif, capture motif, and block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a recognition motif, a stall motif, capture motif, and block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif , a recognition motif, a block motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif , a recognition motif, a block motif, a stall motif, and a capture motif. In some WSGR Docket Number: 64828-710.601 embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a recognition motif, a block motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a recognition motif, a block motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a capture motif, a block motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a capture motif, a block motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a capture motif, a block motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a capture motif, a block motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a capture motif, a recognition motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a capture motif, a recognition motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a capture motif, a recognition motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a coupling motif, a capture motif, a recognition motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a capture motif, a stall motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a capture motif, a stall motif, a block motif, and a recognition motif. [0757] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a coupling motif, a capture motif, a stall motif, recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a coupling motif, a capture motif, a stall motif, recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a block motif, a recognition motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a block motif, a recognition motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a block motif, a recognition motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a block motif, a recognition motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a block motif, a capture motif, a recognition motif, and a coupling motif. In some WSGR Docket Number: 64828-710.601 embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a block motif, a capture motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a block motif, a capture motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a block motif, a capture motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a block motif, a coupling motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a block motif, a coupling motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a block motif, a coupling motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a block motif, a coupling motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a recognition motif, a block motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a recognition motif, a block motif, a capture motif, and a coupling motif. [0758] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a recognition motif, a block motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a recognition motif, a block motif, a coupling motif, and a capture motif. [0759] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a recognition motif, a capture motif, a block motif, and coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a recognition motif, a capture motif, a block motif, and coupling motif. [0760] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a recognition motif, a capture motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a recognition motif, a capture motif, a coupling motif, and a block motif. [0761] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a recognition motif, a coupling motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a recognition motif, a coupling motif, a block motif, and a capture motif. WSGR Docket Number: 64828-710.601 [0762] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a recognition motif, a coupling motif, a capture motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a recognition motif, a coupling motif, a capture motif, and a block motif. [0763] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a capture motif, a block motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a capture motif, a block motif, a recognition motif, and a coupling motif. [0764] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a capture motif, a block motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a capture motif, a block motif, a coupling motif, and a recognition motif. [0765] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a capture motif, a recognition motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a capture motif, a recognition motif, a block motif, and a coupling motif. [0766] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a capture motif, a recognition motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a capture motif, a recognition motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a capture motif, a coupling motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a capture motif, a coupling motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a capture motif, a coupling motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a capture motif, a coupling motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a coupling motif, a block motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a coupling motif, a block motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a coupling motif, a block motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a coupling motif, a block motif, a capture motif, and a WSGR Docket Number: 64828-710.601 recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a coupling motif, a recognition motif, a capture motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a coupling motif, a recognition motif, a capture motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a coupling motif, a recognition motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a stall motif, a coupling motif, a recognition motif, a block motif, and a capture motif. [0767] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a coupling motif, a capture motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a coupling motif, a capture motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a stall motif, a coupling motif, a capture motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a stall motif, a coupling motif, a capture motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a stall motif, a recognition motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a stall motif, a recognition motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a stall motif, a recognition motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a stall motif, a recognition motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3; orientation: a block motif, a stall motif, a capture motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a stall motif, a capture motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a stall motif, a capture motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a stall motif, a capture motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3; orientation: a block motif, a stall motif, a coupling motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a block motif, a stall motif, a coupling motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3; orientation: a block motif, a stall motif, a coupling motif, a capture motif, and a recognition motif. In some embodiments, the leader construct WSGR Docket Number: 64828-710.601 can comprise the following N-terminal to C-terminal orientation: a block motif, a stall motif, a coupling motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a recognition motif, a stall motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a recognition motif, a stall motif, a capture motif, and a coupling motif. [0768] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a recognition motif, a stall motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a recognition motif, a stall motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a recognition motif, a capture motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a recognition motif, a capture motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a recognition motif, a capture motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a recognition motif, a capture motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a recognition motif, a coupling motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a recognition motif, a coupling motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a recognition motif, a coupling motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following N- terminal to C-terminal orientation: a block motif, a recognition motif, a coupling motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a capture motif, a stall motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a capture motif, a stall motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a capture motif, a stall motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a capture motif, a stall motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a capture motif, a recognition motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a capture motif, a recognition motif, a stall motif, and a coupling motif. WSGR Docket Number: 64828-710.601 [0769] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a capture motif, a recognition motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a capture motif, a recognition motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a capture motif, a coupling motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a capture motif, a coupling motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a capture motif, a coupling motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a capture motif, a coupling motif, a recognition motif, and a stall motif. [0770] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a coupling motif, a capture motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a coupling motif, a capture motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a coupling motif, a capture motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a coupling motif, a capture motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a coupling motif, a stall motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a coupling motif, a stall motif, a recognition motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a coupling motif, a stall motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a coupling motif, a stall motif, a capture motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a coupling motif, a recognition motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a coupling motif, a recognition motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a block motif, a coupling motif, a recognition motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a block motif, a coupling motif, a recognition motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a stall motif, a block motif, a capture motif, WSGR Docket Number: 64828-710.601 and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a recognition motif, a stall motif, a block motif, a capture motif, and a coupling motif. [0771] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a stall motif, a block motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a stall motif, a block motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a stall motif, a capture motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a recognition motif, a stall motif, a capture motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a stall motif, a capture motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a stall motif, a capture motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a stall motif, a coupling motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a stall motif, a coupling motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a stall motif, a coupling motif, a capture motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a stall motif, a coupling motif, a capture motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a block motif, a stall motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a block motif, a stall motif, a capture motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a block motif, a stall motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a recognition motif, a block motif, a stall motif, a coupling motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a block motif, a capture motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a block motif, a capture motif, a stall motif, and a coupling motif. [0772] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a block motif, a capture motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a block WSGR Docket Number: 64828-710.601 motif, a capture motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a block motif, a coupling motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a recognition motif, a block motif, a coupling motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a block motif, a coupling motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a block motif, a coupling motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a coupling motif, a block motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a coupling motif, a block motif, a stall motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a coupling motif, a block motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a coupling motif, a block motif, a capture motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a coupling motif, a stall motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a coupling motif, a stall motif, a block motif, and a capture motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a coupling motif, a stall motif, a capture motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a recognition motif, a coupling motif, a stall motif, a capture motif, and a block motif. [0773] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a coupling motif, a capture motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a coupling motif, a capture motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a coupling motif, a capture motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a recognition motif, a coupling motif, a capture motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a capture motif, a block motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a capture motif, a block motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a capture motif, a block motif, a coupling motif, and a stall motif. In some WSGR Docket Number: 64828-710.601 embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a capture motif, a block motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a capture motif, a stall motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a capture motif, a stall motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a capture motif, a stall motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a capture motif, a stall motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a capture motif, a coupling motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a recognition motif, a capture motif, a coupling motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a recognition motif, a capture motif, a coupling motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a recognition motif, a capture motif, a coupling motif, a block motif, and a stall motif. [0774] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a recognition motif, a stall motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a recognition motif, a stall motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a recognition motif, a stall motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a recognition motif, a stall motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a recognition motif, a block motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a recognition motif, a block motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, recognition motif, a block motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, recognition motif, a block motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a recognition motif, a coupling motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following N- terminal to C-terminal orientation: a capture motif, a recognition motif, a coupling motif, a stall motif, and a WSGR Docket Number: 64828-710.601 block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a recognition motif, a coupling motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a recognition motif, a coupling motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a stall motif, a block motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a stall motif, a block motif, a recognition motif, and a coupling motif. [0775] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a stall motif, a block motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a stall motif, a block motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, the stall motif, the recognition motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, the stall motif, the recognition motif, a block motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a stall motif, a recognition motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a stall motif, a recognition motif, a coupling motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a stall motif, a coupling motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a stall motif, a coupling motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a stall motif, a coupling motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following N- terminal to C-terminal orientation: a capture motif, a stall motif, a coupling motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a block motif, a stall motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a block motif, a stall motif, a recognition motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a block motif, a stall motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a block motif, a stall motif, a coupling motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a block motif, a recognition motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can WSGR Docket Number: 64828-710.601 comprise the following N-terminal to C-terminal orientation: a capture motif, a block motif, a recognition motif, a stall motif, and a coupling motif. [0776] In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a block motif, a recognition motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a block motif, a recognition motif, a coupling motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a block motif, a recognition motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a block motif, a recognition motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a block motif, a recognition motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a block motif, a recognition motif, a stall motif, and a coupling motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a coupling motif, a stall motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a coupling motif, a stall motif, a block motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a coupling motif, a stall motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following N- terminal to C-terminal orientation: a capture motif, a coupling motif, a stall motif, a recognition motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a coupling motif, a block motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a coupling motif, a block motif, a stall motif, and a recognition motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a coupling motif, a block motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C- terminal orientation: a capture motif, a coupling motif, a block motif, a recognition motif, and a stall motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a coupling motif, a recognition motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a coupling motif, a recognition motif, a stall motif, and a block motif. In some embodiments, the leader construct can comprise the following 5’ to 3’ orientation: a capture motif, a coupling motif, a recognition motif, a block motif, and a stall motif. In some embodiments, the leader construct can comprise the following N-terminal to C-terminal orientation: a capture motif, a coupling motif, a recognition motif, a block motif, and a stall motif. In some WSGR Docket Number: 64828-710.601 embodiments, the leader construct can be configured to couple the analyte to one or more translocases. In some embodiments, the leader construct can be coupled to the analyte via the coupling motif. In some embodiments, the capture motif can be configured to couple the analyte with one or more translocases. [0777] In some embodiments, the leader construct can comprise a stall motif. In some cases, the stall motif can comprise nucleic acids. In some cases, the nucleic acids can comprise DNA, RNA, LNA, PNA, BNA, GNA, TNA, or HNA, or any combination thereof. In some cases, the stall motif can comprise one or more peptides or proteins. In some cases, the stall motif can comprise nucleic acids, proteins, or peptides, or fragments thereof, or any combination thereof. [0778] In some embodiments, the leader construct can comprise a block motif. In some cases, the block motif can comprise nucleic acids. In some cases, the nucleic acids can comprise DNA, RNA, LNA, PNA, BNA, GNA, TNA, HNA, or any combination thereof. In some cases, the block motif can comprise one or more peptides or proteins. In some cases, the block motif can comprise nucleic acids, proteins, or peptides, or any combination thereof. [0779] In some embodiments, the leader construct can comprise a coupling motif. In some cases, the coupling motif can comprise nucleic acids. In some cases, the nucleic acids can comprise DNA, RNA, LNA, PNA, BNA, GNA, TNA, or HNA, or any combination thereof. In some cases, the coupling motif can comprise one or more peptides or proteins. In some cases, the coupling motif can comprise nucleic acids, proteins, peptides, or any combination thereof. [0780] In some embodiments, the leader construct can comprise a recognition motif. In some cases, the recognition motif can comprise nucleic acids. In some cases, the nucleic acids can comprise DNA, RNA, LNA, PNA, BNA, GNA, TNA, or HNA, or any combination thereof. In some cases, the recognition motif can comprise one or more peptides or proteins. In some cases, the recognition motif can comprise nucleic acids, proteins, or peptides, or fragments thereof, or any combination thereof. In some embodiments, the recognition motif can be configured to bind to one or more translocases. [0781] In some embodiments, the leader construct can comprise a capture motif. In some cases, the capture motif can comprise nucleic acids. In some cases, the nucleic acids can comprise DNA, RNA, LNA, PNA, BNA, GNA, TNA, or HNA, or any combination thereof. In some cases, the capture motif can comprise one or more peptides or proteins. In some cases, the capture motif can comprise nucleic acids, proteins, or peptides, or fragments thereof, or any combination thereof. [0782] In some embodiments, an analyte can be conjugated at its N- terminus, or its C-terminus, or both its N- and C-terminus to a leader of similar composition, so that when added to a pore system, capture and translocation can be possible for one or both directions of N-to-C and C-to-N depending on what end the captured leader was attached. Analytes can be characterized both in an N-to-C direction and/or a C-to-N WSGR Docket Number: 64828-710.601 direction. As a protein or peptide analyte translocating in the N-to-C direction and the C-to-N direction can produce different signals, this can be used to improve the accuracy of characterization. [0783] In some embodiments, an analyte can be conjugated to the leader using chemical or enzymatic approaches. In some embodiments, a method may comprise conjugating an analyte to a leader. In some embodiments, protein conjugation to a leader can be performed prior to contacting an analyte with protein translocase. In some embodiments, a leader can be pre-loaded with protein translocase prior to conjugation to the protein. [0784] In some embodiments, a leader may contain a barcode motif/sequence that produces a unique signal when passing through a pore so as to uniquely identify a species of a leader from a mixture of other barcoded leaders. A barcode can be used to label multiple separate samples that can then combined and run as a single pool in a pore system. The separate barcoded samples can be separated bioinformatically based on the barcode signals. Barcode motifs can be created from different amino-acid sequences, non-natural amino acids, modified amino acids, or other polymer moieties or combinations thereof to create unique signals when passed through a pore. [0785] In some embodiments, a leader can comprise a membrane- or pore- or translocase- binding motif to direct the binding of a leader and attaching an analyte either to a membrane or a pore or a translocase, respectively. A membrane binding molecule can be, for example, a hydrophobic or amphipathic molecules such as cholesterol that can be attached to a leader via a side chain. In some embodiments, a leader can comprise a sequence that forms an amphipathic structure, e.g. an amphipathic alpha helix for membrane association. [0786] In some embodiments, denaturation can be performed by heating, providing extreme pH conditions, providing chemical denaturants (e.g., urea, guanidinium hydrochloride, detergents, or any combination thereof), or any combination thereof. Denatured protein can be diluted and added to a cis compartment of a pore system under non-denaturing run conditions but remain denatured and/or unable to refold. In some embodiments, pre- denatured analytes may be added to a pore system that retain mild or moderate denaturing conditions to prevent any refolding of the analyte. Pores can tolerate mild or moderate denaturing conditions, so that mild or moderate of denaturing conditions can be employed in the running system to maintain the denatured or partially denatured state of the analytes while retaining the integrity of the pore. For example, some pores can withstand temperatures up to about 100ºC or urea concentrations up to 4M. [0787] In some embodiments, a method comprises adding to the first side (e.g., cis side) of a pore system a partially denatured or non-denatured (label-free) analyte or a mixture of partially denatured and/or non- denatured (label-free) analytes. In some embodiments, an analyte can be added in its native form. [0788] In some embodiments, a method comprises isolating analytes from a sample. In some embodiments, isolating can comprise separating impurities (e.g., a type of analyte) from a sample. In some embodiments, an WSGR Docket Number: 64828-710.601 impurity can be a nucleic acid. For instance, when processing the sample to prepare for identifying proteinaceous analytes, nucleic acids can be separated from the sample in a preparation step. In another example, when processing the sample to prepare for identifying nucleic acid analytes, proteinaceous analytes can be separated from the sample in a preparation step. In some embodiments, separation can be performed using magnetic beads. In some embodiments, magnetic beads can be used to specifically bind or to non- specifically bind to the impurities. The magnetic beads can then be separated from the solution, e.g., by a magnetic field. In some embodiments, magnetic beads can be paramagnetic beads. [0789] In some embodiments, the present disclosure provides a novel modular nanopore sensor allowing for the stochastic sensing of (label-free) protein targets, which does not suffer from the drawbacks of known nanopore-based sensors. In some embodiments, the present disclosure provides a generic and versatile system which allows for specific and sensitive detection of proteins and protein-containing pathogens. The present disclosure provides a novel modular nanopore sensor allowing for the stochastic sensing of analytes (e.g., proteins), In some embodiments, the nanopore sensor can be a generic and versatile system which allows for specific and sensitive detection of proteins and protein-containing analytes (e.g. viruses, bacteria), including analytes larger than the pore diameter, in complex samples, such as blood or serum. [0790] A complex sample can comprise one or more analytes. In some embodiments, a complex sample can comprise at least about 5 analytes, at least about 10 analytes, at least about 20 analytes, at least about 30 analytes, at least about 40 analytes, at least about 50 analytes, at least about 100 analytes, at least about 250 analytes, at least about 500 analytes, at least about 750 analytes, at least about 1,000 analytes, at least about 5,000 analytes, at least about 10,000 analytes, at least about 25,000 analytes, at least about 50,000 analytes, at least about 100,000 analytes, or greater than about 100,000 analytes. In some embodiments, a complex sample can comprise at most about 100,000 analytes, at most about 50,000 analytes, at most about 25,000 analytes, at most about 10,000 analytes, at most about 5,000 analytes, at most about 1,000 analytes, at most about 750 analytes, at most about 500 analytes, at most about 250 analytes, at most about 100 analytes, at most about 50 analytes, at most about 40 analytes, at most about 30 analytes, at most about 20 analytes, at most about 10 analytes, at most about 5 analytes, or less than about 5 analytes. In some embodiments, a complex sample can comprise between about 5 analytes to about 100,000 analytes. In some embodiments, a complex sample can comprise between about 5 analytes to about 10 analytes, about 5 analytes to about 50 analytes, about 5 analytes to about 100 analytes, about 5 analytes to about 500 analytes, about 5 analytes to about 1,000 analytes, about 5 analytes to about 2,500 analytes, about 5 analytes to about 5,000 analytes, about 5 analytes to about 10,000 analytes, about 5 analytes to about 25,000 analytes, about 5 analytes to about 50,000 analytes, about 5 analytes to about 100,000 analytes, about 10 analytes to about 50 analytes, about 10 analytes to about 100 analytes, about 10 analytes to about 500 analytes, about 10 analytes to about 1,000 analytes, about 10 analytes to about 2,500 WSGR Docket Number: 64828-710.601 analytes, about 10 analytes to about 5,000 analytes, about 10 analytes to about 10,000 analytes, about 10 analytes to about 25,000 analytes, about 10 analytes to about 50,000 analytes, about 10 analytes to about 100,000 analytes, about 50 analytes to about 100 analytes, about 50 analytes to about 500 analytes, about 50 analytes to about 1,000 analytes, about 50 analytes to about 2,500 analytes, about 50 analytes to about 5,000 analytes, about 50 analytes to about 10,000 analytes, about 50 analytes to about 25,000 analytes, about 50 analytes to about 50,000 analytes, about 50 analytes to about 100,000 analytes, about 100 analytes to about 500 analytes, about 100 analytes to about 1,000 analytes, about 100 analytes to about 2,500 analytes, about 100 analytes to about 5,000 analytes, about 100 analytes to about 10,000 analytes, about 100 analytes to about 25,000 analytes, about 100 analytes to about 50,000 analytes, about 100 analytes to about 100,000 analytes, about 500 analytes to about 1,000 analytes, about 500 analytes to about 2,500 analytes, about 500 analytes to about 5,000 analytes, about 500 analytes to about 10,000 analytes, about 500 analytes to about 25,000 analytes, about 500 analytes to about 50,000 analytes, about 500 analytes to about 100,000 analytes, about 1,000 analytes to about 2,500 analytes, about 1,000 analytes to about 5,000 analytes, about 1,000 analytes to about 10,000 analytes, about 1,000 analytes to about 25,000 analytes, about 1,000 analytes to about 50,000 analytes, about 1,000 analytes to about 100,000 analytes, about 2,500 analytes to about 5,000 analytes, about 2,500 analytes to about 10,000 analytes, about 2,500 analytes to about 25,000 analytes, about 2,500 analytes to about 50,000 analytes, about 2,500 analytes to about 100,000 analytes, about 5,000 analytes to about 10,000 analytes, about 5,000 analytes to about 25,000 analytes, about 5,000 analytes to about 50,000 analytes, about 5,000 analytes to about 100,000 analytes, about 10,000 analytes to about 25,000 analytes, about 10,000 analytes to about 50,000 analytes, about 10,000 analytes to about 100,000 analytes, about 25,000 analytes to about 50,000 analytes, about 25,000 analytes to about 100,000 analytes, or about 50,000 analytes to about 100,000 analytes. [0791] In some embodiments, the nanopore can be functionalized at the top (or mouth) of a large vestibule nanopore with small (up to about 50 kDa) recognition elements (e.g., proteinaceous recognition elements), such as nanobodies, that can move in and out of the pore to provoke a blocking current. Addition of an analyte (e.g., target analyte) to the solution outside (at the first or cis end) of the pore and the formation of a target-recognition element complex leads to a change in the capture of the recognition elements inside the nanopore, which in turn leads to a change in the ionic current through the open pore. The nanobody (or other small binding molecule) occludes the pore in the resting state, limiting the entry of other proteins. Importantly, in the resting state, the recognition element can be mostly lodged inside the nanopore and proteins and other unwanted non-specific background molecules from solution cannot enter the nanopore. Hence, this approach does not suffer from background noise from non-cognate proteins in solution that might otherwise interrupt the signal or block the nanopore. WSGR Docket Number: 64828-710.601 [0792] In some embodiments, the recognition element can be small recognition elements (e.g., proteinaceous recognition elements), this approach can be highly specific for detection of a wide range of entities, such as proteins. Secondly, the recognition elements are suitably tethered to the nanopore as replaceable modules, for example by complementary strand hybridization. Thus, nanopores functionalized with different recognition elements (e.g., nanobodies) can be easily acquired and the preparation process can be less laborious compared to existing methods. Thirdly, this nuclease-tolerant nanopore design enables the sensing of proteins in biofluids independently of their sizes, in which large proteins will be detected outside of the nanopore, while small proteins will be detected inside the lumen of the pore. [0793] In some embodiments, the recognition element can move through an external region of the nanopore and an internal region of the nanopore. In some cases, the internal region of the nanopore can be a channel of the nanopore. In some cases, the internal region of the nanopore can be an internal lumen of the nanopore on the first side of the fluid chamber. In some instances, the external region can be any region outside of the channel of the nanopore or the internal lumen of the nanopore. In some cases, the recognition element can move freely between the internal region and the external region of the nanopore. In some instances, moving freely can comprise movement of the recognition element that may not be hindered by the linker. [0794] In some embodiments, movement of the recognition element between the internal region of the nanopore and the external region of the nanopore can effect a change in an ionic current of the nanopore. In some embodiments, the movement of the recognition element into the internal region of the nanopore can decrease the ionic current (e.g., magnitude or noise of ionic current) moving through the channel of the nanopore. In some instances, the decrease in the ionic current moving through the channel of the nanopore can be measured. [0795] In some embodiments, the movement of the recognition element into the internal region of the nanopore can block at least a portion of the channel of the nanopore. In some cases, the movement of the recognition element into the internal region of the nanopore can increase the ionic current moving through the channel of the nanopore. [0796] In some embodiments, the nanopore system can exist in two states: (i) the recognition element can be in the internal region of the nanopore and (ii) the recognition element can be in the external region of the nanopore. In some cases, a change of the nanopore system from (i) to (ii) can be measured. In some cases, a frequency of the nanopore system can be a measurement of the movement between (i) and (ii). In some cases, the nanopore system can measure a change in the frequency of the movement of the recognition element. In some instances, a change in the frequency can indicate presence of the analyte. In some instances, a change in the frequency can indicate absence of the analyte. In some cases, the change in frequency can be used to determine the concentration of the analyte in solution. In some cases, the frequency of the movement of the WSGR Docket Number: 64828-710.601 recognition element in the nanopore system can be between 0.1 kilo Hertz (kHz) to about 1 mega Hertz (MHz). In some cases, the frequency of the nanopore system can be between 0.1 kHz to about 1 kHz, between about 1 kHz to about 100 kHz, or between about 100 kHz to about 1 MHz. In some cases, the frequency of the movement of the recognition element in the nanopore system can be at least about 0.1 kHz, at least about 1 kHz, at least about 5 kHz, at least about 10 kHz, at least about 20 kHz, at least about 30 kHz, at least about 40 kHz, at least about 50 kHz, at least about 60 kHz, at least about 70 kHz, at least about 80 kHz, at least about 90 kHz, at least about 100 kHz, at least about 200 kHz, at least about 300 kHz, at least about 400 kHz, at least about 500 kHz, at least about 600 kHz, at least about 700 kHz, at least about 800 kHz, at least about 900 kHz, at least about 1,000 kHz, at least about 1 MHz, or more than 1 MHz. In some cases, the frequency of the movement of the recognition element in the nanopore system can be at most about 1 MHz, at most about 1,000 kHz, at most about 900 kHz, at most about 800 kHz, at most about 700 kHz, at most about 600 kHz, at most about 500 kHz, at most about 400 kHz, at most about 300 kHz, at most about 200 kHz, at most about 100 kHz, at most about 90 kHz, at most about 80 kHz, at most about 70 kHz, at most about 60 kHz, at most about 50 kHz, at most about 40 kHz, at most about 30 kHz, at most about 20 kHz, at most about 10 kHz, at most about 5 kHz, at most about 1 kHz, at most about 0.1 kHz, or less than 0.1 kHz. In some cases, the frequency of the movement of the recognition element in the nanopore system can be about 0.1 kHz, about 1 kHz, about 5 kHz, about 10 kHz, about 20 kHz, about 30 kHz, about 40 kHz, about 50 kHz, about 60 kHz, about 70 kHz, about 80 kHz, about 90 kHz, about 100 kHz, about 200 kHz, about 300 kHz, about 400 kHz, about 500 kHz, about 600 kHz, about 700 kHz, about 800 kHz, about 900 kHz, about 1,000 kHz, or about 1 MHz. [0797] In some embodiments, the recognition element can couple to an analyte. In some cases, the recognition element can couple to a small analyte (e.g., 0.1 nm to 10 nm). In some instances, the recognition element coupled to the small analyte can move between the internal region of the nanopore and the external region of the nanopore. In some instances, the recognition element coupled to the small analyte can move into the internal region of the nanopore. In some cases, the recognition element can couple to a large analyte (e.g., 10 nm or greater ). In some instances, the recognition element coupled to the large analyte cannot move between the internal region of the nanopore and the external region of the nanopore. In some instances, the recognition element coupled to the large analyte cannot move into the internal region of the nanopore. [0798] In some embodiments, the recognition element can couple to an analyte. In some cases, the recognition element can specifically couple to the analyte. In some embodiments, the coupling of the recognition element to the analyte can affect the movement of the recognition element. In some cases, the recognition element may not be able to move into the internal region of the nanopore when coupled to the analyte. [0799] In some embodiments, the recognition element coupled to the analyte can be larger than the internal diameter (e.g., internal lumen diameter) of the nanopore. In some cases, the recognition element coupled to the WSGR Docket Number: 64828-710.601 analyte can be between about 0.1% to about 500% larger than the internal diameter (e.g., internal lumen diameter) of the nanopore. In some instances, the recognition element coupled to the analyte can be between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 5% to about 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, between about 40% to about 45%, between about 45% to about 50%, between about 50% to about 55%, between about 55% to about 60%, between about 60% to about 65%, between about 65% to about 70%, between about 70% to about 75%, between about 75% to about 80%, between about 80% to about 85%, between about 85% to about 90%, between about 90% to about 95%, between about 95% to about 100%, between about 100% to about 110%, between about 110% to about 120%, between about 120% to about 130%, between about 130% to about 140%, between about 140% to about 150%, between about 150% to about 160%, between about 160% to about 170%, between about 170% to about 180%, between about 180% to about 190%, between about 190% to about 200%, between about 200% to about 210%, between about 210% to about 220%, between about 220% to about 230%, between about 230% to about 240%, between about 240% to about 250%, between about 250% to about 260%, between about 260% to about 270%, between about 270% to about 280%, between about 280% to about 290%, between about 290% to about 300%, between about 300% to about 310%, between about 310% to about 320%, between about 320% to about 330%, between about 330% to about 340%, between about 340% to about 350%, between about 350% to about 360%, between about 360% to about 370%, between about 370% to about 380%, between about 380% to about 390%, between about 390% to about 400%, between about 400% to about 410%, between about 410% to about 420%, between about 420% to about 430%, between about 430% to about 440%, between about 440% to about 450%, between about 450% to about 460%, between about 460% to about 470%, between about 470% to about 480%, between about 480% to about 490%, or between about 490% to about 500% larger than the internal diameter (e.g., internal lumen diameter) of the nanopore. [0800] In some embodiments, when the recognition element may not be coupled to the analyte, the recognition element can move between the internal region of the nanopore and the external region of the nanopore. In some cases, when the recognition element can be in the internal region of the nanopore, the recognition element can decrease the ionic current (e.g., magnitude or noise of ionic current) passing through the nanopore. In some cases, when the recognition element can be in the internal region of the nanopore, the recognition element can increase the ionic current passing through the nanopore. In some instances, the increase and/or decrease in the ionic current can be measured. In some cases, the increase and/or decrease in the ionic current can indicate the absence of the analyte. [0801] In some embodiments, when the recognition element can be coupled to the analyte, the internal region of the nanopore can be open. In some cases, when the internal region of the nanopore can be open, an ionic current can pass through the nanopore. In some cases, the passage of the ionic current through the nanopore can WSGR Docket Number: 64828-710.601 be measured. In some instances, measuring an ionic current through the nanopore can indicate the presence of the analyte. [0802] In some embodiments, the recognition element can be coupled to the nanopore in the first side of the nanopore system. In some embodiments, the recognition element can be coupled to the nanopore in the second side of the nanopore system. In some embodiments, the recognition element can be coupled to the nanopore in the first side of the nanopore system and the second side of the nanopore system. [0803] In some embodiments, the analyte can be added to the first side of the nanopore system. In some embodiments, the analyte can be added to the second side of the nanopore system. In some embodiments, the analyte can be added to the first side of the nanopore system and the second side of the nanopore system. [0804] In some embodiments, the recognition element can be coupled to the nanopore in the first side of the nanopore system and the analyte can be added to the first side of the nanopore system. In some embodiments, the recognition element can be coupled to the nanopore in the second side of the nanopore system and the analyte can be added to the second side of the nanopore system. In some embodiments, the recognition element can be coupled to the nanopore in the first side of the nanopore and the second side of the nanopore and the analyte can be added to the first side of the nanopore system and the second side of the nanopore system. [0805] In some embodiments, the recognition element can be coupled to the nanopore. In some cases, the recognition element can be reversibly coupled to the nanopore. In some cases, the recognition element can be irreversible coupled to the nanopore. [0806] In some embodiments, the recognition element can be directly coupled to the nanopore. In some cases, the recognition element can be directly coupled to the nanopore via a covalent bond. In some instances, the covalent bond can be a non-polar covalent bond. In some instances, the covalent bond can be a polar covalent bond. In some cases, the recognition element can be directly coupled to the nanopore via a non-covalent bond. In some instances, the non-covalent bond can be hydrophobic interactions, van der Waals interactions, electrostatic interactions, hydrogen bonds, or any combination thereof. [0807] In some embodiments, the recognition element can be indirectly coupled to the nanopore. In some cases, the recognition element can be indirectly coupled to the nanopore via linker. In some instances, the linker can be a flexible linker. In some cases, the nanopore can be coupled to the linker via a conjugation reaction. In some instances, the conjugation reaction can be a sulfide based conjugative reaction, an ester reaction, a thioester reaction, an amide reaction, a native chemical ligation reaction, or any combination thereof. In some cases, the nanopore can be coupled to the linker via a bioconjugation reaction. In some instances, the bioconjugation reaction can comprise reacting lysine with a N-hydroxysuccinimidyl (NHS) ester, a lysine acylation reaction, reacting lysine with isocyanates, reacting lysine with isothiocyanates, reacting lysine with benzoyl fluorides, reacting cysteine with maleimides, reacting cysteine with iodoacetamides, reacting cysteine WSGR Docket Number: 64828-710.601 with 2-thiopyridine, reacting cysteine with 3-arylpropiolonitrile, an electrophilic aromatic substitution reactions, reacting tyrosine with diazonium salts, reacting tyrosine with 4-phenyl-1,2,4-triazole-3,5-dione (PTAD), a mannich reaction, reacting a N-terminal serine or threonine with NaIO4, reacting N-terminal cysteine with iodoacetamides, reacting N-terminal of analyte with pyridoxal phosphate, staudinger ligation with azides, huisgen cyclization of azides, strain promoted huisgen cyclization of azides, cysteine or Tryptophan RH-catalyzed alkylation, lysine or N-terminal Ir-catalyzed alkylation, tyrosine Pd-catalyzed O-alkylation. cysteine Au-catalyzed alkylation, tryptophan arylation, cysteine arylation, or lysine arylation, or any combination thereof. [0808] In some embodiments, the recognition element (e.g., proteinaceous recognition element) can be tethered atop of the nanopore and dynamically moves in and out of the nanopore lumen (vestibule) to provoke transient current blockage events. Binding of the recognition element to the analyte (e.g., target analyte) modulates this dynamic movement, thereby inducing a change in the frequency and/or magnitude of the current blockage events, wherein the change in the frequency and/or magnitude of current blockage events indicates the presence of the analyte (e.g., target analyte) in the sample. Typically, binding of the recognition element to the analyte (e.g., target analyte) increases the time of the recognition element staying outside of the pore, thereby decreasing the frequency of the current blockage events. [0809] In some embodiments, to allow for dynamic movement in and out of the vestibule of a nanopore, the recognition element moiety for use in the present invention can be much smaller than conventional IgG antibodies, having a molecular weight of approximately 150 kDa, composed of two different kinds of polypeptide chains. The typical dimensions of IgG are approximately 14.5 nm × 8.5 nm × 4.0 nm, with antigen binding sites separated by 13.7 nm. The molecular weight of R can be in the range of 5 to 50 kDa, preferably 10 to 40 kDa, 10 to 35 kDa, 10 to 30 kDa, more preferably 12-15 kDa. The the recognition element moieties may have dimensions in the single digit nanometer range, for example 1-5 nanometers (nm) x 1-5 nm. [0810] In some embodiments, the molecular weight of the recognition element can be at least about 5 kDa, at least about 10 kDa, at least about 15 kDa, at least about 20 kDa, at least about 25 kDa, at least about 30 kDa, at least about 35 kDa, at least about 40 kDa, at least about 45 kDa, at least about 50 kDa, or greater than about 50 kDa. In some embodiments, the molecular weight of recognition element can be at most about 50 kDa, at most about 45 kDa, at most about 40 kDa, at most about 35 kDa, at most about 30 kDa, at most about 25 kDa, at most about 20 kDa, at most about 15 kDa, at most about 10 kDa, at most about 5 kDa, or less than about 5 kDa. [0811] In some embodiments, the molecular weight of recognition element can be from about 5 kDa to about 60 kDa. In some embodiments, the molecular weight of recognition element can be from about 5 kDa to about 10 kDa, about 5 kDa to about 15 kDa, about 5 kDa to about 20 kDa, about 5 kDa to about 25 kDa, about 5 kDa to about 30 kDa, about 5 kDa to about 35 kDa, about 5 kDa to about 40 kDa, about 5 kDa to about 45 kDa, WSGR Docket Number: 64828-710.601 about 5 kDa to about 50 kDa, about 5 kDa to about 55 kDa, about 5 kDa to about 60 kDa, about 10 kDa to about 15 kDa, about 10 kDa to about 20 kDa, about 10 kDa to about 25 kDa, about 10 kDa to about 30 kDa, about 10 kDa to about 35 kDa, about 10 kDa to about 40 kDa, about 10 kDa to about 45 kDa, about 10 kDa to about 50 kDa, about 10 kDa to about 55 kDa, about 10 kDa to about 60 kDa, about 15 kDa to about 20 kDa, about 15 kDa to about 25 kDa, about 15 kDa to about 30 kDa, about 15 kDa to about 35 kDa, about 15 kDa to about 40 kDa, about 15 kDa to about 45 kDa, about 15 kDa to about 50 kDa, about 15 kDa to about 55 kDa, about 15 kDa to about 60 kDa, about 20 kDa to about 25 kDa, about 20 kDa to about 30 kDa, about 20 kDa to about 35 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 45 kDa, about 20 kDa to about 50 kDa, about 20 kDa to about 55 kDa, about 20 kDa to about 60 kDa, about 25 kDa to about 30 kDa, about 25 kDa to about 35 kDa, about 25 kDa to about 40 kDa, about 25 kDa to about 45 kDa, about 25 kDa to about 50 kDa, about 25 kDa to about 55 kDa, about 25 kDa to about 60 kDa, about 30 kDa to about 35 kDa, about 30 kDa to about 40 kDa, about 30 kDa to about 45 kDa, about 30 kDa to about 50 kDa, about 30 kDa to about 55 kDa, about 30 kDa to about 60 kDa, about 35 kDa to about 40 kDa, about 35 kDa to about 45 kDa, about 35 kDa to about 50 kDa, about 35 kDa to about 55 kDa, about 35 kDa to about 60 kDa, about 40 kDa to about 45 kDa, about 40 kDa to about 50 kDa, about 40 kDa to about 55 kDa, about 40 kDa to about 60 kDa, about 45 kDa to about 50 kDa, about 45 kDa to about 55 kDa, about 45 kDa to about 60 kDa, about 50 kDa to about 55 kDa, about 50 kDa to about 60 kDa, or about 55 kDa to about 60 kDa. [0812] In some embodiments, the molecular weight of recognition element can be about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, or about 50 kDa. [0813] In some embodiments, the recognition element (e.g., R) can be a single domain antibody, also known as a nanobody. For example, nanobodies derived from heavy-chain antibodies found in camelids (also known as VHH fragments), or nanobodies derived from the heavy-chain antibodies of Cartilaginous fish (also known as variable new antigen receptor VNAR fragments). Alternatively the recognition element can be a Fab fragment, such as an IgG based moiety, for example a single-chain variable fragment (scFv). Alternatively, the recognition element can be a non-IgG based moiety, such as those based on affimers, affibodies (based on the Z domain of protein A from Staphylococcus aureus), monobodies and Adnectins (based on the fibronectin type III domain), DARPins (designed ankyrin repeat proteins) or anticalins (based on lipocalins). [0814] In one aspect, the recognition element can be a Fab fragment. In some embodiments, the fragment antigen-binding region (Fab region) can be a region on an antibody that binds to antigens. It can be composed of one constant and one variable domain of each of the heavy and the light chain. Fab fragment antibodies can be generated by papain digestion of whole IgG antibodies to remove the entire Fc fragment, including the hinge region. These antibodies are monovalent, containing only a single antigen binding site. The molecular weight a Fab fragment can be about 50 kDa. The variable domain contains the paratope (the antigen-binding site), WSGR Docket Number: 64828-710.601 comprising a set of complementarity-determining regions, at the amino terminal end of the monomer. Each arm of the Y thus binds an epitope on the antigen. [0815] In another embodiment, the recognition element can be based on a single-chain variable fragment (scFv), which are fusion proteins of about 30-35 kDa and 2×3 nm that link the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. See Asaadi et al. (Biomarker Research volume 9, Article number: 87 (2021), which is incorporated by reference herein in its entirety). [0816] In some embodiments, the recognition element can be a nanobody or a so-called VHH antibody, originally referred to as a heavy chain antibody (HCAb), also known as a single-domain antibody. Nanobodies, which consist of the variable domain of camelid-derived heavy-chain-only antibodies, have emerged as a rapidly growing family of strong protein binders. The nomenclature of “nanobody” originally adopted by the Belgian company Ablynx® stemmed from its nanometric size, (e.g., 4 nm in length, 2.5 nm in width, and only 12-14 kD in molecular weight). Nanobodies are nuclease-tolerant, which make them more favorable for the indirect protein sensing compared to aptamers. Apart from that, nanobodies can easily be produced as recombinant protein in bacterial expression system and can readily be equipped with customized tags without affecting their function. Besides, it has been reported that nanobody multimerization can improve its binding affinity and enhance detection sensitivity. [0817] In some embodiments, the recognition element can be a non-IgG based moiety, such as an affimer. Affimer® molecules are small proteins that bind to analytes (e.g., target analytes) with affinity in the nanomolar range. At 12-14 kDa, Affimer reagents are small non-antibody binding proteins, ∼10 times smaller than IgG antibodies and they are less than 4 nm in length. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. [0818] In some embodiments, the recognition element can be a nanobody or a so-called VHH antibody, originally referred to as a heavy chain antibody (HCAb), also known as a single-domain antibody, as described herein above. In some embodiments, the recognition element can be a non IgG based moiety, such as an affimer. Affimer® molecules are small proteins that bind to target proteins with affinity in the nanomolar range, as described herein above. See also Bedford et al. (Biophysical Reviews volume 9, pg.299–308; 2017, which is incorporated by reference herein in its entirety) providing examples of smaller size immunoglobulin G (IgG) and non-IgG-based binding reagents that are suitably used for nanopore functionalization. [0819] In some embodiments, one nanopore may be conjugated to the same type of recognition element moiety, e.g. all being nanobodies, scFv or affimers, or to mixed type of recognition element moieties, e.g. a combination of two or more types of those listed herein above, such as scFv and affimers or scFv and nanobodies. WSGR Docket Number: 64828-710.601 [0820] In some embodiments, the nanopore can be coupled to one or more recognition elements. In some embodiments, the nanopore can be coupled to at least about 1 recognition element, at least about 2 recognition elements, at least about 3 recognition elements, at least about 4 recognition elements, at least about 5 recognition elements, at least about 10 recognition elements, at least about 12 recognition elements, at least about 15 recognition elements, at least about 18 recognition elements, at least about 20 recognition elements, at least about 25 recognition elements, at least about 30 recognition elements, at least about 35 recognition elements, at least about 40 recognition elements, at least about 45 recognition elements, at least about 50 recognition elements, or greater than about 50 recognition elements. In some embodiments, the nanopore can be coupled to at most about 50 recognition elements, at most about 45 recognition elements, at most about 40 recognition elements, at most about 35 recognition elements, at most about 30 recognition elements, at most about 25 recognition elements, at most about 20 recognition elements, at most about 18 recognition elements, at most about 15 recognition elements, at most about 12 recognition elements, at most about 10 recognition elements, at most about 5 recognition elements, at most about 4 recognition elements, at most about 3 recognition elements, at most about 2 recognition elements, at most about 1 recognition element, or less than 1 recognition element. [0821] In some embodiments, the nanopore can be coupled to from about 1 recognition element to about 50 recognition elements. In some embodiments, the nanopore can be coupled to from about 1 recognition element to about 2 recognition elements, about 1 recognition element to about 3 recognition elements, about 1 recognition element to about 4 recognition elements, about 1 recognition element to about 5 recognition elements, about 1 recognition element to about 10 recognition elements, about 1 recognition element to about 15 recognition elements, about 1 recognition element to about 20 recognition elements, about 1 recognition element to about 25 recognition elements, about 1 recognition element to about 30 recognition elements, about 1 recognition element to about 40 recognition elements, about 1 recognition element to about 50 recognition elements, about 2 recognition elements to about 3 recognition elements, about 2 recognition elements to about 4 recognition elements, about 2 recognition elements to about 5 recognition elements, about 2 recognition elements to about 10 recognition elements, about 2 recognition elements to about 15 recognition elements, about 2 recognition elements to about 20 recognition elements, about 2 recognition elements to about 25 recognition elements, about 2 recognition elements to about 30 recognition elements, about 2 recognition elements to about 40 recognition elements, about 2 recognition elements to about 50 recognition elements, about 3 recognition elements to about 4 recognition elements, about 3 recognition elements to about 5 recognition elements, about 3 recognition elements to about 10 recognition elements, about 3 recognition elements to about 15 recognition elements, about 3 recognition elements to about 20 recognition elements, about 3 recognition elements to about 25 recognition elements, about 3 recognition elements to about 30 WSGR Docket Number: 64828-710.601 recognition elements, about 3 recognition elements to about 40 recognition elements, about 3 recognition elements to about 50 recognition elements, about 4 recognition elements to about 5 recognition elements, about 4 recognition elements to about 10 recognition elements, about 4 recognition elements to about 15 recognition elements, about 4 recognition elements to about 20 recognition elements, about 4 recognition elements to about 25 recognition elements, about 4 recognition elements to about 30 recognition elements, about 4 recognition elements to about 40 recognition elements, about 4 recognition elements to about 50 recognition elements, about 5 recognition elements to about 10 recognition elements, about 5 recognition elements to about 15 recognition elements, about 5 recognition elements to about 20 recognition elements, about 5 recognition elements to about 25 recognition elements, about 5 recognition elements to about 30 recognition elements, about 5 recognition elements to about 40 recognition elements, about 5 recognition elements to about 50 recognition elements, about 10 recognition elements to about 15 recognition elements, about 10 recognition elements to about 20 recognition elements, about 10 recognition elements to about 25 recognition elements, about 10 recognition elements to about 30 recognition elements, about 10 recognition elements to about 40 recognition elements, about 10 recognition elements to about 50 recognition elements, about 15 recognition elements to about 20 recognition elements, about 15 recognition elements to about 25 recognition elements, about 15 recognition elements to about 30 recognition elements, about 15 recognition elements to about 40 recognition elements, about 15 recognition elements to about 50 recognition elements, about 20 recognition elements to about 25 recognition elements, about 20 recognition elements to about 30 recognition elements, about 20 recognition elements to about 40 recognition elements, about 20 recognition elements to about 50 recognition elements, about 25 recognition elements to about 30 recognition elements, about 25 recognition elements to about 40 recognition elements, about 25 recognition elements to about 50 recognition elements, about 30 recognition elements to about 40 recognition elements, about 30 recognition elements to about 50 recognition elements, or about 40 recognition elements to about 50 recognition elements. [0822] In some embodiments, the nanopore can be coupled to about 1 recognition element, about 2 recognition elements, about 3 recognition elements, about 4 recognition elements, about 5 recognition elements, about 10 recognition elements, about 12 recognition elements, about 15 recognition elements, about 18 recognition elements, about 20 recognition elements, about 25 recognition elements, about 30 recognition elements, about 35 recognition elements, about 40 recognition elements, about 45 recognition elements, or about 50 recognition elements. [0823] In some embodiments, the one or more recognition elements can couple to the same region of the analyte. In some embodiments, the one or more recognition elements can couple to different regions of the analyte. In some embodiments, the one or more recognition element can couple to different analytes. WSGR Docket Number: 64828-710.601 [0824] In some embodiments, the recognition element can be coupled or positioned via a flexible tether atop of the nanopore (e.g., at the cis side atop of the nanopore) allowing for contacting the analyte that can be added to the first side (e.g., cis chamber). The site of linker coupling to R can be chosen on a surface, loop or termini of the protein such that it leaves the binding domain motifs of the recognition element free and sterically unhindered. Common conjugation sites (e.g. for binding R to beads or surfaces) are well known for many suitable the recognition element binders. The site of nanopore modification with the recognition element can be chosen such that it allows the recognition element to dynamically move in and out of the interior of the nanopore, or at least to provoke transient current blockage events in the absence of analyte (e.g. target analyte), and wherein binding of analyte (e.g. target) to the recognition element modulates its dynamic movement, thereby inducing a change in the frequency and/or magnitude of the current blockage events. [0825] In some embodiments, binding of the recognition element to the analyte (e.g. target analyte) increases the time of the recognition element staying outside of the pore, for example through steric or electrostatic effects that reduce the ability of the recognition element-analyte complex to enter the nanopore cavity, thereby decreasing the frequency of the current blockage events. Alternatively, binding of the recognition element to the analyte (e.g. target analyte) reduces the time of the recognition element staying outside of the pore, thereby increasing the frequency of the current blockage events. For example, binding to a highly charged analyte may aid in internalization through changes to electrophoretic forces acting on the recognition element-analyte complex. [0826] In other embodiments, binding of the recognition element to the analyte (e.g. target analyte) alters the ionic current flow through the nanopore when the recognition element-analyte complex can be inside the nanopore. For example, in embodiments where the recognition element-analyte complex can be able to enter the nanopore cavity, the presence of the analyte either increases or decreases the ionic current flowing through nanopore relative to the unbound recognition element current level as a result of a change in excluded volume or electrostatics. In some embodiments the recognition element-analyte complex exhibits multiple current levels as a result of the complex being located at different positions within the nanopore. The changes in current level can be used to detect the presence of the analyte. The changes and absolute values of the current levels associated with the recognition element-analyte complex inside the nanopore can also be used to determine other properties of the analyte, such as for example the presence and type of one or more post-translational modifications (e.g. phosphorylations, glycosylations, or any combination thereof). For example, the recognition element may be designed to universally bind to a specific analyte (e.g., specific target protein analyte) that can be present in multiple post-translationally or otherwise modified forms in a mixture, for example binding to unmodified epitope region of the protein, so that the modified regions of the protein analyte facing into the nanopore alter the ionic current in a distinctive manner. WSGR Docket Number: 64828-710.601 [0827] In some embodiments, the nanopore (e.g., biological nanopore) may be functionalized with one type of recognition element allowing for sensing one analyte (e.g., target analyte), or it may be functionalized with at least two different recognition elements (e.g., proteinaceous recognition elements) R’ and R’’. In one embodiment, the nanopore can be functionalized with at least recognition element prime (e.g., R’) and recognition element double prime (e.g., R’’), each of which binds specifically to a different analyte (e.g., target analyte), thus enabling a single nanopore to detect multiple different analytes (e.g., target analytes). In preferred embodiments, the nanopore can be functionalized with at least R’ and R’’, each binding specifically to distinct sites (epitopes) of the same analyte (e.g., target analyte). In this way the binding strength and duration of the analyte bound state can be increased, as well as increasing the specificity for binding the given analyte (e.g., target analyte) over other background analytes. [0828] In some embodiments, the recognition element can be positioned via a flexible tether at the first side (or at the cis side) atop of the nanopore. This flexible tether allows the recognition element to move in and out of the pore as described herein. [0829] In one aspect, the recognition element can be directly coupled to the nanopore. In one aspect, flexibility, e.g., allowing rotation or bending of the recognition element so that it can move in and out of the pore as described herein, may be achieved by the bond via which the recognition element can be attached to the nanopore. In one aspect, such flexibility may be achieved by flexibility within the recognition element or within the nanopore. In some examples, the nanopore may be conjugated to a flexible region of the recognition element such as flexible N- or C-termini, or flexible loops on the outer surface of the recognition element. Alternatively or in combination, the recognition element may be coupled to flexible regions of the nanopore, such as flexible N- or C- termini, or flexible loops on the outer surface of the nanopore. [0830] In some embodiments, the methods described herein may comprise providing a sample. The sample may comprise an analyte, or at least a portion of an analyte, or any combination thereof. In some embodiments, the sample comprises one or more analytes. In some embodiments, one or more properties of the sample may be determined. The one or more properties of the sample may be determined from one or more characteristics of one or more analytes. The sample may comprise an analyte and an additional analyte (e.g., the sample may comprise at least about two analytes). An additional analyte can comprise an additional analytes as described herein. In some embodiments, the additional analyte (e.g., an additional non-nucleic acid based polymer analyte) can comprise at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or any combination thereof. An analyte and an additional analyte of a sample may be the same species. An analyte and an additional analyte of a sample may be different species. An analyte and an additional analyte of a sample may be of the same type of analyte. One or analytes of a sample may be determined. In some embodiments, a structure of an analyte may be determined. A method WSGR Docket Number: 64828-710.601 described herein can comprise determining one or more of a number of analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or any combination thereof. [0831] In some embodiments, one or more advantages of the methods and systems described herein is the ability to measure one or more long analytes (e.g., peptides, polypeptides, or proteins, or fragments thereof, or any combination thereof). The long analyte can be a long non-nucleic acid based polymer analyte. For example, the long analyte (e.g., long peptide, polypeptide, or protein, or fragments thereof, or any combination thereof) can comprise a length of greater than about 1000 amino acids. The long analyte can comprise a mass of greater than about 100 kDa. In some embodiments, a long analyte can comprise an one or more analytes (e.g., analyte units) that may be concatenated together. For example, a long non-nucleic acid based polymer analyte can comprise one or more analyte units (e.g., peptide units, polypeptide units, or protein units, or any combination thereof) linked together to compose the long non-nucleic acid based polymer analyte. In some embodiments, the methods and/or systems described herein can comprise measuring one or more long analytes (e.g., peptides, polypeptides, or proteins, or any combination thereof) composed of one or more concatenated analyte units as opposed to adding one or more analyte units separately to a system and/or measuring the one or more analyte units individually. [0832] In some embodiments, one or more barcodes (e.g., one or more peptide units, polypeptide units, or protein units, or any combination thereof) can be concatenated to an analyte. The one or more barcodes may be concatenated during a sample preparation. For example, the one or more barcodes may be concatenated during a sample preparation so that a mixture of one or more concatemer analytes (e.g., analytes comprising one or more concatenated units) may be added to a system described herein. One or more signals (e.g., one or more unique signals) can be measured in the system. In some embodiments, the one or more measured signals (e.g., one or more unique signals) can be from the one or more barcodes concatenated to the analyte. The one or more measured signals (e.g., one or more unique signals) can be used to identify the analyte. In some embodiments, the one or more measured signals (e.g., one or more unique signals) can be used to link (e.g., informatically link) the analyte to a sample of origin. [0833] The one or more concatenated units of an analyte may be linked. The one or more concatenated units of an analyte may be linked physically. In some embodiments, the one or more concatenated units of an analyte may be linked and may be grouped in an analysis. The analysis may determine information on an origin of one or more molecules of a sample. For example, a sample may comprise two or more different types of concatemer polypeptides. One or more concatenated units of a first concatemer polypeptide can be prepared from a first sample. One or more concatenated units of a second concatemer polypeptide can be prepared from a second sample. Analysis of the one or more concatenated units may comprise analysis of an identity, a number, or a WSGR Docket Number: 64828-710.601 characteristic, or any combination thereof. Analysis of the one or more concatenated units may allowing for linking of the concatemer polypeptide types to an origin sample when measured in the system. [0834] In some embodiments, concatenation may provide improved throughput of one or more analytes. In some embodiments, concatenation can refer to one or more units translocating through a nanopore. The one or more units translocating through a nanopore may be read as a single molecule (e.g., not read as separate reads and/or translocations). Without wishing to be bound by theory, the one or more units of a concatemer analyte (e.g., a non-nucleic acid based polymer analyte comprising one or more concatenated units) being read as a single molecule may lead to higher throughput as time can be lost between separate nanopore translocations (e.g., two or more translocations). For example, time can be lost between separate nanopore translocations between one translocation finishing of a first molecule, and a capture and/or translocation of a second molecule. Concatenating one or more units together can remove time in which the pore does not comprise an analyte (e.g., “open-pore” time). The removal of time in which the pore does not comprise an analyte can result in higher throughput. [0835] In some embodiments, as described herein a translocation can be controlled by a motor protein (e.g., a translocase). The motor protein may be located outside of a nanopore. The motor protein may be located outside (e.g., located above or located below) of a constriction region of the nanopore. The constriction region can refer to an area of the pore in which an analyte may be read. In some embodiments, a portion of translocation of an analyte may occur when a motor protein reaches the end of the analyte. Translocation of one or more termini of an analyte may be faster than a non-terminal end of the analyte. Translocation of one or more termini of an analyte may be slower than a non-terminal end of the analyte. Faster translocation of an analyte with a motor protein may result in an inability to resolve one or more ionic current signals (e.g., one or more sufficient ionic current signals). Inability to resolve one or more ionic current signals may result in an inability to measure one or more characteristics of the analyte (e.g., the one or more termini of the analyte). [0836] As an example, the proportion of the molecule information that can be lost for a long analyte (e.g., a long peptide, a long polypeptide, a long protein, or any combination thereof) may be small. The proportion of the molecule information that can be lost for a long analyte may be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or greater than about 50%. The proportion of the molecule information that can be lost for a long analyte may be at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 15%, at most about 10%, at most about 5%, or less than about 5%. [0837] In some embodiments, a proportion of lost molecule information may be larger for one or more smaller analytes (e.g., an analyte ranging from 1-55 amino acid residues in size) than for one or more larger analyte. Molecular information for one or more smaller analytes may be lost due to dimensions of the nanopore, WSGR Docket Number: 64828-710.601 dimensions of the motor protein, or any combination thereof. Concatenation of one or more units of an analyte may reduce and/or elimination the proportion of molecular information lost. One or more small peptide units (e.g., peptide units comprising at most about 50 amino acids, at most about 40 amino acids, at most about 30 amino acids, at most about 20 amino acids, at most about 30 amino acids, at most about 30 amino acids, at most about 30 amino acids, or less than about 5 amino acids) may be concatenated to provide one or more longer analytes. For example, one or more small peptide units may be concatenated to provide one or more longer analytes comprising at least about 1 concatenated unit, at least about 2 concatenated units, at least about 5 concatenated units, at least about 10 concatenated units, at least about 50 concatenated units, at least about 100 concatenated units, at least about 200 concatenated units, or greater than about 200 concatenated units long. One or more small peptide units may be concatenated to provide one or more longer analytes comprising at most about 200 concatenated units, at most about 100 concatenated units, at most about 50 concatenated units, at most about 10 concatenated units, at most about 5 concatenated units, at most about 2 concatenated units, at most about 1 concatenated unit, or less than about 1 concatenated unit long. Concatenation may allow for measurement of a larger proportion of the concatenated units (e.g., small peptide units, small polypeptide units, small protein units, or any combination thereof) compared to measurement of the units (e.g., small peptide units, small polypeptide units, small protein units, or any combination thereof) not concatenated (e.g., not linked). As another example, one or more tails (e.g., polypeptide tails, peptide tails, protein tails, or any combination thereof) may be of a length to span a pore and/or motor protein. The one or more tails may be added to an end (e.g., terminal) of an analyte to allow for the concatenated terminal of the analyte to be under a control of the motor protein when the analyte can be translocating through a pore. KITS [0838] Provided herein are various kits and kit components that can be used to prepare a pore, a membrane, a device, or a sample; or process one or more analytes and/or a sample using a pore, membrane, nanopore system, or device, or any combination thereof. Kits can comprise one or more elements disclosed herein in relation to any of the various aspects, in any combination. Reagents and other materials in a kit may be contained in any suitable container, and may be in an immediately usable form or require combination with other reagents in the kit or reagents supplied by a user (e.g. dilution of a concentrated composition). In some embodiments, the kit comprises instructions for use of the kit in accordance with one or more methods disclosed herein. The kit according to any aspects or embodiments disclosed herein can comprise one or more buffers disclosed herein, one or more preservative components, one or more stabilizer components, one or more electrolytes disclosed herein, one or more translocases disclosed herein, one or more preloading solutions (e.g., co-factors) disclosed herein, one or more crowding agents disclosed herein, one or more leaders disclosed herein, one or more motor WSGR Docket Number: 64828-710.601 proteins (e.g., translocases, unfoldases, protease) disclosed herein, one or more beads disclosed herein, one or more analyte adaptors disclosed herein, or one or more recognition elements disclosed herein, or any combination thereof. [0839] In some cases, the kit can comprise one or more chips disclosed herein, one or more membranes disclosed herein, one or more sensors disclosed herein, one or more sensor arrays disclosed herein, one or more nanopores disclosed herein, one or more nanopore systems disclosed herein, or one or more devices disclosed herein, or any combination thereof. [0840] In some cases, any of the aforementioned kit components (e.g., electrolytes, biomolecules such as motors and cofactors) can be packaged in a container (e.g., tubes). This can either be separate from the chip or can be provided preloaded on the chip. [0841] In some cases, the one or more chips can be provided with pre-formed membranes and pre-inserted pores. In other cases, the one or more chips may be provided with one or more membrane materials and/or one or more pores that are provided in one or more containers to enable formation of membranes and/or insertion of the one or more pores respectively (e.g., when added to blank chips). [0842] In some embodiments, a kit comprises a buffer. In some embodiments, analytes can still be detected or identified near physiological pH, such as about 7.0. In some embodiments, analytes can be detected or identified at a pH between 4.5 and 9.5, 5 and 9, or 6 and 8. In some embodiments, analytes can be detected or identified under low pH conditions, such as lower than pH 4.5. In some embodiments, analytes can be detected or identified at a pH of lower than about 4, 3.5, 3, 2.5, 2, 1.5, or about 1. In some embodiments, analytes can be detected or identified under high pH conditions, such as higher than pH 9.5. In some embodiments, analytes can be detected or identified at a pH of higher than about 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or about 14. Under physiological pH conditions, naturally occurring peptides can have a wide range of charge distributions and net charges (e.g., both net positive and net negative) as a result of their highly variable composition of acidic, basic and neutral amino acids. This diversity of charge can complicate the ability to capture and detect all the peptides in a diverse mixture of different peptides in a pore sensing system when a fixed applied potential can be applied, since not all peptides will experience the same direction of net electrophoretic force. Depending on the polarity of the applied potential and the specific charge composition of each peptide, some peptides may experience net electrophoretic force into the pore, while other oppositely charged peptides may experience net electrophoretic force away from the pore. By implementing low pH conditions on the side of the pore sensing system containing a complex peptide analyte mixture, and in some cases, on both the first side and second side (e.g., cis side and trans side) of the membrane, the amino acids of the peptide analytes can become protonated. The increased protonation can both 1) increase the net positive charge of all peptides in a diverse mixture, and 2) create a more uniform distribution of charges in the peptide mixture. The increased net positive charge can WSGR Docket Number: 64828-710.601 allow for an improved electrophoretic capture of the peptides in a pore system held under an appropriate polarity applied potential (e.g. when a negative potential can be applied to the electrode on the opposite side of the membrane to the peptide analytes). [0843] An improved uniformity of charge in an analyte mixture (e.g., the peptide mixture, polypeptide mixture) or sample can be advantageous when all molecules experience more similar electrophoretic forces acting upon them under an applied potential. Since electrophoresis can determine the efficiency of analyte capture into a pore, non-uniform charges can reduce capture efficiency biases between different analyte compositions in mixtures. [0844] Implementing low pH conditions can alter the charge characteristics of the pore in a sensor by protonating some of the pore’s amino acids. For example, increased positive charge inside the pore channel, and inside the lumen recognition region, can alter the capture and subsequent detection of peptide analytes. Under low pH conditions the increased positive charge in the pore can electrostatically repel the mostly positively charged peptide analytes, which can in turn reduce capture efficiency and/or reduce the residence time of peptides inside the pore. This can reduce the ability to detect and characterize some peptide analytes. [0845] Accordingly, in some embodiments, a kit comprises a buffer. In some embodiments, a buffer comprises a pH at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or about 14. In some embodiments, a buffer comprises a pH at most about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, a buffer may be configured to provide a pH condition on a trans, cis, or both sides of a pore. In some embodiments, a buffer may be configured to provide a pH condition in combination with an electrolyte or salt solution. In some embodiments, a buffer may be integrated into a device or a sensor. In some embodiments, a buffer may be provided as a reagent configured to be added to a device or a sensor. [0846] In some embodiments, a kit comprises an electrolyte. In some embodiments, analyte detection or identification can be carried out in the presence of charge carriers present in the electrolyte, such as metal salts or ionic liquids. For example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt, or ionic liquids or organic salts such as tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazolium chloride. A salt can be potassium chloride (KC1), sodium chloride (NaCl), or lithium chloride (LiCl). An electrolyte may comprise redox salts to mediate electron transfer at suitable electrodes, for example potassium ferrocyanide and potassium ferricyanide or other redox couples. In some embodiments, an electrolyte can comprise an ionic concentration of 0.1 M to 3 M, 0.1 M to 1.5 M, or 0.15 M to 1.0 M. In some embodiments, an electrolyte can comprise a concentration of at least about 0.01 M, 0.05 M, 0.1 M, 0.5 M, 1 M, 1.5 M, 2 M, 2.5 M, 3 M, 3.5 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10 M, or greater than about 10 M. In some embodiments, an electrolyte can comprise a concentration of at most about 10 M, 9 M, 8 M, 7 M, 6 M, 5 M, 4 M, 3.5 M, 3 M, 2.5 M, 2 M, 1.5 WSGR Docket Number: 64828-710.601 M, 1 M, 0.5 M, 0.1 M, 0.05 M, 0.01 M, or less than about 0.01 M. In some embodiments, an electrolyte can comprise a buffer configured to maintain a substantially constant pH when contacted with a sample. A buffer can comprise bis-tris buffer, citrate buffer, phosphate buffer (PBS), HEPES buffer, Tris-HC1 buffer, ethylenediaminetetraacetic acid (EDTA), tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), glycerol, BES buffer, Bicine, Tris (Tris(hydroxymethyl)aminomethane), MES (2-(N- morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), CAPS (3- (cyclohexylamino)-1-propanesulfonic acid), or Tricine, or any combination thereof. The electrolyte can comprise any buffer disclosed herein. [0847] In some embodiments, a kit can comprise a translocase. A translocase can be any translocase provided herein. In some embodiments, a translocase and an analyte may be incubated at a higher concentration, and can be diluted to a lower concentration when being contacted with a pore. In some embodiments, preloading can be performed. In some embodiments, preloading can be performed prior to loading in a device. In some embodiments, preloading can be performed after loading in a device. In some embodiments, preloading can be performed in solutions that may be closer to the optimal salt concentration and salt types, the optimal pH, the optimal temperature, and in the presence of optimal co-factors (e.g. NTP, M2+ ions, or any combination thereof) that favor binding between a translocase and an analyte. In some embodiments, preloading can be performed in combination with an accessory cofactor that aids in binding. In some embodiments, an accessory cofactor comprises analyte adaptors, e.g., NblA/B, ClpS, ClpF. In some embodiments, an accessory cofactor comprises engineered binding cofactors such as ones derived from antibodies, nanobodies, or affimers. In some embodiments, the kit can comprise one or more crowding agents (e.g., PE and/or dextrans) to aid preloading. Accordingly, in some embodiments, a kit can comprise a diluent for diluting a sample with the translocase. In some embodiments, a kit can comprise a reagent or a buffer comprising an accessory cofactor. [0848] In some embodiments, a kit can comprise a leader. A leader can be any leader provided herein. In some embodiments, a kit can comprise beads. A bead can be any bead provided herein. In some embodiments, a kit can comprise a protease. A protease can be any protease provided herein. In some embodiments, a kit can comprise a sensor, a sensor array, or a device. A sensor, a sensor array, or a device can be any sensor, sensor array, or device disclosed herein. In some embodiments, a kit can comprise a chip. In some embodiments, a chip can comprise a sensor, a sensor array, or a device, or any combination thereof. In some embodiments, a chip can comprise one or more membranes. In some embodiments, a chip can comprise one or more nanopores disposed in one or more membranes. In some embodiments, a kit can comprise a dried composition. In some embodiments, a kit can comprise a reconstitution reagent configured to reconstitute a dried composition. In some embodiments, a dried composition can comprise dried lipids, one or more pores, one or more salts, one or more electrolytes, one or WSGR Docket Number: 64828-710.601 more buffers, one or more leaders, one or more beads, one or more analyte adaptors, one or more preloading solutions (e.g., co-factors), one or more crowding agents, one or more leaders, one or more motor proteins (e.g., translocases, unfoldases, protease), or one or more recognition elements, or one or more beads, or combinations thereof. In some embodiments, a kit can comprise a concentrated liquid composition. In some embodiments, a kit can comprise a dilution agent configured to dilute a concentrated liquid composition. In some embodiments, a concentrated liquid composition can comprise one or more lipids, one or more pores, one or more salts, one or more electrolytes, one or more buffers, one or more leaders, one or more beads, one or more analyte adaptors, one or more preloading solutions (e.g., co-factors), one or more crowding agents, one or more leaders, one or more motor proteins (e.g., translocases, unfoldases, protease), or one or more recognition elements, or one or more beads, or combinations thereof. ANALYSIS [0849] Provided herein are various systems and methods for measuring signals generated by an analyte when the analyte translocates through a pore. [0850] A nanopore system as described herein may find its use in various applications, ranging from analytical detection methods in a research setting, high throughput drug development to real-time diagnostic applications. In some aspects, the nanopores, methods, and system provided herein comprise detecting and/or characterizing one or more characteristics of an analyte. Characteristics of the analyte (e.g., biopolymer) comprise a shape of the biopolymer, a structure of the biopolymer, one or more mutations of the biopolymer, a sequence of the non- nucleic acid polymer analyte, a surface charge of the biopolymer, one or more post-translation modifications of the biopolymer, or one or more ligands coupled to the biopolymer, or any combination thereof. [0851] In an aspect of the present disclosure, provided herein is a method comprising: providing a nanopore system. The nanopore system may comprise a membrane comprising a nanopore. In some cases, the membrane may separate the fluidic chamber into a first side (e.g., cis side) and a second side (e.g., trans side). A biopolymer may also be provided. In some cases, the first side (e.g., cis side) can have a first solution. In some cases, the second side (e.g., trans side) can have a second solution. The first solution and the second solution may be configured to translocate the biopolymer. The system can further comprise a controller. In some cases, the controller can be operatively coupled to the fluidic chamber and the nanopore. The controller may be configured to detect one or more signals associated with at least one characteristic of a leader construct. The controller may be configured to detect one or more signals associated with at least one characteristic of the biopolymer. The controller may be configured to detect one of more signals associated with at least one characteristic of a leader construct and one or more signals associated with at least one characteristic of the biopolymer. In some cases, the one or more signals may be detected during translocation of the biopolymer. In WSGR Docket Number: 64828-710.601 some cases, the one or more signals may be detected subsequent the translocation of the biopolymer. In some cases, the one or more signals may be detected during or subsequent the translocation of the biopolymer. [0852] A method according to present disclosure may further comprise measuring ionic current changes caused by translocation of the analyte through the nanopore. Current changes may be measured for states of (i) open channel, (ii) capture of the analyte by the nanopore, and /or(iii) passage of an analyte from (ii) through the nanopore. For example, the method of measuring ionic current changes comprises detecting differences between states (i), (ii) and (iii). In a specific aspect, the measuring comprises measuring differences during state (iii) caused by amino acid composition or structure of the analyte passing through the nanopore. The method suitably comprises taking one or more measurements characteristic of the analyte. The one or more measurements may be characteristic of one, two, three, four or five or more characteristics of the analyte. One or more characteristics are selected from (i) length of the analyte; (ii) analyte identity; (iii) analyte sequence; (iv) secondary or tertiary structures of the analyte; and (v) whether the analyte was modified or not. Any combination of (i) to (v) may be measured in accordance with the present disclosure. [0853] A further embodiment of the present disclosure relates to a nanopore system for translocating an analyte through a nanopore, comprising: (a) a membrane having nanopore therein, said membrane separating a chamber into a first side (e.g., cis side) and a second side (e.g., trans side), wherein the analyte can be to be added to the first side (e.g., cis side) and translocated through the nanopore to the second side (e.g., trans side); (b) on the first side (e.g., cis side) of said chamber an analyte captured by a protein translocase, which can bind and translocate the analyte through the nanopore in a sequential order; and (c) element for providing a voltage difference between the first side (e.g., cis side) and the second side (e.g., trans side) of the membrane. In some cases, the element in (c) can comprise a pair of electrodes. In some embodiments, the nanopore system can be further characterized by a cis to trans electro-osmotic force (EOF) resulting from a net ionic current flow cis- to-trans, so that the analyte can be captured in the nanopore with on top of the nanopore the translocase controlling the translocation. [0854] Various states of a pore, or events occurring at a pore, can be measured using a signal. Open-pore current, average or median current of an analyte translocation, the duration (dwell) time of the analyte translocation (the entire analyte, or portion thereof), the frequency of analyte translocation (which can provide a measure of concentration of the analyte), noise (which can be used to diagnose and/or troubleshoot defective pores, membranes, sensor, or device), or the shape of the signal during analyte translocation (e.g., smooth or stepwise changes), or any combination thereof can be measured or determined from a signal. In some embodiments, a signal comprises characteristics or information of a translocation event. A range of analytical tools can be used to extract characteristics or information of a translocation event. For example, edge-detecting algorithms can be used to segment a raw signal into parts. In some embodiments, a raw signal can be analyzed WSGR Docket Number: 64828-710.601 directly, with or without the application of filters, for example using sliding window features and algorithms with long range memory, to extract characteristic metrics. [0855] The arrangement of electrodes connected to a measurement circuit is used to detect any measurement disclosed herein. With another arrangement of electrodes adjacent to the nanopore and/or membrane it may also be possible to measure the voltage at the nanopore. In some cases, the signal may comprise a measurement of ionic current or change thereof ,voltage or change thereof, or resistance or change thereof, or any combination thereof. The current or change thereof, voltage or change thereof, or resistance or change thereof, or any combination thereof at the nanopore may be measured electrically via a circuit. The measurements can also occur by optical means (e.g., fluorophores). In some cases, an optical sensor, such as CCD sensor or a CMOS sensor or other sort of camera, either co-located on chip (e.g. individual sensors per compartment containing a nanopore on a chip) or externally sited (e.g. imaging via lens elements) may be used to detect current or change thereof, voltage or change thereof, resistance or change thereof, or any combination thereof (e.g. via local FET measurements of changes at the nanopore) via optical (e.g. fluorescence, raman, etc.) means. The measurement turned into an electrical signal via a controller and/or a detection circuit. The detection from the camera may be turned into an electrical signal. The signal may be raw electrical data. The signal may be digitized signal as recorded by the attached instrument. The signal may be digitized electrical signal. The signal may comprise an amplitude and/or one or more time points. The signal may comprise amplitude versus one or more time points. [0856] In some cases, the signal may be analyzed. The analysis may occur in near real time. For example, the signal may be processed and analyzed as it streams off the measurement device without recording the raw signal to memory. The analysis may occur once the signal has been recorded. The analysis may occur in real-time. The analysis may occur without the signal being recorded. The signal may be recorded independent of analysis. The analysis may occur prior to the signal being recorded and the signal recorded separately from the analysis. The analysis may occur on a different device from where the signal is recorded (such as the signal being recorded on a database, in a cloud computing architecture, or on a personal computer and the analysis occurring on different database, cloud computing architecture, or personal computer). [0857] In some cases, the signal may be preprocessed. The signal may undergo signal preprocessing. The signal may be processed by hardware configured to preprocess the signal. The signal may be preprocessed by software configured to preprocess the signal. The preprocessing may comprise denoising, segmentation, scaling, or any method disclosed herein. In some cases, analysis may occur on a preprocessed signal. [0858] In some cases, one or more characteristics disclosed herein may be assigned to at least portion of the analyte and/or at least another analyte (or a fragment thereof) based on one or more signals (e.g., electrical signal) and a database. The database may comprises one or more reference signals associated with a wide variety of molecules. The database can comprise one or more reference signals for one or more polypeptides, one or WSGR Docket Number: 64828-710.601 more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. [0859] In some cases, the one or more variants thereof may be the one or more polypeptides, one or more proteins, or one or more peptides coupled to a leader, adapter, or other molecular entity. The molecular entity may comprise a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, or protein, or any combination thereof. In some cases, the one or more variants may comprise post translational modifications (PTMs). The PTMs may comprise phosphorylation, acetylation, amidation, deamidation, glycosylation, oxidation, ubiquitination, sumolation, lipidation, or carbonylation, or any combination thereof. [0860] In some cases, the electrical signal or change thereof may comprise (1) one or more reads; and/or (2) one or more additional portions of the electrical signal or change thereof. The one or more additional portions of the electrical signal or change thereof may comprise one or more blocks of impurities. [0861] The electrical signal or change thereof can comprise measurements of (1) current or change thereof or (2) voltage or change thereof over a period of time. In some cases, the period of time can comprise one or more portions which can be used to determine a measurement of concentration associated with a sample comprising the at least the portion of the at least portion of the analyte and/or at least another analyte (or a fragment thereof). [0862] In one case, the electrical signal or change thereof may be pre-processed (e.g., denoising, segmenting), thereby generating a pre-processed electrical signal or change thereof. In some cases, the one or more characteristics disclosed herein can be assigned using the pre-processed electrical signal or change thereof. In some cases, a read (e.g., putative read) may be a contiguous portion of the signal relating to an event of interest. This may further be divided into segments and/or denoised. The signal was preprocessed to determine the location and length of putative reads related to putative events and then the corresponding region of measurements extracted. The read may be filtered based on a set of metrics to filter out reads that do not meet one or more criteria for one or more good nanopore translocation events. The remaining reads may be split into regions with similar characteristics and/or optionally smoothed, with the processes of segmentation and denoising, respectively. [0863] Information about regions of the current that are not putative reads may also be stored for further reference. For example, useful information can comprise, but are not limited to, estimates of open-pore current from the smoothing of a contiguous chunk of current can be helpful in determining how a read should be scaled, the time between reads provides a measure of sample concentration, location length and noise of current blocks or regions of high frequency noise are informative about the performance of system, or the time of voltage flicks or membrane ruptures than may affect measurements in other wells, or any combination thereof. WSGR Docket Number: 64828-710.601 [0864] The one or more reads can be extracted from (1) the electrical signal or change thereof and/or (2) the pre-processed electrical signal or change thereof. The extracted reads may be pre-processed to generate one or more pre-processed reads. The extracted reads may be pre-processed by denoising, filtering, segmenting, or scaling, or a combination thereof. [0865] To determine whether a putative read may be processed further, one or more of the following may be calculated median current, median absolute deviation of the current, the median absolute deviation of the difference between adjacent current samples, the number of runs of current values above or below the median current, and the length of read. In some cases, reads with unusual values of metrics may be filtered out and/or discarded as outliers. [0866] In some cases, segmentation of one or more reads into regions of current, voltage, or resistance, or any combination thereof with similar properties may proceed by wavelet decomposition to at least a level and/or discarding any unwanted components (e.g., high frequency components). For example, segmentation of one or more reads into regions of current, voltage, or resistance, or any combination thereof with similar properties may proceed by performing a Haar discrete wavelet decomposition to the fifth level and/or discarding the detail (high frequency) component. Segments may be an ordered series of regions covering the read, Segments may be formed from an ordered series of piecewise constant values resulting from a wavelet transformation (e.g., an inverse discrete wavelet transform). The segmentation may associate a read with a length. The segmentation may associate parameters summarizing characteristics of the segment (e.g., mean or median of the current, measures of noise or range like the standard deviation or median absolute deviation, the autocorrelation between samples or other frequency derived components). [0867] In some cases, neighboring segments can be merged by similarity. One or more pairs of neighboring segments can be scored for similarity and then the most similar were merged (e.g., bottom up fashion). The resulting segment can comprise a equal to the sum of the lengths of the constituent segment lengths and/or a level being the length-weighted average of the levels of the constituent segments. Merging of neighboring segments can continue until the measure of similarity for the most similar neighboring segments reaches a pre- defined threshold signifying that the segments are considered distinct. [0868] In some cases, the one or more characteristics may be assigned using (1) the one or more reads or (2) the one or more pre-processed reads (e.g., one or more segments). [0869] In some cases, (1) the one or more reads or (2) the one or more pre-processed reads may be compared (e.g., aligned) to the one or more reference signals in the database. The comparison can be accomplished by alignment (e.g., soft alignment, time warping). [0870] In some cases, (1) the one or more reads or (2) the one or more pre-processed reads scored against one or more reference signals, thereby assigning the one or more characteristics to the at least portion of the analyte WSGR Docket Number: 64828-710.601 and/or at least another analyte (or a fragment thereof). The scoring can comprises aligning at least a portion of the electrical signal of change thereof with the at least the portion of the one or more reference signals. [0871] Because of the stochastic nature of how an analyte translocates through a pore during measurement, the alignment between a read and a reference squiggle may not be known. Segment values and/or squiggle levels may not be directly compared. To score a read against a reference, multiple different alignments can be considered. However, not all alignments may be equally likely. To determine an alignment between one or more reads and one or more reference squiggles, the score of the highest scoring alignment can be calculated as described herein. [0872] Due to intra- and inter- experimental variation, the absolute level and range of reads for the same substrate may fluctuate relative to each other. It can be desirable to correct for range changes to avoid false positive differences and to reveal differences that may be disguised. [0873] In some cases, to directly compare reads to each other, the one or more reads can be transformed into coordinates that have been time-warped to a common reference squiggle. Such a transformation can enable reads to be piled-up for comparison and/or enable the formation of a consensus read. [0874] In some cases, alignment can assign one or more segments of each read to a position. Since reference squiggle may be common then these positions can form a common coordinate system for the reads. As the reference squiggle has been shifted and/or scaled to the read, the range may not be comparable. The shift and scale may be inverted and applied to the levels of each segment of the read so the values of all segments can be put on a scale that is common to all reads. [0875] In one example, for a read, the levels of all segments aligned to a given position can be averaged to create a new ordered series of levels. The average can be the mean level weighted by the segment lengths. The new ordered series of segment levels can be compared to the original reference squiggle to determine positions of similarity or differences. The differences can be indicative of one or more characteristics disclosed herein (e.g., presence of a mutation, PTM or other variant). [0876] In a similar fashion to that of a single read, the aligned segments from multiple reads can be averaged at each position to create a consensus of multiple reads. The average can be the mean level weighted by the segment lengths. Analogously to the procedure described for a single read, standard statistical tests can be applied at each position to determine whether an estimated level differs from that of the reference squiggle and so whether and where there are significant differences between the read and the reference squiggle. [0877] The consensus of multiple reads can have an interpretation as reference squiggle and methods disclosed above can be reapplied for further refinement. WSGR Docket Number: 64828-710.601 [0878] One or more reads can be visualized by plotting the level of one or more segments against its assigned position in the reference squiggle. For each read, the segments aligned to each squiggle position can be assigned time-warped coordinates that are uniformly spaced between that squiggle position and the next one. [0879] In some cases, (1) the one or more reads or (2) the one or more pre-processed reads may be aggregated to assign the one or more characteristics to the at least portion of the analyte and/or at least another analyte (or a fragment thereof). [0880] In some cases, the average accuracy can be identified. The number of reads correctly identified can be counted and then divided by the total number of reads to obtain the proportion of reads correctly identified. In addition or instead of estimating the identity of individual reads, it may be desirable to aggregate information from all reads known to or believed to be from the same substrate into a single estimate of identity. For example, analysis of a sample containing a single protein, or analysis of a sample containing a mixture of proteins where the reads have been clustered by similarity. The scores for all reads can be aggregated together for each putative reference squiggle before estimating the identity from these aggregated scores. Methods of aggregation include, but are not limited to: the sum of the scores, the weighted sum of scores, the median of the scores, the maximum or minimum of the scores, a L-statistic (linear combination of order statistics), and a M-estimator. The scores may be normalized by read length or substrate length before aggregation. [0881] In other cases, when given a sample containing a mixture of analytes, it may be desirable to determine what analytes are present in the sample and to quantify their absolute or relative abundance. The counts of identity may be normalized to create an estimate of the composition of the sample. One method of normalization is to divide the count of each identity by the total over all identities. The normalization can be constrained by prior knowledge of the sample composition or biology. As an example: it might be known that two or more peptide chains must be present in a known ratio and so their counts may be combined during normalization; or a previous measurement of the composition may be available. The estimation of quantification can be determined and/or be iteratively improved by reapplying the process to identify each read using the estimated composition as a prior probability to weight the reference squiggles and hence their score, and/or discouraging or preventing assignment to identities that are not supported by other reads in the sample. A new estimate of quantification, and hence composition, can be derived from these identities and this could be used for further refinement of the estimate. [0882] In some cases, the one or more characteristics may be assigned using the electrical signal or change thereof in combination with any of the aforementioned processes. [0883] In some cases, the at least portion of the analyte and/or at least another analyte (or a fragment thereof) may be among a sample disclosed herein. One or more properties of the sample may be determined using the one or more characteristics of at least portion of the analyte and/or at least another analyte (or a fragment WSGR Docket Number: 64828-710.601 thereof). The one or more properties may involve a summation of analyte identities. The summation may indicate the absolute abundance. The summation may be used to calculate the count of one or more proteins. The properties may involve a division of the summation by the total count of analytes (e.g relative abundance or percent composition). The properties may involve a normalizing the counts of one or more proteins. The normalization may comprise weighting. The weighting may comprise a multiplication of a property by a number. The weighting may comprise masking (e.g., binary masking, Boolean masking). The weighting may comprise a posterior probability (e.g., Bayesian). The weighting may comprise calculating a bias. [0884] In some cases, the characterizing the one or more properties comprises determining the number of analytes in the first type of analyte and determining the number of analytes in at least the second type of analyte. It may be determined in the first type of analyte or at least the second type of analyte, one or more of a number of analytes, relative abundance of analytes in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, one or more impurities in the sample, or a combination thereof. In some cases, the sample may comprise one type of analyte. [0885] Various characteristics of an analyte can be determined or approximated by analyzing a signal that the analyte produces during translocation. In some cases, the one or more characteristics are determined using the electrical signal or change thereof. A characteristic can comprise the length of the analyte (e.g., a contour length, in the case of polymeric analyte), the volume of the analyte, the mass of the analyte, the shape of the analyte, the secondary structure of the analyte, the tertiary structure of the analyte, the charge distribution of the analyte, the identity of the analyte, the sequence of the analyte, or any chemical modifications of the analyte, or any combination thereof. [0886] In some cases , the one or more characteristic of the at least the portion of an analyte and/or at least a portion of an at least an additional analyte may be determined and/or assigned. The determining and/ or assigning may be based, at least in part on, at least an electrical signal and a database. The electrical signal or change thereof, may be of a current or change thereof and/or voltage or change thereof. In some cases, the database may comprise one or more reference signals for at least one of one or more polypeptides, one or more peptides, one or more proteoforms thereof, one or more variants thereof, or any combination thereof. In some cases the database may comprise a reference signal associated with the at least a portion of an analyte and/or at least a portion of an at least an additional analyte. In some cases the database may not comprise a reference signal associated with the at least a portion of an analyte and/or at least a portion of an at least an additional analyte. The one or more reference signals may be direct reference signals (e.g., related to the raw data), signals, preprocessed signals, or generated by a machine learning model. The electrical signal or change thereof may comprise one or more reads and/or one or more additional portions of the signal or change thereof. The one or WSGR Docket Number: 64828-710.601 more additional portions of the electrical signal or change thereof may comprise one or more blocks of impurities. The electrical signal or change thereof may comprise a measurement. The measurement may comprise a current or change thereof, and/or a voltage or change thereof over a period of time, or any combination thereof. The period of time may comprise one or more portions associated with a measurement of a concentration associated with a sample comprising the at least thea portion of an analyte and/or at least a portion of an at least an additional analyte. In some cases, the at least a portion of an analyte and/or at least a portion of an at least an additional analyte comprises a first analyte and a second analyte wherein the first analyte comprises one or more first characteristics, and the second analyte comprises one or more second characteristics. In some embodiments, an analysis described herein may comprise a plurality of analytes (e.g., a plurality of non-nucleic acid based polymer analytes). At least a subset of the plurality of analytes may be used in the methods and/or systems described herein. For example, a plurality of analytes may comprise at least about 100 analytes and a subset of the plurality of analytes may be used to determine a plurality of characteristics. In some embodiments, the plurality of characteristics and/or a subset of the plurality of characteristics may be used in the methods and/or systems described herein to characterize one or more properties of a sample. [0887] In some embodiments, the signal comprises an ionic current, a change in ionic current, an ionic conductance, an electron flow, a change in ionic conductance, an impedance, a current rectification, a conductivity, or a tunneling current, or combinations thereof. In some embodiments, the signal comprises a first, second, or third order derivative of ionic current over time, a change in ionic current over time, an ionic conductance over time, an electron flow over time, a change in ionic conductance over time, an impedance over time, a current rectification over time, a conductivity over time, or a tunneling current over time. In some embodiments, the detected change in ionic current may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or about 150 pA. In some embodiments, the detected change in ionic current may be greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or about 150 pA. In some embodiments, the detected change in ionic current may be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or about 150 pA. [0888] A time resolution can refer to a digitization of a signal into units of a finite time (for example, frequency). The signal can be relative to ions flowing through a nanopore while the analyte (e.g., non-nucleic acid based polymer analyte) resides within. In some embodiments, the analyte may be moving in the nanopore. In some embodiments, the analyte may not be moving in the nanopore. The signal can be measured in discreet WSGR Docket Number: 64828-710.601 time units (e.g., digitized) at a given frequency. In some embodiments, a time-resolution of a signal can be at least about 0.1 kHz, at least about 0.5 kHz, at least about 1 kHz, at least about 5 kHz, at least about 10 kHz, at least about 20 kHz, at least about 30 kHz, at least about 40 kHz, at least about 50 kHz, at least about 75 kHz, at least about 100 kHz, at least about 200 kHz, or greater than about 200 kHz. In some embodiments, a time- resolution of a signal can be at most about 200 kHz, at most about 100 kHz, at most about 75 kHz, at most about 50 kHz, at most about 40 kHz, at most about 30 kHz, at most about 20 kHz, at most about 10 kHz, at most about 5 kHz, at most about 1 kHz, at most about 0.5 kHz, at most about 0.1 kHz, or less than about 0.1 kHz. [0889] In some embodiments, the time-resolution of a signal is greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 nanoseconds. In some embodiments, the time-resolution of a signal can be greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 microseconds. In some embodiments, the time-resolution of a signal can be greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 milliseconds. In some embodiments, the time-resolution of a signal can be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 nanoseconds. In some embodiments, the time-resolution of a signal can be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 microseconds. In some embodiments, the time-resolution of a signal can be less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000 milliseconds. The time resolution may be the digitization of the signal into units of a finite time and/or frequency. The signal may be relative to ions flowing through the nanopore while the polypeptide resides within (e.g., whether moving or not). The signal may be measured in discreet time units (e.g., digitized) at a given frequency. Common acquisition may be in the range 0.1-100KHz (e.g., 10,000,000 nanosecond increments to 10,000 nanosecond increments), there may also be instruments in the 0.1-10MHz range (e.g., 10,000 ns to 100 ns). [0890] In some cases, the determining of one or more characteristics may comprise pre-processing (e.g., denoising, smoothing, frequency manipulations, fast Fourier transform (FFT), Fourier transform, wavelets, or wavelet transformation) the electrical signal or change thereof. The preprocessing may generate a preprocessed electrical signal or change thereof. The one or more characteristics may be determined using the preprocessed electrical signal or change thereof. [0891] The preprocessing may comprise extracting one or more reads form the electrical signal or change thereof, and/or the preprocessed electrical signal or change thereof. The preprocessing may comprise one or WSGR Docket Number: 64828-710.601 more reads, thereby generating one or more preprocessed reads. The pre-processing may comprise denoising, filtering, segmenting, scaling, or a combination thereof. [0892] In some embodiments, signal analysis can comprise comparison of a signal produced during translocation to a database of known signals produced during translocation. In some embodiments, a signal may comprise a measured property over time. In some embodiments, the signals may comprise spectra. In some embodiments, a spectra may comprise a measured property over time. In some embodiments, the signals may comprise spectra of current, change in current, ionic conductance, electron flow, changes in ionic conductance, impedance, current rectification, conductivity, or tunneling current, or combinations thereof. In some embodiments, the signals may comprise further information derived from the signals. In some embodiments, signal analysis may comprise a collection of multiple associated signals. In some embodiments, signal analysis comprises a multi-dimensional analysis of signals, device operating parameters, or measurement conditions, or combinations thereof. In some embodiments, the database may comprise information on measurement conditions. The open pore may be characterized by the highest current level in the signal, and/or the least noisy portions of the signal, and/or may fall within a calibrated range based, at least in part, on previous standards defined for the system (e.g., in testing without any analyte added to the system). In some embodiments, conditions can comprise pH, temperature, type and/or concentration of any components of the run solution (e.g., ions, co-factors, buffers, or impurities, or any combination thereof), or applied voltage, or any combination thereof. In some embodiments, measurement conditions may comprise electrolyte concentrations, electrolyte concentration differences across a membrane, electric potential across a membrane, open pore current, open pore current noise, blocked pore current, blocked pore current noise, residual current, or residual current noise, or combinations thereof. In some embodiments, signal analysis may comprise spectral matching, or fingerprinting. In some embodiments, a database of known signals may comprise signals of analytes. In some embodiments, a database of known signals may comprise signals of expressible polypeptides, or proteoforms thereof, or isoforms thereof, or digests thereof, or tryptic digests thereof, or post-translationally modified variants thereof, or patterns of post-translation modifications thereof, or alternative protein splicings thereof, or chiral variants thereof, chemical degradations thereof, oxidation products thereof, residue tagged versions thereof, chemically modified versions thereof, signals from polypeptides translocated in a C-to-N or N-to-C direction, or combinations thereof. In some embodiments, a database of known signals may comprise signals of small molecules, biomolecules, biopolymers, metabolites, organic molecules, steroids, carbohydrates, amino acids, nucleotides, hormones, fatty acids, vitamins, flavins, protein-cofactors, lipids, or phenolic compounds, or combinations thereof. In some embodiments, a database of known signals may comprise signals of proteins complexed with other molecules, such as protein-DNA complexes, protein-RNA complexes, protein-ligand complexes, protein-carbohydrate complexes, protein-metabolite complexes, or protein-polymer complexes, or WSGR Docket Number: 64828-710.601 combinations thereof. In some embodiments, a database may comprise sequence information. In some embodiments, a database may comprise data collection conditions, device operating parameters, type of pore used during collection, or orientation of analyte translocation, or combinations thereof. In some embodiments, a database may comprise Edman degradation products. In some embodiments, a database can be used to train or optimize a machine learning model or other statistical or machine learning methods. [0893] In some embodiments, the signal may be denoised. In some embodiments, the denoising may comprise wavelet transformation. In some embodiments, the wavelet transformation may alter the signal based on a calculation of frequency of signal components. In some embodiments, the signal may comprise one or more components. In some embodiments, the one or more components may be one or more waveforms. In some embodiments, the one or more waveforms may comprise a frequency. In some embodiments, the denoising may comprise removing one or more waveforms with a frequency above a threshold. In some embodiments, the wavelet transformation may comprise removing high frequency components of the signal. In some embodiments, the wavelet transformation may comprise a Haar wavelet transformation. In some embodiments the wavelet transformation may be inverted. In some embodiments the inversion of the wavelet transformation may produce an ordered series of constant levels (i.e. waveforms). In some embodiments one or more segments are formed from the series of constant levels. [0894] In some embodiments, the signal may comprise one or more regions. In some embodiments, the one or more regions may comprise one or more reads. The read may be one or more portions of the raw signal or pre- processed raw signal. The read may be one or more portions of the aforementioned signals that relates to the event of interest (e.g., putative molecular interaction of interest, molecular event, interaction of nanopore with molecule). The read may comprise a chunk of current vs. time for an analyte translocation. The read may be based on the signal and/or one or more portions of the time period related to the putative event. In some cases, the read may be a subset of the signal if the signal is continuous and/or spans the event of interest. In some cases, the read may be concatenated chunks of signal if the signal has gaps, interleaved or off periods. [0895] In some cases, other measurements of the aforementioned signal can also be useful for determining properties of the sample (e.g., outside of translocation reads). This may comprise other events (e.g., impurity blocks) and/or the time between reads can be a measure of the concentration of the sample. In some cases, a segment may be at least a portion of a read or a signal as disclosed herein. In some cases, a squiggle can be derived from signal but with one or more different types of processing. Reads can be derived from signal. In some cases, reads can be compared to predicted/reference signals or squiggles in the database. [0896] In some embodiments, the one or more regions may comprise one or more impurities. Impurities may be derived from one or more sources. Examples of impurity sources may include, but are not limited to background, unwanted proteins, peptides, polypeptides, fragmented proteins, denatured proteins, albumins, WSGR Docket Number: 64828-710.601 immunoglobulins, fibrinogen, transferins, polymers (e.g. PEG, dextrans, etc.), sugars (e.g., single, complex, polymerics or branched), carbohydrates, saccharides (e.g., glucoses, sucroses, lactoses, maltoses, fructoses), starches, celluloses, chitins, molecules, vitamins, hormones, lipids, fatty acids, sterols, and/or glycerides. In some embodiments, impurities may be used to determine the concentration of the sample. In some embodiments, the sample comprises one or more of the same molecule. In some embodiment, concentration of impurities may be determined based at least in part on a count of impurities. In some embodiments, concentration of analyte may be determined based at least in part on a count of analytes. In some embodiments, the type of impurity may be determined based at least in part on duration of current blockades from impurities and from the characteristics of the impurity signals. In some embodiments, concentration may be determined based at least in part on average duration of impurities. In some embodiments, concentration may be determined based at least in part on percent of the signal due to of impurities. Concentration of the impurities may be calculated by count of impurities over time. Concentration of analytes of interest may be determined by count of analytes over time. [0897] In some embodiments, the signal may be segmented. In some embodiments, the segmentation may extract one or more reads from the signal. In some embodiments, the segmentation may extract one or more impurities from the signal. In some embodiments, the impurities may be discarded. In some embodiments, the signal may be preprocessed. In some embodiments, the reads may be preprocessed. In some embodiments, the reads may be one or more portions of the signal. In some embodiments, the one or more portions of the signal relate to one or more events. In some embodiments, the one or more events of interest may be an indication of one or more proteins translocating the pore protein. In some embodiments, one or more reads are determined by detecting one or more regions of signal indicating an empty pore. An empty pore may be an open pore. An open pore may be determined as the current falling within a certain current range (e.g., pre-calibrated for any disclosed system herein). In some cases, the read’s current range may be well separated from the open pores current range. In some cases a simple change in current may be used to extract putative reads from the open- pore (e.g., threshold triggers). In some cases, when the two are close or overlap the edges between reads and an open-pore may be determined by features of either the read of the open pore (see points below on the pattern of current changes, noise, complexity of signal). [0898] In some embodiments, one or more reads are determined from a signal without an indication of an empty pore, the one or more reads being after analysis (e.g., after sequencing or alignment of sequences to references to determine the segments that define reads and read boundaries. One or more motifs can be introduced between the concatemers to create a unique signal to split the reads. In some embodiments, an open pore is indicated in the signal by a change in the signal above a threshold. In some embodiments the change may be an increase in the signal. In some embodiments, the change may be a decrease in the signal. In some WSGR Docket Number: 64828-710.601 embodiments, the change may be an indication of one or more squiggle like properties. Examples of squiggle like properties may be, but are not limited to, range of the signal (e.g. measured by volatility), characteristic level of noise (e.g. volatility of neighboring differences), measures of complexity (e.g. independence), and or number of runs of samples above / below the median. In some cases, signatures may be added to the leader and/or tail of components of one or more analytes which may be used to create a unique current change (e.g., of unique magnitude of current change, pattern of change, noise). In some embodiments, segmentation comprises scoring one or more reads for similarity, a distance metric, a correlation coefficient, a statistical measure ,a rank-sum, a likelihood ratio, a sequential independence measure, a machine learning model. Examples of methods to perform segmentation and/or denoising include, but are not limited to, change-point detection methods, total variation denoising techniques, linear filters, median filters, iterated median filters, Kalman filters, wavelet denoising techniques. [0899] In some embodiments, the one or more reads may be characterized by one or more metric, for example median current, median absolute deviation of the current, the median absolute deviation of the difference between adjacent current samples, the number of runs of current values above or below the median current, the length of read, or a combination thereof. [0900] In some embodiments, reads may be segmented to produce segments. In some embodiments, the signal is segmented to produce segments. In some embodiments the segments may be merged. In some embodiments merging may comprise producing a similarity score for one or more reads. In some embodiments, the one or more reads may be adjacent to each other in the signal. In some embodiments, the one or more segments may be merged when a similarity score is above a threshold. In some cases, the neighboring segments may be merged. In some cases merging may happen in a bottom up fashion. In some embodiments, a similarity score may be calculated for segments that are neighboring one another in a read or signal. In some embodiments, segments may be merged based on length. In some embodiments, segmentation comprises scoring one or more reads for similarity, a distance metric, a correlation coefficient, a statistical measure, a rank-sum, a likelihood ratio, a sequential independence measure, comprise a machine learning model. Examples of scoring metrics may be, but are not limited to, the difference in mean and/or median of the consistent samples of the two segments, statistics which adjust for sample-size and variation in the background noise such as the t-statistic, order-based statistics such as Wilcoxon Rank-Sum or Mann-Whitney statistics, distribution based statistics such as likelihood-ratio test statistic or score statistic or Lagrange multiplier statistic, tests of sequential independence such as Wald-Wolfowitz test, classification techniques such as that from a machine learning model (examples include but are not limited to, convolutional neural network, hidden Markov model, recurrent neural network, or transformer model or any combination thereof), or any combination thereof. model. WSGR Docket Number: 64828-710.601 [0901] In some embodiments, the one or more segments may be associated with one more metadata. In some embodiments, the one or more metadata may comprise an average, a median, or a standard deviation, or any combination thereof. [0902] In some embodiments, one or more reads may be filtered. In some embodiments, one or more segments may be filtered. In some embodiments, one or more segments may result from segmentation as described herein. In some embodiments, filtering removes one or more segments. In some embodiments, filtering removes one or more segments. In some embodiments, filtering comprises a criteria for removal. In some cases, filtering may be of whole reads (e.g., pre- or post- segmentation) to remove the entire read. In some cases, filtering may be or portions of the reads (e.g., pre or post segmentation). Filtering may include filters for non-squiggle like blockades (e.g.,. from impurities), poor squiggles (e.g., event relates to target molecule of interest but read did not translocate correctly). In some cases, filtering portions of squiggle may include removal of artefacts, including temporary blocks (e.g.,. pore shutdown or impurities), shot noise, periods of stall, and/or high noise. [0903] In some embodiments, the database may comprise one or more squiggles In some embodiments, the one or more squiggles may be one or more reads. In some embodiments, the one or more squiggles may be one or more segments. In one or more embodiments, the one or more squiggles may be portions of one or more reads. In one or more embodiments, the one or more squiggles may be portions of one or more segments. In one or more embodiments, the one or more squiggles may have one or more protein identifications associated with each of the one or more squiggles. [0904] In some embodiments, one or more segments may be aligned to one or more squiggles from the database. In some embodiments, the alignment may be pairwise. In some embodiments, the alignment may be multiple alignment. In some embodiments, the alignment may be partial alignment. In some embodiments the alignment may be to one or more squiggle levels of the one or more squiggles in the database. A squiggle may be an ordered series of levels. In some cases, there may be one level per position. A squiggle level may be a level at one position. [0905] In some embodiments, the alignment may progress in a monotonic fashion through the one or more segments and/or the one or more series of squiggles or squiggle levels. In some embodiments, the alignment may be performed using a dynamic programming algorithm (such as a Pair Hidden Markov Model, for example). In some embodiments the dynamic programming algorithm may comprise one or more emission weights. In some embodiments, the one or more emission weights may form an emission density. In some embodiments, the emission density may be continuous. In some embodiments, the emission density may be a location-scale. In some embodiments the location-scale may comprise the formula where d is the difference between the segment level and the level of the position of reference and s is a scaling constant and k is a kernel density WSGR Docket Number: 64828-710.601
Figure imgf000261_0001
log ^ [0907] In some embodiments, the kernel density may be gaussian, such as in the following formula 1 log ^(^) = − ^^ + ^ 2 [0908] In some embodiments, the kernel density may be laplacian, such as in the following formula, [0909] log ^(^) = −|^| + ^ [0910] In some embodiments, the kernel density may be fair such as in the following formula, [0911] log ^(^) = log(1 + |^|) − |^| + ^ [0912] In the previous example formulas, K is the normalizing constant for the distribution. [0913] In some cases the determining may comprise a score. In some cases the scoring may comprise a comparison. In some cases the determining the one or more characteristics may comprise scoring the one or more reads or the one or more preprocessed reads to the one or more reference signals. The scoring may comprise aligning at least a portion of the electrical signal or change thereof with the at least the portion of the one or more reference signals. The comparing may comprise soft alignment. [0914] In some embodiments, aligning may comprise a time-warping algorithm. In some embodiments the time-warping algorithm may be dynamic time warping. In some embodiments, aligning may comprise a machine learning model. In some embodiments, the machine learning model may produce one or more scores. In some embodiments, the one or more scores may be arranged into one or more matrices matrix. In some embodiments, the alignment may comprise an approximation technique (such as beam search and/or prefix search). [0915] In some embodiments, a squiggle may need to be adjusted (such as when there may be differences that are not relevant to the method being performed, for example: experimental variation, changes in pore or membrane, the presence of variants or PTMs in a two-sample comparison. In some embodiments, adjustment comprises a measure of fit. In some embodiments, the measure of fit is relative to one or more reads. In some embodiments, the one or more reads are aligned to a reference squiggle. In some embodiments, the level of the reference squiggle is adjusted based at least in part on the measure of fit to the one or more reads. In some embodiments, the measure of fit is used to optimize the adjustment of the reference squiggle. In some embodiments, the adjustment may be at one or more positions in the squiggle. In some embodiments, the adjustment at one or more positions may be 0. In some cases, the adjustment at one or more positions may be fixed at 0. In some embodiments, the adjustments at one or more positions may be greater than 0. In some embodiments, the measure of fit may be one or more scores obtained through alignment of the one or more reads to an adjusted squiggle. In some embodiments, the measure of fit may be one or more scores obtained WSGR Docket Number: 64828-710.601 through alignment of the one or more reads to a squiggle. In some embodiments, the measure of fit may comprise a sum of squares difference. [0916] In some cases, the sum of squares difference is calculated between the segment and the level being adjusted. The sum of squares may comprise the following formula
Figure imgf000262_0001
[0917] The measure of fit may incorporate a penalty term (e.g., sum of squares of the adjustments, the sum of absolute value of adjustments, the weighted sum of squares of the adjustments, the weighted sum of absolute value of adjustments, the weighted or unweighted sum of the squares or absolute values of the difference between neighboring adjustments, the continuous exact L0 penalty, applied to the adjustments or difference between neighboring adjustments, group-sparse penalty applied to the adjustments or difference between neighboring adjustments, an indicator function that the adjustment at some positions should to be equal to zero). The squiggle may be adjusted using Expectation-Maximisation and Majorize-Minimization. In some embodiments, the measure of fit may comprise a stopping criteria (such as fit failing to improve, fit improving below a threshold, magnitude of change in fit falling below a threshold). In some embodiments, the measure of fit may be weighted. [0918] A non-limiting example of how the contribution of each segment to each position might be weighted include, but not limited to: the posterior probability that the read matches the squiggle being adjusted, the posterior probability that a segment of the read is aligned to the position of squiggle, the weighted contribution of a segment to the position over all possible alignments, the weighted contribution of a segment to the position over all possible alignments determined by the forward-backward algorithm, the length of the segment; the estimated noise of the segment, a weight representing the confidence that the segment is not a measurement of a pore blockage, stall, or other undesired system noise, a binary weight representing whether the segment is aligned to the position, some combination of one or more of the previous examples. [0919] In some cases, the contribution of segment i to level j is weighted by wij, the measure of fit Mw of the adjustment and may be written as
Figure imgf000262_0002
[0920] The fit of adjusted squiggle to the reads may comprise aligning the reads to the adjusted squiggle and applying the squiggle adjustment method with the previously determined adjusted squiggle taking the place of the reference squiggle. The squiggle adjustment method may be applied iterative until no further adjustment. Examples of stopping criteria include, but are not limited to: the measure of fit failing to improve; the measure WSGR Docket Number: 64828-710.601 of fit improving by a less than a small absolute amount; the measure of fit improving by less than a small relative amount; the magnitude of adjustments changing by less than a small absolute amount. [0921] In some cases, the one or more characteristics may be determined using the one or more reads, and/or the one or more preprocessed reads (e.g., one or more segment). The determining may comprise comparing (e.g., aligning) the one or more reads, and/or the one or more preprocessed reads to one or more reference signals in the database. The comparing may comprise alignment. The alignment may comprise time warping. In some embodiments, the alignment may be used to estimate the identification of one or more reads. In some embodiments, the identification of one or more reads may be aggregated. In some embodiments, the aggregation maybe used to identify a sample identity (such as sample source) In some embodiments, the sample identification may comprise a clustering method. In some embodiments, aggregation may comprise at least one of a sum of scores, a median of scores, a maximum of scores, a minimum of scores, a L-statistic, a M-statistic. In some embodiments, the aggregation may be normalized. In some embodiments, the identification may comprise a confidence score. In some embodiments, the confidence score may be calculated by a bootstrap method, a sampling method, a resampling method, or any combination thereof. Confidence in the estimate of identity may be assessed by bootstrap method. The bootstrap method may comprise resampling the reads with replacement to produce pseudo-replicate experiments for which the identity of the substrate can be estimated and the proportion of pseudo-replicates where the protein is identified correctly gives an accuracy of identity over repeated experiments. In some cases, the proportion of pseudo-replicates that agree with the estimate of identity of the protein is a measure of confidence in that estimate. [0922] In some cases, the determining the one or more characteristics may comprise aggregating the one or more read and/or the one or more preprocessed reads. In some embodiments, the sample comprises a plurality of proteins. In some embodiments, the identification of one or more reads may be quantified. In some cases, cases, the quantification may be the aggregation. In some embodiments, the quantification may be the aggregation. In some embodiments, the quantification may comprise a summation. In some embodiments, the quantification may be normalized. In some embodiments, the quantification may be a relative abundance. In some embodiments, the quantification may be an absolute abundance. [0923] In some cases, the individual reads may be identified by searching against a set of reference squiggles and their identities tabulated. The tabulated identities may be normalized to create an estimate of the composition of the sample (e.g., to divide each element of the tabulation by the total over all elements). The normalization may be constrained by prior knowledge of the sample composition or biology (e.g., it might be known that two or more peptide chains must be present in a known ratio and so their counts may be combined during normalization, or previously measurement of the composition may have been made). WSGR Docket Number: 64828-710.601 [0924] In some cases, the individual reads may be assigned a weighting over the set of references. Examples of weighting include, but are not limited to: a posterior probability of that reference being the identity of the read, a weighting to correct for known capture biases of the system, a weighting informed by prior knowledge about the sample composition or the biology, a binary weight to include or exclude individual or groups of references; error, any combination of the examples provided. [0925] In some cases, the tabulated identities or estimate of the composition may be used to improve the assignment of identity to reads. Examples of how the assignment of identity may be improved include, but are not limited to: removing reference squiggles corresponding to proteins estimated to have zero abundance or low abundance, removing reference squiggles corresponding to proteins that have low counts of tabulated identity, using the tabulated identities or estimate of composition to weight the scores of each read against each squiggle before assignment, where the estimate of composition is used to weight the assignment and the assignment of identity is made by scores representing the (mathematical) likelihood of the squiggle given the read, the technique known as Bayes Empirical Bayes may be used to assign identity. Non-limitative examples of when iteration may be stopped include: when the identities assigned to reads no longer change, when the tabulated identities no longer change or change by less than a threshold, when no reference squiggles are discarded, when the estimate of quantification changes by less than a threshold. [0926] In some cases, after adjusting a reference squiggle to a set of reads, it may be referred to as an empirical squiggle (e.g., its levels are now derived from observed values of a particular experiment or set of experiments). [0927] In some cases reads may be directly compared to each other. In some cases reads may be converted into coordinates that have been time-warped to a common squiggle. The reads may be aligned to the squiggle and the segments, or raw current samples, corresponding to each squiggle position are assigned time-warped coordinates that are uniformly spaced between that squiggle position and the next, preserving the original time ordering. The reads may be plotted using these time-warped coordinates results in the features corresponding to each position being overlaid, revealing differences and similarities in their surrounding context. The time- warped coordinates may be used for other analyses. [0928] In some cases, one or more properties of the sample may be characterized using a plurality of characteristics associated with the plurality of analytes. The one or more properties may comprise an absolute or relative abundance, absolute concentration, relative concentration, and/or origin of one or more analyte types in the sample. The one or more properties may comprise an absolute or relative abundance, absolute concentration, relative concentration, and/or origin of one or more analytes in the sample. The one or more properties may comprise differences in sequence (e.g., differences in sequence is of at most 10 amino acids, at most 5 amino acids, etc.) of at most 10 units between at least a subset of analytes of the plurality of analytes. WSGR Docket Number: 64828-710.601 The one or more properties may comprise a percentage of modified and/or different analytes in the plurality of analytes. [0929] In some embodiments, one or more databases may contain information. In some embodiments, the information may be one or more signals. In some embodiments, the information may be one or more reads. In some embodiments, the information may be one or more squiggles. In some embodiments, one or more squiggles may be one or more reads in a database. In some embodiments, one or more squiggles may be one or more reads in a database produced through a machine learning model. In some embodiments, one or more squiggles may be one or more signals in a database. In some embodiments, one or more squiggles may be one or more signals in a database produced through a machine learning model. In some embodiments, one or more squiggles may be one or more segments in a database. In some embodiments, one or more squiggles may be one or more segments in a database produced through a machine learning model. [0930] In some cases, the machine learning model may comprise a neural network. In some embodiments the neural network may be a convolutional neural network, a recurrent neural network, an autoencoder, a u-net, a variational autoencoder, a generative adversarial network, a transformer, or any combination thereof. The neural network may comprise one or more layers (e.g input, convolutional, recurrent, attention, normalization, flat, fully connected, pooling, dropout). The neural network may comprise residual connections. The one or more layers may comprise nodes. The one or more layers may comprise the same number of nodes. The one or more layers may comprise different numbers of nodes. The neural network may comprise one or more activations (e.g., sigmoid, rectified linear unit (ReLU), sigmoidal linear unit (SiLU), and/or linear). [0931] In some cases, the machine learning model may comprise a hidden Markov model, a Bayesian network, a regression, a clustering method, or any combination thereof. [0932] In some cases, the machine learning model is pretrained. In some cases, the machine learning model is trained. The machine learning model may be trained using a training dataset. The training dataset may comprise one or more reference sequences. The training dataset may comprise one or more signals. The training dataset may comprise one or more reads. The training dataset may comprise one or more segments. The training dataset may comprise one or more squiggles. The training dataset may be an aggregation of data from a single experiment with a single nanopore system. The training dataset may be an aggregation of data from a single experiment with a one or more nanopore systems. The training dataset may be an aggregation of data from one or more experiments with a one or more nanopore systems. The training dataset may be an aggregation of data from one or more experiments with a single nanopore system. [0933] In some cases, the machine learning model may be validated. The machine learning model may be validated using a validation dataset. The validation dataset may comprise one or more reference sequences. The validation dataset may comprise one or more signals. The validation dataset may comprise one or more reads. WSGR Docket Number: 64828-710.601 The validation dataset may comprise one or more segments. The validation dataset may comprise one or more squiggles. The validation dataset may be an aggregation of data from a single experiment with a single nanopore system. The dataset may be an aggregation of data from a single experiment with a one or more nanopore systems. The validation dataset may be an aggregation of data from one or more experiments with a one or more nanopore systems. The validation dataset may be an aggregation of data from one or more experiments with a single nanopore system. [0934] In some cases, the machine learning model may be tested. The machine learning model may be tested using a test dataset. The test dataset may comprise one or more reference sequences. The test dataset may comprise one or more signals. The test dataset may comprise one or more reads. The test dataset may comprise one or more segments. The test dataset may comprise one or more squiggles. The test dataset may be an aggregation of data from a single experiment with a single nanopore system. The test dataset may be an aggregation of data from a single experiment with a one or more nanopore systems. The test dataset may be an aggregation of data from one or more experiments with a one or more nanopore systems. The test dataset may be an aggregation of data from one or more experiments with a single nanopore system. [0935] In some cases, the training dataset, validation dataset, and the test dataset may be a part of the same dataset. The training dataset, validation dataset, and the test dataset may be from different datasets. The training dataset, validation dataset, and the test dataset may be different sizes. The training dataset, validation dataset, and the test dataset may be the same sizes. The training dataset, validation dataset, and the test dataset may comprise data from one or more individuals. The training dataset, validation dataset, and the test dataset may comprise data from one or more organisms. The training dataset, validation dataset, and the test dataset may comprise data from one or more proteins. The training dataset, validation dataset, and the test dataset may comprise data with impurities. The training dataset, validation dataset, and the test dataset may comprise data from one or more analytes. The training dataset, validation dataset, and the test dataset may comprise data of the same length. The training dataset, validation dataset, and the test dataset may comprise data of different lengths. The training dataset, validation dataset, and the test dataset may comprise data that has been batch normalized. The training dataset, validation dataset, and the test dataset may comprise data that has been processed to remove batch effect. The training dataset, validation dataset, and the test dataset may comprise data that has been augmented. The training dataset, validation dataset, and the test dataset may comprise data that is synthetic. The training dataset, validation dataset, and the test dataset may comprise data that is produced by a machine learning model. The training dataset, validation dataset, and the test dataset may comprise data may comprise kmer information. The training dataset, validation dataset, and the test dataset may utilized variable kmer length. The training dataset, validation dataset, and the test dataset may comprise a dictionary of a plurality of amino-acid words. WSGR Docket Number: 64828-710.601 [0936] The model may comprise one or more parameters. The parameters may be initialized with one or more values. The one or more values may be the same. The one or more values may be different. The one or more values may be initialized randomly. The one or more values may be loaded into the model. The loaded values may be from another model. The loaded values may be from a previous version of the model. The loaded values may be partially trained. The loaded values may be fully trained. The loaded values may be masked. The loaded values may be normalized. [0937] In some cases, the model may be trained iteratively (e.g., epochs).The iterations may comprise an optimizer (e.g., Adam, AdaDelta,,Stochastic Gradient Decent (SGD), RMSPropm ADAGrad, NADAM, FTRL, Nesterov movement, Gauss-newton algorithm, momentum, AdaFactor, AdamW). The optimizer may comprise a learning rate. The learning rate may be static. The learning rate may be dynamic. The learning rate may be controlled by a learning rate scheduler (e.g., StepLR, ExponentialLR, CyclicLR, Multi-StepLR, ReduceLROnPlateau, Cosine annealing, LinearLR, PolynomialLR, ConstantLR, LambdaLR, OneCycleLR). [0938] In some cases, the model may be implemented using a programming package (e.g., Scikit-learn, Pytorch, Iml, Tensorflow, Gmodels, OneR, Range, XGboost, H20, Theano, keras, nlp, seaborn). [0939] In some cases, the machine learning model may generate one or more outputs. The one or more outputs may be compared to one or more expected outputs. The comparison may be a loss calculation. The loss may be calculated using an loss method (e.g., L1, Crossentro-py, Poisson, VAEloss, Mean Squared Error, Triplet loss, cosine embedding loss). [0940] The machine learning model may be configured to stop training upon satisfying a stopping parameter. The stopping parameter may be based at least in part on the magnitude of the loss. The stopping parameter may be based at least in part on the number of epochs. The stopping parameter may be based at least in part on the magnitude of the loss. The stopping parameter may be based at least in part on a patience (e.g., number of epochs without improvement). [0941] In an embodiment, a signal may be detected. The signal may be denoised. One or more reads may be extracted from the signal. The one or more reads may be preprocessed. The one or more reads may be denoised. The one or more reads may be filtered. The one or more reads may be segmented (e.g., chunks of raw or preprocessed signals may be merged into a segment). The segments may be filtered. The reads may be filtered. The extraction may extract a read from a signal. The extraction may comprise detecting one or more characteristics of the read from the open-pore level. The one or more characteristics may be a change in current above a certain level. The one or more characteristic may have squiggle like properties. The Denoising may comprise applying one or more filters to the signal of one or more reads to remove high noise (e.g., shot noise or random noise artifacts). Denoising may partially smooth the signal. Segmentation may detect one or more segment portions of the signal or preprocessed signal with similar properties and segment the signal into one or WSGR Docket Number: 64828-710.601 more segments. The one or more segments may be associated with metadata representative of the segmentation (e.g., average, median, standard deviation). The segmentation may be an edge detecting algorithm. The segmentations may be a segmentation algorithm. The filtering may remove segments (e.g., unwanted segments), and/or entire reads. One or more reads may be adjacent to each other. Merging may merge one or more adjacent reads based, at least in part on similarity criteria. [0942] In another embodiment, a signal may be detected. The signal may be denoised. The signal may be segmented into one or more segments. One or more reads may be extracted from the one or more segments. The one or more reads may be filtered. [0943] In some embodiments, a characteristic of an analyte can be determined with a degree of accuracy. In some embodiments, the analyte characteristic comprises a sequence. In some embodiments, accuracy can be evaluated as a sequence accuracy, as a consensus accuracy, as an amino acid accuracy, an identity, or as combinations thereof. [0944] In some cases, the database is generated from one or more reference sequences. The reference signals may be direct reference signals (e.g., related to the raw data by some means), or artificial reference signals (e.g., generated by a machine learning algorithm) The one or more reference sequences may be derived with, and/ or from, genomic information or transcriptomic information of the sample. The genomic information may comprise genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. The transcriptomic information may comprise genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. [0945] In some cases, the database may be generated from the one or more reference sequences using one or more machine learning algorithms. The database may comprise one or more reference signals for the at least a portion of an analyte and/or at least a portion of an at least an additional analyte, at least one proteoform thereof, at least one variant thereof, or combination thereof. The one or more polypeptides may comprise one or more expressible polypeptides. The one or more variants thereof may comprise one or more post-translationally modified variants thereof. The data may be generated in a dynamic fashion (e.g., additional reference signals may be added or removed during an analysis) [0946] In some cases properties may be characterized for a sample. The properties may be characterized by determining at least one feature of a proteome associated with the sample. The proteome coverage may be at least 1%. The methods of this disclosure may be used to characterize properties of a sample. WSGR Docket Number: 64828-710.601 [0947] In some cases, the database is formed by a machine learning model. In some cases the database is created by a machine learning model. In some cases, the machine learning model generates the one or more reference sequences. In some cases, the one or more reference sequences are created by a machine learning model and then compiled into a set of references on the database. [0948] In some embodiments, a characteristic of an analyte can be determined by comparison of a signal to a database, signal analysis with a statistical learning method or model, Edman degradation, time-of-flight mass spectrometry, SDS-PAGE analysis, ELISA analysis, or any combination thereof. [0949] In some embodiments, analysis of multiple characteristics can be combined to improve identification of an analyte or the characteristic thereof. In some embodiments, signal measurements from multiple pore types can be combined to improve identification of an analyte or characteristic thereof. [0950] In some embodiments, an analysis of an analyte using the pores (e.g., nanopore), systems, methods, or any combination thereof described herein may comprise a sequence accuracy. A sequence accuracy may comprise a percentage accuracy of an analyte sequence obtained from an analysis compared to a reference sequence of the analyte. A consensus accuracy of one or more analytes may be obtained by comparing the one or more reads from the one or more analytes to one or more references. A consensus accuracy may also be obtained for an analyte if the analyte can be read multiple times passing through (e.g., translocating through) a pore described herein. For example, a consensus accuracy can be measured by at least two or more reads (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10 reads) from an analyte translocating through a nanopore. In some cases, a consensus accuracy may be obtained by comparing multiple analytes to a reference and/or obtaining some at least a blended, median, blended accuracy, blended score, blended probability, cumulative average, cumulative median, cumulative accuracy, cumulative score, and/or cumulative probability across the multiple molecules. In some cases, a consensus accuracy may be obtained from a single molecule in the case it is read multiple times in a multi-pass manner many times across the nanopore. [0951] In some embodiments, a sequence can be determined with a sequence accuracy of about 50% to about 99.9%. In some embodiments, a sequence can be determined with a sequence accuracy of 50% to 55%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to 99.9%. In some embodiments, the sequence can be determined with a sequence accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or at least about 99.9%. [0952] In some embodiments, a sequence can be determined with a consensus accuracy of about 50% to about 99.9%. In some embodiments, a sequence can be determined with a consensus accuracy of about 50% to 55%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to 99.9%. In some embodiments, the sequence can be determined with a consensus accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or at least about 99.9%. WSGR Docket Number: 64828-710.601 [0953] In some embodiments, a sequence can be determined with an amino acid accuracy of about 50% to about 99.9%. In some embodiments, a sequence can be determined with an amino acid accuracy of 50% to 55%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to 99.9%. In some embodiments, the sequence can be determined with an amino acid accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or at least about 99.9%. [0954] In some embodiments, an analyte can be determined with a true negative rate, or specificity. In some embodiments, an analyte can be determined with a true negative rate of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or about 100%. In some embodiments, an analyte can be determined with a true negative rate of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 99.9%. In some embodiments, an analyte can be determined with a true negative rate of less than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or about 100%. [0955] In some embodiments, an analyte can be determined with a true positive rate, or sensitivity. In some embodiments, an analyte can be determined with a true positive rate of about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or about 100%. In some embodiments, an analyte can be determined with a true positive rate of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 99.9%. In some embodiments, an analyte can be determined with a true positive rate of less than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or about 100%. [0956] In some embodiments, an analyte can be detected at a concentration of about 1 analyte/L, 10 analytes/L, 1,000 analytes/L, 10,000 analytes/L, 100,000 analytes/L, 1,000,000 analytes/L, 10,000,000 analytes/L, 100,000,000 analytes/L, 1,000,000,000 analytes/L, 10,000,000,000 analytes/L, 100,000,000,000 analytes/L, or 1,000,000,000,000 analytes/L. In some embodiments, an analyte can be detected at a concentration of at least 1 analyte/L, 10 analytes/L, 1,000 analytes/L, 10,000 analytes/L, 100,000 analytes/L, 1,000,000 analytes/L, 10,000,000 analytes/L, 100,000,000 analytes/L, 1,000,000,000 analytes/L, 10,000,000,000 analytes/L, or 100,000,000,000 analytes/L. In some embodiments, an analyte can be detected at a concentration of at most 10 analytes/L, 1,000 analytes/L, 10,000 analytes/L, 100,000 analytes/L, 1,000,000 analytes/L, 10,000,000 analytes/L, 100,000,000 analytes/L, 1,000,000,000 analytes/L, 10,000,000,000 analytes/L, 100,000,000,000 analytes/L, or about 1,000,000,000,000 analytes/L. [0957] In some embodiments, an analyte can be determined with a positive predictive value, or precision. In some embodiments, an analyte can be determined with a positive predictive value of about 1%, 5%, 10%, 15%, WSGR Docket Number: 64828-710.601 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or about 100%. In some embodiments, an analyte can be determined with a positive predictive value of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 99.9%. In some embodiments, an analyte can be determined with a positive predictive value of less than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or about 100%. [0958] In some embodiments, a mutation in a biological analyte can be detected. In some embodiments, a post- translational modification of a proteinaceous analyte can be detected. In some embodiments, analytes can be distinguished based on chirality. For example, peptide fragments that differ by a single amino acid residue, a phosphorylation, a glycosylation, a methylation, and/or a L or D center, can be distinguished by their translocation signals. [0959] The characteristics of the analytes can be determined by various analytical methods, including for example signal libraries, statistical methods, machine learning methods. Statistical or machine learning methods can comprise using a model trained or optimized with model analytes, or can be built from first principles. For example, the identity of peptides can be determined by comparison to previously acquired data using training data. A machine learning algorithm can be trained by translocating a corpus of different analytes (e.g., different peptide sequences) through a pore, measuring the output signals, and then using analyte identification-signal pairs to train a machine learning algorithm to identify an analyte or a characteristic thereof from the signal. In some embodiments, the machine learning algorithm can be trained on data from a database. In some embodiments, determining the identity or a characteristic of an analyte can be based on a library of theoretically predicted or experimentally measured signals. In some embodiments, the machine learning algorithm can predict an identity or characteristic of an analyte that is not listed in the database used to train the machine learning algorithm. In some embodiments, multiple measurements of an analyte under different conditions can be used in high dimensional analysis (e.g. by combined comparison of 2, 3, 4, 5, 6, or more separate event metrics; e.g., in autoencoders or PCA) to discriminate different analytes that may not be separable by any one metric alone. A collection (spectra) of multiple analyte translocation events can be analyzed as a population ensemble for a discrete population of analytes in a sample. The discrete populations can be resolved (e.g. in multiple dimensions using multiple metrics as axes) and identified using any number of advanced fitting and/or classification tools. Further, unique data from the populations, for example fingerprints, can be used in analytical methods to determine an analyte composition, e.g., assembling the identity of a protein from signals measured from peptide fragments of the protein. In some embodiments, spectral matching can be performed. In some embodiments, measured spectra may be compared to a library of reference spectra. WSGR Docket Number: 64828-710.601 [0960] In some embodiments, translocation through a pore can generate electrical signals that comprise noise from sources other than the translocation. In some embodiments, an electrical signal (or spectrum) can be compared with a reference electrical signal (or spectrum). For example, a squared first derivative Euclidean cosine correlation can be used for the comparison. This comparison can be sensitive to the position of the peaks observed in the spectra. In some cases, it may not be as sensitive to a shifting baseline.
Figure imgf000272_0001
[0961] Where Ai and A2 equal the vectors of excluded current counts and A1,i and A2,i represent the individual bins of the excluded current spectrum. In a more detailed description, A1 and A2 are set as the vector of counts observed for each residual current bin (e.g. An= counts (40-41%), counts(41- 42%), ..., counts (94-95%)). ΔAn, is the derivative of An (difference between bins). In the numerator, each element ΔAn is multiplied with the corresponding ΔAn of the comparing spectrum and take the squared sum of all items. In the denominator, the squared sum of each element in AAn is multiplied with that with the squared sum of each element in the spectrum to compare. So, if the two vectors A1 and A2 are equal, the correlation is 1, else it is less than 1, and because the derivative of A1 and A2 is taken, linear baseline sloping is less impactful. [0962] In some embodiments, identity of the signal can be determined by machine learning methods with an accuracy of about 50% to 55%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 98%, 50% to 99%, 50% to 99.9%. In some embodiments, the identity of a signal can be determined with an accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 99.9%. In some embodiments, a first pass accuracy of a machine learning analysis may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 99.9%. In some embodiments, a first pass accuracy of a machine learning analysis may be at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9%. In some embodiments, a multi-pass accuracy of a machine learning analysis may be about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 99.9%. In some embodiments, a multi-pass accuracy of a machine learning analysis may be at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or about 99.9%. [0963] In some embodiments, sample analysis can result in identification of multiple analytes or multiple counts of analytes. In some embodiments, identified analytes and derived information such as analyte counts can be used for further analysis. In some embodiments, further analysis comprises statistical analysis, proteome analysis, population analysis, phenotypic analysis, metabolomic analysis, disease profiling, health profile analysis, environmental analysis, or combinations thereof. In some embodiments, proteomic analysis comprises determining number of peptides per sample, number of proteins per sample, number of modifications per sample, dynamic range per group, coefficient of variation per group, proteome coverage, sample stratification, WSGR Docket Number: 64828-710.601 differential expression of proteins, protein-protein correlation; interaction networks, drug associations, disease associations, phenotype associations, metabolic pathway associations, or combinations thereof. [0964] In some embodiments, sample analysis can identify a phenotypic profile, a metabolic profile or state, a disease profile, a health profile, an environmental profile. In some embodiments, a phenotypic profile can be determined by a degree of similarity with a previously identified phenotypic profile. In some embodiments, a metabolic profile or state can be determined by a degree of similarity with a previously identified metabolic profile or state. In some embodiments, a disease profile can be determined by a degree of similarity with a previously identified disease profile. In some embodiments, a health profile can be determined by a degree of similarity with a previously identified health profile. In some embodiments, a degree of similarity can be evaluated by proteomic coverage. [0965] In some embodiments, a proteome coverage may be at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50 %, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or greater than about 99%. In some embodiments, proteome coverage may about 100%. In some embodiments, a proteome coverage may be at most about 1%, at most about 2%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50 %, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 99%, at greater than about 99%. In some embodiments, the proteome coverage may be about 100%. In some embodiments, a proteome coverage may be between about 1% to about 100%. In some embodiments, proteome coverage may be between about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 75%, about 1% to about 100%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 10%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 75%, about 2% to about 100%, about 3% to about 4%, about 3% to about 5%, about 3% to about 10%, about 3% to about 20%, about 3% to about 30%, about 3% to about 40%, about 3% to about 50%, about 3% to about 75%, about 3% to about 100%, about 4% to about 5%, about 4% to about 10%, about 4% to about 20%, about 4% to about 30%, about 4% to about 40%, about 4% to about 50%, about 4% to about 75%, about 4% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 75%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about WSGR Docket Number: 64828-710.601 75%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 100%, about 40% to about 50%, about 40% to about 75%, about 40% to about 100%, about 50% to about 75%, about 50% to about 100%, or about 75% to about 100%. [0966] In some embodiments, a sequence coverage may be at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50 %, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or greater than about 99%. In some embodiments, proteome coverage may about 100%. In some embodiments, a sequence coverage may be at most about 1%, at most about 2%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50 %, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95%, at most about 99%, at greater than about 99%. In some embodiments, the sequence coverage may be about 100%. In some embodiments, a sequence coverage may be between about 1% to about 100%. In some embodiments, a sequence coverage may be between about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 75%, about 1% to about 100%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 10%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 75%, about 2% to about 100%, about 3% to about 4%, about 3% to about 5%, about 3% to about 10%, about 3% to about 20%, about 3% to about 30%, about 3% to about 40%, about 3% to about 50%, about 3% to about 75%, about 3% to about 100%, about 4% to about 5%, about 4% to about 10%, about 4% to about 20%, about 4% to about 30%, about 4% to about 40%, about 4% to about 50%, about 4% to about 75%, about 4% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 75%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 75%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 75%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 75%, about 30% to about 100%, about 40% to about 50%, about 40% to about 75%, about 40% to about 100%, about 50% to about 75%, about 50% to about 100%, or about 75% to about 100%. [0967] In some embodiments, sequence coverage may be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 13, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, WSGR Docket Number: 64828-710.601 or about 500 amino acids. In some embodiments, sequence coverage may be greater than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 13, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or about 500 amino acids. In some embodiments, sequence coverage may be less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 13, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or about 500 amino acids. In some embodiments, a sequence coverage may comprise identification of amino acid identity, volume, mass, post translation modification, charge, hydrophobicity, hydrophilicity, chemical modification, oxidation state, reduction state, chirality, isotope labeling, alkylation, or combinations thereof. The data generated and disclosed herein can come from multiple different nanopores, from one nanopore, or from different experimental runs from different devices or systems. The data from different runs can be combined. In the context of analyzing samples, when determining a property of a sample, the analysis can be from multiple or different data sets that may all be aggregated together. The one or more signals or changes thereof in that data set can come from various sources. The methods and systems disclosed herein can be utilized for de novo identification of the one or more analytes. COMPUTER SYSTEMS [0968] The present disclosure provides computer systems that are programmed to implement methods of determining one or more characteristics of an analyte. FIG. 116 shows a computer system 1601 that is programmed or otherwise configured to determine one or more characteristics of an analyte. The computer system 1601 can regulate various aspects of detecting presence or absence of one or more characteristics of the analyte, such as, for example, determining the sequence of the analyte. The computer system 1601 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device. [0969] The computer system 1601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1601 also includes memory or memory location 1610 (e.g., random- access memory, read-only memory, flash memory), electronic storage unit 1615 (e.g., hard disk), communication interface 1620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1625, such as cache, other memory, data storage and/or electronic display adapters. The memory 1610, storage unit 1615, interface 1620 and peripheral devices 1625 are in communication with the CPU 1605 through a communication bus (solid lines), such as a motherboard. The storage unit 1615 can be a data storage unit (or data repository) for storing data. The computer system 1601 can be operatively coupled to a computer network (“network”) 630 with the aid of the communication interface 1620. The network 1630 WSGR Docket Number: 64828-710.601 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 1630 in some cases is a telecommunication and/or data network. The network 1630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1630, in some cases with the aid of the computer system 1601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1601 to behave as a client or a server. [0970] The CPU 1605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1610. The instructions can be directed to the CPU 1605, which can subsequently program or otherwise configure the CPU 1605 to implement methods of the present disclosure. Examples of operations performed by the CPU 1605 can include fetch, decode, execute, and writeback. [0971] The CPU 1605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC). [0972] The storage unit 1615 can store files, such as drivers, libraries and saved programs. The storage unit 1615 can store user data, e.g., user preferences and user programs. The computer system 1601 in some cases can include one or more additional data storage units that are external to the computer system 1601, such as located on a remote server that is in communication with the computer system 1601 through an intranet or the Internet. [0973] The computer system 1601 can communicate with one or more remote computer systems through the network 1630. For instance, the computer system 1601 can communicate with a remote computer system of a user (e.g., a personal computer). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1601 via the network 1630. [0974] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1601, such as, for example, on the memory 1610 or electronic storage unit 1615. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1605. In some cases, the code can be retrieved from the storage unit 1615 and stored on the memory 1610 for ready access by the processor 1605. In some situations, the electronic storage unit 1615 can be precluded, and machine-executable instructions are stored on memory 1610. WSGR Docket Number: 64828-710.601 [0975] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion. [0976] Aspects of the systems and methods provided herein, such as the computer system 1601, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution. [0977] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD- ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier WSGR Docket Number: 64828-710.601 wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution. [0978] The computer system 1601 can include or be in communication with an electronic display 1635 that comprises a user interface (UI) 1640 for providing, for example, the identification of the target nucleic acid sequence. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface. [0979] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1605. [0980] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein. [0981] Another aspect of the present disclosure provides a system comprising one or more computer processors and the computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein. COMPUTER PROGRAMS [0982] In some aspects, the present disclosure describes a computer program product comprising a computer- readable medium having computer-executable code encoded therein, the computer-executable code adapted to be executed to implement any one of the methods disclosed herein. In some embodiments, the platforms, systems, media, and methods disclosed herein include at least one computer program, or use of the same. [0983] A computer program includes a sequence of instructions, executable by one or more processor(s) of the computing device’s CPU, GPU, FPGA, or other processing units, written to perform a specified task. Computer readable instructions may be implemented as program modules, such as functions, objects, Application Programming Interfaces (APIs), computing data structures, and the like, that perform particular tasks or implement particular abstract data types. A computer program may be written in various versions of various languages. In some embodiments, APIs may comprise various languages, for example, languages in various releases of TensorFlow, Theano, Keras, PyTorch, or any combination thereof which may be implemented in various releases of Python, Python3, C, C#, C++, MatLab, R, Java, or any combination thereof. [0984] In some embodiments, a nanopore system described herein may comprise one or more computer programs. For example, a device described herein may comprise one or more computer programs on-chip when WSGR Docket Number: 64828-710.601 using nanopores imbedded on ASICS, on PCB interfacing the chip to the device, on the device, on recording/analysis computer, or in the Cloud, or any combination thereof. In some embodiments, one or more computer programs can perform signal processing on hardware to process one or more signals or changes thereof. [0985] In some embodiments the signal is recorded to media then analyzed at a later time. In other embodiments the signal is analyzed in real-time as it is streamed off an acquisition system [0986] The functionality of the computer readable instructions may be combined or distributed as desired in various environments. In some embodiments, a computer program comprises one sequence of instructions. In some embodiments, a computer program comprises a plurality of sequences of instructions. In some embodiments, a computer program can be provided from one location. In other embodiments, a computer program can be provided from a plurality of locations. In various embodiments, a computer program includes one or more software modules. In various embodiments, a computer program includes, in part or in whole, one or more web applications, one or more standalone applications, one or more web browser plug-ins, extensions, add-ins, or add-ons, or combinations thereof. SOFTWARE MODULES [0987] In some embodiments, the platforms, systems, media, and methods disclosed herein include software, server, and/or database modules, or use of the same. In view of the disclosure provided herein, software modules may be created by various techniques using machines, software, and languages. The software modules disclosed herein may be implemented in a multitude of ways. In various embodiments, a software module comprises a file, a section of code, a programming object, a programming structure, a distributed computing resource, a cloud computing resource, or combinations thereof. In further various embodiments, a software module comprises a plurality of files, a plurality of sections of code, a plurality of programming objects, a plurality of programming structures, a plurality of distributed computing resources, a plurality of cloud computing resources, or combinations thereof. In various embodiments, the one or more software modules comprise, by way of non-limiting examples, a web application, a mobile application, a standalone application, and a distributed or cloud computing application. One or more software modules may comprise firmware application for code stored on hardware (e.g., Field Programmable Gate Arrays (FPGAs)). The firmware application may be used for controlling and/or processing one or more signals. In some embodiments, software modules may be in one computer program or application. In other embodiments, software modules may be in more than one computer program or application. In some embodiments, software modules may be hosted on one machine. In some embodiments, software modules may be encoded to the hardware. In other embodiments, software modules may be hosted on more than one machine. In further embodiments, software modules may be hosted WSGR Docket Number: 64828-710.601 on a distributed computing platform such as a cloud computing platform. In some embodiments, software modules may be hosted on one or more machines in one location. In other embodiments, software modules may be hosted on one or more machines in more than one location. NON-TRANSITORY COMPUTER READABLE STORAGE MEDIUM [0988] In some aspects, the present disclosure describes a non-transitory computer-readable storage media encoded with a computer program including instructions executable by one or more processors to prepare a sample for analysis, process a sample to generate a signal from an analyte in the sample, analyze a signal to identify an analyte, store a signal in a database, or any combination thereof using any one of the methods disclosed herein. In some embodiments, a non-transitory computer-readable storage media may comprise instructions for preparing a sample for analysis, processing a sample to generate a signal from an analyte in the sample, analyzing a signal to identify and/or characterize an analyte, storing a signal in a database, or any combination thereof. In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more non-transitory computer readable storage media encoded with a program including instructions executable by the operating system of an optionally networked computing device. [0989] In further embodiments, a computer readable storage medium can be a tangible component of a computing device. In still further embodiments, a computer readable storage medium can be optionally removable from a computing device. In some embodiments, a computer readable storage medium includes, by way of non-limiting examples, random access memory, flash memory devices, solid state memory, magnetic disk drives, magnetic tape drives, optical disk drives, distributed computing systems including cloud computing systems and services, and the like. In some embodiments, the program and instructions may be permanently, substantially permanently, semi-permanently, or non-transitorily encoded on the media. DATABASES [0990] In some embodiments, the platforms, systems, media, and methods disclosed herein include one or more databases, or use of the same. Many databases are suitable for storage and retrieval of information about analytes, analyte translocation signals, associations between analytes and analyte translocation signals, program instructions for identifying analytes, device conditions, device operating instructions, or any combination thereof. In various embodiments, suitable databases include, by way of non-limiting examples, relational databases, non-relational databases, object oriented databases, object databases, entity-relationship model databases, associative databases, XML databases, document oriented databases, key-value databases, vector databases, and graph databases. Further non-limiting examples include SQL, SQLite, PostgreSQL, MySQL, WSGR Docket Number: 64828-710.601 Oracle, DB2, Sybase, and MongoDB. In some embodiments, a database can be Internet-based. In further embodiments, a database can be web-based. In some embodiments, a database can be cloud computing-based. In some embodiments, a database can be a distributed database. In other embodiments, a database can be based on one or more local computer storage devices. In other embodiments, the database may reside in temporary storage. In a particular embodiment, the temporary storage is random access memory. In some embodiments, the database and/or portions of the database may be generated dynamically. For example, a database may be generated and/or updated dynamically during the course of a measurement on a sample over a period of time. This period of time can be at least about 1 second, at least about 1 minute, at least about 1 hour, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, or greater than about 48 hours. For example, from the measurements may be on preceding analytes (e.g., calibration analytes and/or other analytes). In some embodiments, the database can be implicit in the weights of a machine learning model configured to act like a database. In some embodiments, the machine learning model can be a large language model. In other embodiments, the large language model can be a protein large language model. [0991] In some embodiments, one or more reference signals may be trained from one or more analytes. The detected signal or change thereof (e.g., an electrical signal or change thereof, a current or change thereof, or any combination thereof) of at least a portion of an analyte translocated through a pore may be measured against the one or more reference signals. One or more reference signals can comprise raw signal, denoised signal, segmented signal, or machine learning-generated signal, or any combination thereof. A difference between the detected signal or change thereof and the one or more reference signals may identify a modification (e.g., variant) of the at least a portion of the analyte. As an example, the modification may be one or more residue changes, one or more isoforms, one or more natural and/or unnatural post-translational modifications (PTMs), internal cross-links, or one or more conjugations to the analyte (e.g., drug conjugate, barcode, polynucleotide, leader construct, or any combination thereof), or any combinations thereof. An adjustment may be made to the one or more reference signals in the databases, or a new reference signal may be generated to use in future experiments. [0992] The database described herein may be generated from one or more reference sequences. The reference sequences may be derived from information of a sample. In some embodiments, one or more reference sequences may be derived from genomic information, transcriptomic information, or any combination thereof. The genomic information and/or transcriptomic information may be obtained from one or more other technologies (e.g., sequencing technologies). The one or more reference signals may be trained from a plurality of analytes. In some embodiments, data may be generated from one or more sequences using a machine learning algorithm (e.g., one or more machine learning algorithms). The genomic information may comprise genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of WSGR Docket Number: 64828-710.601 sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. The transcriptomic information may comprise genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. WEB APPLICATIONS [0993] In some embodiments, a computer program includes a web application. In some embodiments, a user may enter a query for saving, retrieving, and/or processing translocation signal data through a web application. In some embodiments, a user may save, retrieve, and/or process translocation signal data through a web application. A web application, in various embodiments, utilizes one or more software frameworks and one or more database systems. In some embodiments, a web application can be created upon a software framework such as Microsoft® .NET or Ruby on Rails (RoR). In some embodiments, a web application utilizes one or more database systems including, by way of non-limiting examples, relational, non-relational, object oriented, associative, XML, and document oriented database systems. In further embodiments, suitable relational database systems include, by way of non-limiting examples, Microsoft® SQL Server, mySQL™, and Oracle®. A web application, in various embodiments, can be written in one or more versions of one or more languages. A web application may be written in one or more markup languages, presentation definition languages, client- side scripting languages, server-side coding languages, database query languages, or combinations thereof. In some embodiments, a web application can be written to some extent in a markup language such as Hypertext Markup Language (HTML), Extensible Hypertext Markup Language (XHTML), or eXtensible Markup Language (XML). In some embodiments, a web application can be written to some extent in a presentation definition language such as Cascading Style Sheets (CSS). In some embodiments, a web application can be written to some extent in a client-side scripting language such as Asynchronous JavaScript and XML (AJAX), Flash® ActionScript, JavaScript, or Silverlight®. In some embodiments, a web application can be written to some extent in a server-side coding language such as Active Server Pages (ASP), ColdFusion®, Perl, Java™, JavaServer Pages (JSP), Hypertext Preprocessor (PHP), Python™, Ruby, Tcl, Smalltalk, WebDNA®, or Groovy. In some embodiments, a web application can be written to some extent in a database query language such as Structured Query Language (SQL). In some embodiments, a web application integrates enterprise server products such as IBM® Lotus Domino®. MOBILE APPLICATIONS WSGR Docket Number: 64828-710.601 [0994] In some embodiments, a computer program includes a mobile application provided to a mobile computing device. In some embodiments, the mobile application can be provided to a mobile computing device at the time it can be manufactured. In other embodiments, the mobile application can be provided to a mobile computing device via the computer network described herein. [0995] In view of the disclosure provided herein, a mobile application can be created by various techniques using hardware, languages, and development environments. Mobile applications can be written in several languages. Suitable programming languages include, by way of non-limiting examples, C, C++, C#, Objective- C, Java™, JavaScript, Pascal, Object Pascal, Python™, Ruby, VB.NET, WML, and XHTML/HTML with or without CSS, or combinations thereof. [0996] Suitable mobile application development environments may be available from several sources. Commercially available development environments include, by way of non-limiting examples, AirplaySDK, alcheMo, Appcelerator®, Celsius, Bedrock, Flash Lite, .NET Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other development environments may be available without cost including, by way of non- limiting examples, Lazarus, MobiFlex, MoSync, and Phonegap. Also, mobile device manufacturers distribute software developer kits including, by way of non-limiting examples, iPhone and iPad (Ios) SDK, Android™ SDK, BlackBerry® SDK, BREW SDK, Palm® OS SDK, Symbian SDK, webOS SDK, and Windows® Mobile SDK. STANDALONE APPLICATIONS [0997] In some embodiments, a computer program includes a standalone application, which can be a program that can be run as an independent computer process, not an add-on to an existing process, e.g., not a plug-in. Standalone applications can be compiled. A compiler can be a computer program(s) that transforms source code written in a programming language into binary object code such as assembly language, byte code, machine code, or any combination thereof. Suitable compiled programming languages include, by way of non-limiting examples, C, C++, Objective-C, COBOL, Delphi, Eiffel, Java™, Lisp, Python™, Visual Basic, and VB .NET, or combinations thereof. Compilation can be often performed, at least in part, to create an executable program. In some embodiments, a computer program includes one or more executable complied applications. EXAMPLES [0998] The following examples are provided to further illustrate some embodiments of the present disclosure, but are not intended to limit the scope of the disclosure; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used. WSGR Docket Number: 64828-710.601 Example 1. Dominant cis-to-trans electro-osmotic flow [0999] Examples 1.1 – 1.4 used the following methods and materials. [1000] Chemicals and reagents. Ampicillin sodium salt (Fisher Bio Reagents), chloramphenicol (≥98.0% (TLC), Sigma Life Science), LB medium (Roth), 2xYT medium (Roth), NaCl (≥99.5%, p.a., ACS, ISO, Roth), HEPES (PUFFERAN® CELLPURE® ≥99.5%, Roth), imidazole (≥99%, Roth), guanidinium chloride (≥99.5%, biochemistry, Roth), citric acid (≥99.6%, ACS reagent, anhydrous, Acros Organics), BIS-TRIS propane (BTP) (≥99.0%, Sigma Life Sciences), urea (≥99.5%, cryst., Roth), KCl (≥99.5%, p.a., ACS, ISO, Roth), isopropylthio-β-galactoside (IPTG) (≥99.0%, bioscience-grade, dioxin-free, animal-free, Roth), protease inhibitors (Pierce™ Protease inhibitor Mini tablets, EDTA-free, Thermo Scientific), Ni-NTA agarose (Qiagen), Strep Tactin® Sepharose® (IBA Lifesciences), D-desthiobiotin (IBA Lifesciences), mPEG-mal 5k (Laysan Bio, Inc.), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (≥98.0%, biochemistry, Roth), DPhPC (Avanti lipids), n-pentane (Sigma-Aldrich), n-hexadecane (99% pure, Acros organics). [1001] Cloning and production of the CytK mutants. Plasmids containing the mutant CytK nanopores were constructed by using USER cloning using uracil-containing primers (Integrated DNA Technologies). The mutation was included in the homology region and the upstream and downstream fragments constructed to be rejoined together with the empty vector in the USER reaction. The two gene fragments and the linearized pT7- sc1 (AmpR) were generated using in-house made PfuX7 DNA polymerase (Norholm paper), with the mention that the extension time was lowered to 30 s/kb. The PCR products were either gel extracted using a GeneJET gel extraction kit (Thermo scientific), or directly cleaned up from the PCR mix using the GeneJET PCR purification kit (Thermo scientific). The gene fragments and the linearized vector were mixed in a molar ratio F1:F2:V of 3:3:1 (recent USER paper) and the USER reaction was performed 25 min at 37 C, followed by a 60 C incubation step of 10 min and lastly, the mixture was cooled down to room temperature (22 or 20 C) for 15 min and subsequently stored on ice/ at 4 C until the transformation step. The circularized plasmids were transformed into chemically competent E. coli cells using the heat shock procedure and the cells were selected on a LB-agar plate supplemented with 100 μg/mL ampicillin. Plasmids from individual colonies were isolated using a GeneJET Plasmid Miniprep kit (Thermo scientific) and the introduction of the mutations was confirmed by Sanger sequencing (Macrogen). [1002] The plasmids encoding for the CytK mutants were electroporated into BL21(DE3) electrocompetent cells using a Bio Rad Micro Pulser (bacterial setting), and the cells were selected on plates containing 100 μg/mL ampicillin. Next day, several transformants were resuspended in LB medium supplemented with 100 μg/mL ampicillin and added to 200 mL LB medium containing 100 μ/mL ampicillin such that the starting optical density at 600 nm (OD600) was 0.05-0.1. Cells were grown at 37 C, 180 rpm until an OD600 of 0.6- 0.8, when the culture was chilled on ice for 5-10 min, followed protein expression being induced with 0.5 mM WSGR Docket Number: 64828-710.601 IPTG. After an incubation of 19-21 h at 25 C, 180 rpm, the cells were harvested (7500 rpm, 5 min). Following a 1 h incubation at -80 C, the cell pellets (100 mL culture) were resuspended in 20-25 mL ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM imidazole, pH 7.4 + 0.02% DDM) supplemented with 1/4 tablet cocktail protease inhibitors per 100 mL cell culture and subsequent steps were performed at 4 C degrees unless stated otherwise. The cell suspension was sonicated (Branson sonifier 450) at 25% duty cycle, 2.5 output control for 2-3 min and the cellular debris was removed (8000 rpm, 20 min). The supernatant was incubated (with shaking) for 20-30 min with 200 μL Ni2+-NTA slurry, pre-equilibrated and prewashed with 1 mL lysis buffer. The beads were briefly pelleted (3000 rpm, 1 min) and transferred to the column (1.2 mL bed volume bio-spin chromatography, BioRad) while allowing the flow through pass, at room temperature. The column was washed in steps with 10 mL wash buffer (50 mM HEPES, 150 mM NaCl, 30 mM imidazole, pH 7.4 + 0.02% DDM). The protein was eluted with 200 μL elution buffer (50 mM HEPES, 150 mM NaCl, 250 mM imidazole, pH 7.4 + 0.02% DDM)() in three elution fractions. The presence of the SDS-stable CytK mutant oligomers was confirmed by SDS-PAGE, omitting the heating step in the sample preparation. [1003] Preparation of the analytes. S1 substrate was as shown (Zhang et al., 2021, Nat Chem., 13(12):1192- 1199). The plasmid bearing tzatziki was prepared from a gblock (Integrated DNA Technologies) introduced in the linearized pT7-sc1 using USER cloning as previously described. The DNA for mujdei was generated by using tzatziki as template. Fusion PCR was used to obtain the full mujdei insert, which would be introduced into pT7-sc1 also by USER cloning. The malE219a, His6-malE219 and strep-malE219 substrates were prepared in a similar fashion. All substrates’ sequences were confirmed by Sanger sequencing (Macrogen). The substrates were expressed in the SG1146a strain (reference) was used, in order to limit protein degradation. The plasmids containing the S1 and malE219a DNA were transformed into chemically competent SG1146a cells and transformants were selected on ampicillin-containing plates. Next day, several colonies were resuspended in LB medium with ampicillin and added to the culture medium (LB supplemented with 100 μg/mL Amp and 25 μg/mL Chloramphenicol) such that the starting OD600 was 0.05-0.1. Cells were grown until OD6000.6-0.8, when the culture was chilled (5-10 min on ice) followed by induction with 0.5 mM IPTG at 25 C, 180 rpm for 18-22 h. The cells were harvested (7500 rpm, 5 min) and stored at -80 C for 1 h, followed by resuspension in 20 mL lysis buffer (50 mM HEPES, 150 mM NaCl, 10 mM imidazole, 6 M GuHCl, pH 7.4) at room temperature and all the subsequent steps were performed at room temperature The cell suspension was sonicated (25% duty cycle, 2.5 output control for 2-3 min). The cellular debris was removed (8000 rpm, 20 min) and the resulting supernatant was incubated with 200 μL Ni2+-NTA slurry per 100 mL culture with shaking for 30 min. The resin was transferred to the column (2 mL, Biorad) and washed with 10 mL wash buffer (50 mM HEPES, 150 mM NaCl, 30 mM imidazole, 1.5 M GuHCl, pH 7.4. Lastly, the protein was eluted four times in 100 μL elution buffer (50 mM HEPES, 150 mM NaCl, 250 mM imidazole, 1.5 M GuHCl, pH 7.4). The WSGR Docket Number: 64828-710.601 presence of the protein was confirmed by SDS-PAGE, followed by aliquoting of the elution fractions and storage at -20 C. [1004] The plasmids containing the tzatziki and mujdei plasmids were transformed into electrocompetent SG1146a cells, which were plated on Amp plates. Several transformants were resuspended in 2YT medium and diluted into 2YT medium supplemented with 100 μg/mL Ampicillin and 20 μg/mL Chloramphenicol to a final OD600 of 0.05-0.1. When OD600 reached 0.6, protein expression was induced with 0.5 mM IPTG at 25 C, 180 rpm for 18-20 h. The proteins were purified as described for S1 and malE219, although under native conditions (thus omitting the GuHCl in the buffers). The presence of the protein was confirmed by SDS-PAGE and using a PEG-maleimide reaction as an additional check. [1005] Electrophysiology measurements. Recordings in planar lipid bilayers were performed using a chamber consisting of two compartments, delimitated by a 25 μm thick TeflonTM membrane which an aperture of approximately 100 μm was sparked into. A droplet (half of a 10 μL capillary) comprising of n-hexadecane dissolved in n-pentane (6.25%) was applied on the TeflonTM membrane, followed by the addition of 500 μL buffer and two droplets of DPhPC lipids in n-pentane (5 mg/mL) in each compartment. Ag/AgCl electrodes were connected to each chamber via agarose bridges (2.5% agarose, 3M KCl solution), grounding the cis compartment. Measurements were performed using an Axon™ Digidata® 1550B digitizer and an Axopatch 200B amplifier (Molecular Devices) and recorded with the Clampex 11.1 software. [1006] Ion selectivity. Buffers: Buffer A 2 M KCl, 15 mM HEPES, pH 7.5, Buffer B 0.5 M KCl, 15 mM HEPES, pH 7.5, Buffer C 0 M KCl, 15 mM HEPES, pH 7.5, Buffer D 2 M KCl, 50 mM citric acid, BTP, pH 3.8, Buffer E 0.5 M KCl, 50 mM citric acid, BTP, pH 3.8, Buffer F 0 M KCl, 50 mM citric acid, BTP, pH 3.8 [1007] The ion selectivity of the CytK mutants was determined in buffers of either pH 7.5 or 3.8. Firstly, both the cis and trans compartments were filled with 2 M buffer (Buffer A/ Buffer D) and a single nanopore was isolated, followed by the pipet-offset adjustment of the current at 0 mV bias to 0 pA. The I/V curve was determined between -140 mV and +140 mV, in steps of 20 mV, using a 10 kHz sampling rate coupled with a 2 kHz Bessel filter. Next, the concentration of KCl in the trans compartment was lowered to approximately 0.5 M by flushing with 0 M buffer (Buffer C/ Buffer F) and repeated flushing with 0.5 M buffer (Buffer B/ Buffer E) ensured that the final concentration of KCl in the trans compartment was correctly fine-tuned to 0.5 M. Similarly, the I/V curve of the nanopore was measured. The reversal potential was determined from the second I/V curve from the linear function fitting the data points between -20 and +20 mV. The ion selectivity was established from triplicate experiments and expressed as the fraction ^^^/ ^^^^ was calculated using the formula below:
Figure imgf000286_0001
WSGR Docket Number: 64828-710.601 [1008] where [a] is the activity of the K+ or Cl- in the cis or trans compartment, Vr is the reversal potential, which is obtained from the experiments, F corresponds to the Faraday constant (96 485 C/mol), R the gas constant (8.3145 J mol-1 K-1) and T the temperature (298 K). [1009] Translocation experiments. The same setup as described above was employed, with the only difference being the KCl concentration in the buffer, namely 1 M. Single pores of the CytK mutants were isolated and the pore orientation was determined from the I/V curve. Provided the pore vestibule was located in cis, the model substrates were added in the cis compartment (1-2 μL S1 elution, or 5-7 μL tzatziki or mujdei elution sample) and translocation was induced by applying a negative bias. These recordings were collected at 50 kHz sampling rate and a Bessel filter of 10 kHz, using a sweep protocol where the first 200-500 ms were used to unclog the pore by applying a positive bias, followed by approximately 2 s of recording at negative bias. In the case of native substrates, urea was introduced into the system after determining the pore orientation and prior to substrate addition, by flushing both cis and trans compartments with 1 M KCl, 4 M urea, 15 mM HEPES, pH 7.5 buffer in 100 μL steps until the aimed concentration was reached (e.g.2 M urea). After addition of urea, the substrate (1-2 μL malE219 or 3 μL GBP H152A sample, pre-unfolded in 1.5 M GuHCl) was added in the cis compartment and translocation was followed like described for the other substrates. Each individual set of conditions was tested in triplicate. [1010] Data analysis. In the case of the translocation experiments, the files containing the recorded sweeps were analyzed using the Clampfit 11.1 software. Firstly, the open pore current (level 0, L0) and the corresponding noise, σ, was determined from the conventional histogram. Secondly, the detection limit, L1, was set at 10σ, which in practice is 5σ (Clampfit sets it half-way, at 5σ). Event detection with the set L0 and L1 was done on those approximately 2 s of recording at negative bias, with a dwell time cut-off of 0.08 ms. The resulting L1 data points were used to construct the log(dwell time) vs amplitude scatter plot, from which the amplitude boundaries of the cluster were defined. Next, using the amplitude boundaries, the logarithmic histogram of the dwell time and the conventional histogram of either the amplitude or the Iex% were constructed. In both cases, the bin value was set such that the distribution within the histogram would resemble as much as possible a Gaussian shape. The values for the log(dwell time) and either the amplitude or the Iex were established by fitting a Gaussian function to the histogram, whose μ is either log(dwell time) or the amplitude/Iex%. Example 1.1. Engineering the electro-osmotic flow in CytK nanopores [1011] This example interrogated whether an electro-osmotic flow (EOF) may be engineered to translocate and stretch polypeptides against an electrophoretic force (EPF), in which CytK nanopores were used (FIG.3). The nanopore was formed by a spherical vestibule ~5 nm in diameter connected to a ~5 nm long by 2 nm diameter cylindrical β-barrel region. The latter dominated the resistance of the nanopore, and the wild-type WSGR Docket Number: 64828-710.601 nanopore had no overall charge (the β-barrel region contains two pairs of opposite charge residues: K128-E139 and K155-E112). Consequently, the measured ion selectivity [(p(K)/p(Cl)] of WT-CytK was nearly one (0.99±0.079), which indicated that the WT pore may be non-selective and thus showed no electroosmotic flow. [1012] An EOF was induced in WT-CytK by lowering the pH to 3.8, which in turn increased the overall positive charge of the nanopore by protonation of the acidic residues (E112 and E139) making the nanopore anion selective [(p(K)/p(Cl)= 0.600±0.000]. Alternatively, an EOF at physiological pH was introduced by removing a positively charged residues near the cis [K128D-CytK, (p(K)/p(Cl) = 2.63±0.02] and trans (K128D- K155Q-CytK and K128D-K155D-CytK) entries of the β-barrel region. These modifications increased the ion selectivity to [(p(K)/p(Cl) = 2.96±0.12 and 3.10±0.08, respectively]. At pH 3.8, the nanopores remained cation selective, but only weakly [(p(K)/p(Cl) = 1.36±0.10 for K128D-K155D-CytK]. [1013] An additional negative charge was introduced at different positions within the β-barrel of K128D- K155D-CytK (2E-2D-CytK) to test how much the ion selectivity may be increased in CytK. It was observed that the greatest effect was obtained when the three rings of charges were distributed more evenly along the length of the beta barrel [i.e. the (p(K)/p(Cl) was 3.7 or 3.8 when the charges were placed at ~1.5 nm apart from the charges at the cis and trans nanopore entry, Table 1, FIG.3]. The addition of a fourth charge increased the ion selectivity further (Table 1). Overall, the pore with the highest ion selectivity was K128D-Q145D-S151D- K155D-CytK (2E-4D-CytK, Table 1), which showed a (p(K)/p(Cl) of 4.04±0.07. At pH 3.8 the effect of the additional aspartate residues was minimal (Table 1). An anion selective nanopore may also be made by replacing the negative charges for positive charges, resulting in the E112K-E139K-Q145K-S151K-CytK mutant (CytK-6K), with an anion selectivity of 0.207±0.008 (pH 7.5), which showed a (p(K)/p(Cl) of (0.213±0.012) at pH 3.8. Table 1. Ion selectivity of nanopores. pH 7.5 pH 3.8 Mutant R.P. avg R.P. Pk/PCl Pk/PCl R.P. avg R.P. Pk/PCl Pk/PCl stdev avg stdev stdev avg stdev 6K -19.3082 0.295947 0.20764 0.008135 -19.0541 0.430281 0.213452 0.012095 WT -0.14499 0.93088 0.991853 0.078977 -6.97445 0.008046 0.603839 0.000512 K128D 12.90689 0.110296 2.637693 0.02426 2.1993 1.201764 1.174445 0.097804 K128D K155Q 14.24263 0.452406 2.959448 0.118611 4.082796 0.168512 1.339853 0.016365 K128D K155D 14.77041 0.276684 3.097926 0.075129 4.333412 0.4065 1.364928 0.039817 K128D K155D T116D 15.80865 0.829636 3.414994 0.2565 6.874349 0.674812 1.645894 0.099883 K128D K155D S120D 16.73486 0.17531 3.710497 0.061888 4.402462 0.094033 1.37116 0.009331 K128D K155D Q122D 16.69164 0.331709 3.697167 0.144758 4.722214 0.275372 1.403646 0.03974 K128D K155D S126D 13.41838 0.276886 2.75487 0.079607 3.729563 0.070704 1.306001 0.008162 WSGR Docket Number: 64828-710.601 K128D K155D T143D 14.66842 0.115902 3.069036 0.038374 3.465806 0.2951 1.281696 0.033533 K128D K155D Q145D 15.69622 0.133073 3.36655 0.050095 3.905657 0.158716 1.32279 0.018525 K128D K155D T147D 16.50404 0.137101 3.629647 0.046791 3.700666 0.071011 1.303281 0.006666 K128D K155D S151D 17.17087 0.673547 3.880897 0.247497 4.346843 0.218138 1.365795 0.021517 K128D K155D T116D T147D 16.70064 0.180842 3.698435 0.077535 3.520646 0.075161 1.286474 0.008517 K128D K155D T116D S151D 16.10496 0.535072 3.501811 0.209061 5.44246 0.335793 1.479538 0.044699 K128D K155D S120D Q122D 17.26572 0.094966 3.905306 0.044557 5.022353 0.692365 1.436375 0.088142 K128D K155D S120D T147D 16.07314 0.08806 3.485588 0.035042 5.92812 0.24134 1.53296 0.033551 K128D K155D S120D S151D 17.2489 0.529757 3.906139 0.198708 4.18201 0.379784 1.349959 0.045391 K128D K155D Q145D S151D 17.60126 0.139616 4.036893 0.068788 4.313304 0.014851 1.362292 0.001796 Example 1.2. Translocation of model substrates across CytK nanopores [1014] Unfolded polypeptide translocation was initially tested with S1, a highly positively charged model peptide of 123 amino acid residues in length, which was designed to be unstructured and to carry large stretches of positive charges (net charge +28, or +23 net charge over 100 amino acids, +23100 at pH 7.5, FIG.4) with an amino acid composition and distribution not found in native proteins. The addition of S1 to the cis side of WT- CytK induced current blockades of two types, showing different dwell times and ionic current during the peptide block Ib. Blockades are indicated herein as the excluded current Iex% = (Io-Ib)/Io x 100, where Io is the open pore current. Type 1 blockades showed an Iex% that increased from 80.78±1.25 % to 91.93±0.22 % from -100 mV to -200 mV. The dwell time increased from 0.35±0.02 ms at -100 mV to 7.00±1.41 ms at -140 mV, and 6.74±2.88 ms at -160 mV, then decreased to 2.11±0.71 ms at -200 mV. Type 2 blockades showed shallower and shorter blockades in which Iex% decreased from 69.24±0.94 % at -120 mV to 62.92±0.06 % at -200 mV. As observed for type 1 blockades, the duration of type 2 blockades first increased (from 0.19±0.05 ms to 0.28±0.09 ms from -120 mV to -140 mV) and then decreased with the applied bias (0.15±0.02 ms at -200 mV). The voltage dependency of the blockades indicated that the electrophoretic force drove S1 translocation across a non-selective nanopore (WT-CytK) above a threshold potential of ~ -160 mV and -140 mV for type 1 and type 2 blockade events, respectively. [1015] Using the nanopores having an enhanced EOF, the threshold bias to obtain translocation was reduced (e.g. to ~-30 mV for 2E-2D-CytK), which indicated that the EOF can further drive the translocation of the polypeptide in combination with the net EPF acting in the same direction. WSGR Docket Number: 64828-710.601 [1016] Above the threshold potential, the dwell time was reduced by the additional EOF (FIG.10). [1017] A new model polypeptide 140 amino acid long was prepared named tzatziki, illustrated in FIG. 4. Tzatziki was designed to be unstructured and to carry a relatively large negative charge density of - 7.0100, so as to test a native-like substrate and establish whether the engineered EOF strength can be used to translocate unfolded polypeptides against an EPF. [1018] When Tzatziki was added to the cis sides, blockades were not observed with the WT-CytK nanopores at up to +200 mV, which indicated that the EPF was too weak to induce polypeptide translocation. In the case of 2E-2D-CytK, capture events were observed above -180 mV against the direction of the EPF that was acting to repel and prevent translocation, although substrate translocation was inconclusive. Tzatziki translocation events were observed in mutant pores with at least three rings of negative residues (cation selectivity >3.5, Table 1) at a bias above -120 mV (FIGs.9A-9H and FIGs.10A-10G). Translocation events were observed in direction of the EOF and against the average EPF force acting on the polypeptide. The dwell time of tzatziki decreased with the EOF of the nanopore (FIGs.9A-9H and FIGs. 10A-10G), further indicating that the EOF drove the translocation of the polypeptide. Hence, a highly negatively charged polypeptide can be translocated against the EPF. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. [1019] It was investigated whether a substrate with several consecutive negative charges could be translocated across 2E-4D-CytK nanopores. Mujdei, which was identical to tzatziki with the exception of a stretch of five negatively charged residues (EDEEE) in the middle region of the polypeptide (FIG. 4) was designed for this purpose. The overall net charge density of this substrate increased to -9.9100. The 2E-4D-CytK nanopore captured and translocated mujdei, showing a slight increase in dwell time compared to tzatziki. The difference in the dwell time between the two substrates decreased with the potential (from 45% at -100 mV to 9% at -140 mV), which suggested that at high bias the electrophoretic effect on the polypeptide can be almost negligible compared to the EOF. [1020] FIGs. 5-8 depict the translocation of analytes S1, tzatziki, and mujdei through the 2E-4D-CytK nanopore. Experiments were performed at pH 7.5 and under 1 M KCl. Traces were collected at 50 KHz sampling rates and filtered at 10 KHz using a Bessel filter. FIGs. 5A, 6A, 7A, and 8A show schematic representations of the pore and analyte. Dots on the analyte of FIGs.5A, 6A, 7A, and 8A correspond to charged residues as denoted in FIG. 4. FIG. 5B shows a voltage dependency of translocation rates for type 1 and type 2 blockades. FIGs.6B, 7B, and 8B depict voltage dependency of translocation rates for S1, tzatziki, and mujdei, respectively. FIGs.5C, 6C, 7C, and 8C illustrate the voltage dependency of the excluded current (Iex%). FIGs. 5D, 6D, 7D, and 8D show representative traces at –160 mV bias. FIGs.5E, 6E, 7E, and 8E show dwell time versus current amplitude at –160 mV bias. Increasing current amplitude resulted in increased dwell times. WSGR Docket Number: 64828-710.601 Example 1.3. Stretching the polypeptides inside the nanopore [1021] In nanopore protein sequencing it may be advantageous for polymer to be linearized during translocation and for there to be enough current associated with the polypeptide blockade to identify individual amino acids. The Iex% may be dominated by the excluded volume of the polymer inside the nanopore. Therefore, if the polypeptide translocates as a linear polypeptide, the Iex% may be expected to be low, while if the polypeptide is folded inside the nanopore the Iex% may be expected to be high. However, the Iex% might also be influenced by the charges of the analyte and the nanopore, which might create additional energy barriers for the translocation of ions from solution. For example, in a nanopore with a highly positively charged lumen the translocation of a polymer with high negative charge density such as DNA may increase the Iex% to almost hundred percent, possibly because the transport of anions is blocked by the charge in the DNA and that of cations by the charge of the nanopore. [1022] For most substrates, a reduced Iex% was observed with the increasing of the applied voltage, which suggested that the polypeptide may be stretched as the electroosmotic pulling force increased. A notable exception was the translocation of S1 through 2E-2D-CytK nanopores. When comparing the two protein-like substrates the Iex% of mujdei was ~1% higher than in tzatziki for all nanopores, which suggested that the increased EPF opposing translocation on the more negatively charged mujdei may have slightly reduced the stretching of the substrate inside the nanopore. EXAMPLE 1.4. Non-enzymatic translocation of native substrates across the 2E-4D mutant [1023] S1, tzatziki and mujdei were designed to contain only disorder-promoting hydrophilic amino acids to minimize the folding of the polymer. Two proteins were chosen to test the non-enzymatic translocation of native proteins. A maltose-binding-protein variant, malE219a (containing the G220D and E221P destabilizing mutations and a total of 412 amino acids, charge density of -2.3100 at pH 7.5, FIG. 11), and a glucose binding protein H152A-GBP (bearing a destabilizing mutation H152A, 341 amino acids, charge density -1.1100 at pH 7.5, FIG.12). As previously reported, malE219 fully unfolds in 0.7 M GuHCl, while H152A-GBP is unfolded in 1 M GuHCl. [1024] The presence of urea (a neutral denaturant) decreased the open pore current of 2E-4D-CytK while retaining the current asymmetry under opposite bias (FIGs. 14A and 14C), which suggested that it may not have changed the ionic properties of the nanopore. In the presence of GuHCl the current symmetry was lost and even reverted at higher concentrations (FIGs. 14B and 14D), which suggested that, as previously shown by MD simulations, the GuH+ may bind to the negative charges within the lumen of the nanopore reducing the ion selectivity. [1025] When adding (pre-unfolded) malE219a substrate to the cis side of 2E-4D-CytK in the presence of 2 M urea, translocation events were observed, which were different from the translocation of the model substrates WSGR Docket Number: 64828-710.601 (FIG. 11). The threshold translocation of MalE219a was ~-60 mV compared to ~-120 mV for tzatziki. MalE219a had a lower negative charge density than tzatziki (-2.3100 vs -7.0100). The dwell times were about two-fold longer compared to the dwell time of the model substrates adjusted for their length, possibly reflecting a stronger interaction between the hydrophobic amino acids and the lumen of the nanopore. During the blockade, a larger residual current was observed during the translocation of the protein (Iex% 70.5±0.57% at - 120 mV vs 88.79%±2.03 (tzatziki) or 90.05±0.50 (mujdei) at - 160 mV, FIGs. 9A-9H and FIGs. 10A-10G), which suggested a more stretched polypeptide. Furthermore, the current signature showed patterns, which may be related to the sequence and/or structure of the translocating proteins. [1026] Pre-unfolded H152A-GBP (added to the cis side) also translocated through 2E-4D-CytK nanopores in the presence of urea (2.44 M), although the threshold for translocation was higher than for malE219a (-80 mV, FIGs.11A-11F and 12A-12F) despite having a lower charge density (-1.1100 vs -2.3100). The Iex% showed two levels: level 1 defined by an Iex% of 70.62±1.08% (at -120 mV), which was similar to the Iex% measured for malE219a, and Level 2 with an Iex% of 89.11±3.50% (at -120 mV, FIGs. 9A-9H and FIGs. 10A-10G), which was similar to the model substrates. This behavior may be explained by the two-step unfolding of H152A- GBP, as observed for thioredoxin, or by the translocation of a partially folded structure. H152A-GBP translocation threshold was similar to that of malE219a (FIGs.11A-11F and 12A-12F). [1027] In some cases, unfolded model proteins might be translocated across β-barrel nanopores, such as wild type α-hemolysin, which is homologous in structure to CytK, in the presence of GuHCl. A 10-residue aspartate tag at the N- or C- termini was added to drive the transport across nanopores in this case. The addition of a D10 tag at the C-terminus (malE219a-D10) allowed malE219a translocation at positive applied potentials (FIG.15). This indicated that the EOF induced by GuHCl alone may not drive polypeptide translocation, and an additional EPF force on the D10 tag needed to be applied. By contrast, the urea-promoted translocation of substrates across 2E-4D-CytK nanopores was only induced by the engineered EOF, which overcame the opposing EPF exerted on the negatively charged native proteins during translocation. [1028] When GuHCl (1 M or 1.8 M) was used with 2E-4D-CytK, translocation events were observed at negative bias but with a reduced frequency and the threshold potential shifted towards higher values with more GuHCl. Hence, the EOF induced by the nanopore was reduced due to the binding of GuH+ ions to the nanopore, but still allowed polypeptide translocation against the EPF. In addition, the Iex% was substantially lower in 2 M urea (70.5±0.57% at -120 mV) compared to 1.8 M GuHCl (88.75±0.31%, at -120 mV) and the dwell time was longer (3.28±0.45 ms vs 1.90±1.02 ms, FIG.10). A possible explanation is that the polypeptides in the presence of urea may be more stretched (higher Iex%) and weaker interactions can occur between the GuH+-coated nanopore and the negative substrates (faster translocation in urea compared to GuHCl). WSGR Docket Number: 64828-710.601 [1029] As shown in FIGs.13A-13E, malE219a showed translocation through the 2E-2D CytK mutant in the presence of 1 M and 1.8 M GuHCl. In FIG.13D, in 1 M GuHCl, increasing amplitude resulted in increased dwell time for malE219a. Voltages were -80 mV (1301), -100 mV (1302), -120 mV (1303), -140 mV (1304), and -160 mV (1305). In FIG.13E, in 1.8 M GuHCl, increasing amplitude resulted in increased dwell time for malE219a. Voltages were -120 mV (1306), -140 mV (1307), -160 mV (1308), and -180 mV (1309). Table. 2. Sequences of Protein Analytes and CytK monomers Description Sequence SEQ ID NO. malE219a MGSSHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEH 1 PDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRY NGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNK DPTAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGLYFQSH malE219 MGSSHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEH 2 PDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRY NGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNK DPTAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQT tzatziki MGCHHHHHHGSSNNQNNDNNNNNEDQQNQQKSSSSSENNNNNKDSSSSSDQQQQQRNNNN 3 NESSSSSDSSSSSKQQNQQESSSSSDNNNNNKQQQQQEENNNNNRSSSSSEQQSQQDDSS SSSRNNSNNAANDENYALAA mujdei MGCHHHHHHGSSNNQNNDNNNNNEDQQNQQKSSSSSENNNNNKDSSSSSDQQQQQRNNNN 4 NESSSSSDSSSSSKQEDEEESSSSSDNNNNNKQQQQQEENNNNNRSSSSSEQQSQQDDSS SSSRNNSNNAANDENYALAA S1 MGHHHHHHSSRRRRRRRRRRSSSSSSSSSSSSSSSFGYGWSSSSSSSSSSSSSSSRRRRR 5 RRRRRSSSSSSSSSSSSSSSFGYGWSSSSSSSSSSSSSSSRRRRRRRRRRSSAANDENYA LAA GBP- MANKKVITLSAVMASMLFGAAAHAADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQL 6 H152A LMNDSQNDQSKQNDQIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSR KALDSYDKAYYVGTDSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGAPDAE ARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAM GAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGK GAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKKGSSHHHHH 6K CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 7 MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEKTTVTSS VSYQLGGSIKASVTPSGPSGKSGATGKVTWSDKVSYKQTSYKTNLIDQTNKHVKWNVFFN GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH WT CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 8 MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS VSYQLGGSIKASVTPSGPSGESGATGQVTWSDSVSYKQTSYKTNLIDQTNKHVKWNVFFN GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH WSGR Docket Number: 64828-710.601 K128D MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 9 CytK MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS VSYQLGGSIDASVTPSGPSGESGATGQVTWSDSVSYKQTSYKTNLIDQTNKHVKWNVFFN GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 10 K155Q MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS CytK VSYQLGGSIDASVTPSGPSGESGATGQVTWSDSVSYQQTSYKTNLIDQTNKHVKWNVFFN GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 11 K155D MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS CytK VSYQLGGSIDASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VFFNGYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSP GMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 12 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVDSS T116D VSYQLGGSIDASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 13 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS S120D VDYQLGGSIDASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 14 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS Q122D VSYDLGGSIDASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 15 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS S126D VSYQLGGDIDASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 16 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS T143D VSYQLGGSIDASVTPSGPSGESGADGQVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 17 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS Q145D VSYQLGGSIDASVTPSGPSGESGATGDVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN WSGR Docket Number: 64828-710.601 KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 18 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS T147D VSYQLGGSIDASVTPSGPSGESGATGQVDWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 19 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS S151D VSYQLGGSIDASVTPSGPSGESGATGQVTWSDDVSYDQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 20 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVDSS T116D/ VSYQLGGSIDASVTPSGPSGESGATGQVDWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN T147D GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA CytK VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 21 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVDSS T116D/ VSYQLGGSIDASVTPSGPSGESGATGQVTWSDDVSYDQTSYKTNLIDQTNKHVKWNVFFN S151D GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA CytK VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 22 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS S120D/ VDYDLGGSIDASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN Q122D GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA CytK VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 23 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS S120D/ VDYQLGGSIDASVTPSGPSGESGATGQVDWSDSVSYDQTSYKTNLIDQTNKHVKWNVFFN T147D GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA CytK VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 24 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS S120D/ VDYQLGGSIDASVTPSGPSGESGATGQVTWSDDVSYDQTSYKTNLIDQTNKHVKWNVFFN S151D GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA CytK VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 25 K155D/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS Q145D/ VSYQLGGSIDASVTPSGPSGESGATGDVTWSDDVSYDQTSYKTNLIDQTNKHVKWNVFFN S151D GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA CytK VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH K128D/ MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSF 26 K155Q/ MKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSS Q122D VSYDLGGSIDASVTPSGPSGESGATGQVTWSDSVSYQQTSYKTNLIDQTNKHVKWNVFFN CytK GYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNSGFSPGMIA VVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKN KKLVEKKGSAHHHHHH WSGR Docket Number: 64828-710.601 Example 2. Pores with Aromatic Groups in the Lumen [1030] Examples 2.1-2.9 used the following chemicals and reagents. [1031] Chemicals. Sphingomyelin (Porcine brain, >99 %, CAS# 383907-91-3) and diphytanoyl-sn-glycero- 3-phosphocholine (DPhPC, >99 %, CAS# 207131-40-6) were retrieved from Avanti Polar Lipids. Ni-NTA resin was obtained from Qiagen. Lysozyme (Albumin free for tryptic digest, CAS# 12650-88-3), Glucose (>99 %, CAS# 50-99-7), Sodium chloride (>99.5 %, CAS# 7647-14-5),Potassium chloride (>99 %, CAS# 7447-40- 7), Dithiothreitol (DTT, >99.0 %, 3483-12-3), Trizma® HC1 (>99 %, CAS# 1185-53-1), Trizma® base (>99.9 %, CAS# 77-86-1), Imidazole (>99 %, CAS# 288-32-4), n-Dodecyl β-D-maltoside (DDM, >99 %, CAS# 69227-93-6), Hydrochloric acid (1 M, CAS# 7647-01-0), Urea (>99.5 %, CAS# 57-13-6), Magnesium chloride (>98.5 %, CAS# 7786-30-3), LB Broth (Luria/Miller), Agar-agar and 2x YT Broth were obtained from Carl Roth. Ampicillin sodium salt (CAS# 69-52-3), Isopropyl β-D-l- thiogalactopyranoside (IPTG, >99 %, CAS# 367-93-1), Ethanol (>99.8 %, CAS# 64-17-5) and all enzymes were received from Fisher Scientific. Lysozyme from chicken egg white (for Lysis, CAS# 12650-88-3), N,N- Dimethyldodecylamine N-oxide (LDAO, >99.0 %, CAS# 1643-20-5), Pentane (>99 %, CAS# 109-66-0), Iodoacetamide (IAA, >99 %, CAS# 144-48-9), Bis- tris propane (>99.0 %, CAS# 64431-96-5) were bought from Sigma-Aldrich. n-Hexadecane (99 %, CAS# 544- 76-3) and Citric acid (99.6 %, CAS# 77-92-9) were purchased from Acros. Trypsin (bovine pancreas, CAS# 9002-07-7) was obtained from Alfa Aesar. [1032] Examples 2.1-2.9 used the following methods. [1033] Fragaceatoxin C (FraC) monomer expression and purification. pT7- SC1 vector containing His6- tagged FraC plasmids were electrochemically inserted into E. coli BL21 (DE3) cells and grown overnight at 37 °C on LB agar plates supplemented with 100 mg/1 ampicillin and 1% glucose. Colonies were used to inoculate 200 ml 2xYT medium supplemented with 100 mg/1 ampicillin and grown at 37 °C until the optical density at 600 nm (OD600) reached 0.6, after which expression was induced using 0.5 mM isopropyl β-D-l- thiogalactopyranoside (IPTG), allowing continued growth overnight at 21 °C. Cell pellets were collected by centrifugation (6,000g, 20 min, 4 °C) and stored at -80 °C for at least one hour. The pellets were resuspended in 10 ml lysis buffer per 50 ml culture, with a lysis buffer consisting of 150 mM NaCl, 15 mM Tris base solution at pH 7.5 supplemented with 1 mM MgCl2, 2 M Urea, 20 mM imidazole, 0.2 mg/ml lysozyme and 0.2 units/ml DNase. [1034] The solution was mixed for 1 hour at room temperature (21°C) using a rotating mixer at 15 RPM. The cells were fully disrupted by sonification, applying 30 sweeps (duty cycle 30%, output control 3) three times using a Branson Sonifier 450. The lysate was centrifuged at 6000g for 20 minutes at 4 °C. The supernatant was incubated for 1 hour, while under constant rotation (15 RPM), with 100 μL resuspended Ni-NTA resin WSGR Docket Number: 64828-710.601 (resuspended in 150 mM NaCl, 15 mM Tris base at pH 7.5 supplemented with 20 mM imidazole). The solution was loaded onto a prewashed Micro Bio-Spin column (Bio-Rad). The Ni-NTA beads were extensively washed with 20 ml WB (150 mM NaCl, 15 mM Tris base at pH 7.5 supplemented with 20 mM imidazole). The column was inserted into a microtube and spin-dried using a centrifuge (13,300g, 1 min) in order to remove residual wash buffer.150 μl of 150 mM NaCl, 15 mM Tris base solution at pH 7.5 supplemented with 300 mM imidazole (EB) was added and left to incubate for 5 minutes before elution. This step was repeated four times to retrieve four fractions containing FraC monomers. The presence and purity of FraC monomers was estimated using SDS-PAGE. Pure fractions were pooled and stored at 4 °C. The concentration of FraC monomers was estimated using a Nano Drop 2000 UV-Vis Spectrophotometer (Thermo Scientific) using the elution buffer as blank. [1035] Sphingomyelin-DPhPC liposomes preparation. 25 mg sphingomyelin (Brain, Porcine) was mixed with 25 mg 1,2-diphytanoyl-sn-glycero-3- phosphocholine (DPhPC) and dissolved in 4 ml pentane containing 0.5% v/v ethanol. The lipid mixture was evaporated while turning inside a round bottom flask by application of a hot air stream to create a thin lipid film over the surface of the flask. The film was reconstituted into 10 ml of Sdex buffer (150 mM NaCl, 15 mM tris, pH 7.5) using a sonication bath. The liposome solution (5 mg/ml) was frozen and stored at -20 °C. [1036] Fragaceatoxin C oligomerisation. Liposomes were thawed and added to FraC monomers in a lipid to protein mass ratio of 10:1. The mixture was incubated for 30 minutes at 37 °C, after which N,N- Dimethyldodecylamine N-oxide (LDAO) was added to a final concentration of 0.6 v/v% to dissolve the liposomes. The solution was diluted 10-fold in 150 mM NaCl supplemented with 15 mM Tris (pH 7.5) and 0.02 v/v% n-Dodecyl β-D- maltoside (DDM). The diluted solution was combined with 100 μl of Ni-NTA, prewashed using WB2 (150 mM NaCl, 15 mM Tris base, pH 7.5 supplemented with 20 mM imidazole and 0.02 v/v% DDM). The mixture was left to incubate for 30 minutes while mixing under constant rotation (15 RPM). The solution was loaded onto a Micro Bio-Spin column (Bio-Rad), prewashed with 500 μl WB2. The Ni-NTA beads were washed extensively using 10 ml WB2. The column was spin-dried in a microtube using a centrifuge (13,300g, 1 min) to remove residual wash buffer.150 μl elution buffer was added onto the column (150 mM NaCl, 15 mM Tris base supplemented with 1M imidazole and 0.02 v/v% DDM) and left to stand for 10 minutes before elution into a clean microtube by centrifugation (13,300g, 2 min). [1037] Construction of Fragaceatoxin C mutants. Fragaceatoxin C mutant DNA was prepared using the MEGAWHOP method6. The megaprimer was constructed using a forward primer synthesized by Integrated DNA Technologies and a T7 reverse primer (5’-GCTAGTTATTGCTCAGCGG-3’). Six reactions were performed per mutation—in order to receive enough DNA for the second PCR—using 25 μl REDTag® Ready Mix™ PCR Reaction Mix (Sigma-Aldrich) combined with 22 μl PCR grade water (Sigma-Aldrich), 1 μl of WSGR Docket Number: 64828-710.601 each forward and reverse primer and 1 μl His6-tagged Fragaceatoxin C template DNA. The PCR protocol comprised a 90 second denaturation step at 95 °C followed by 30 cycles of denaturation at 95 °C (15 seconds), annealing at 55 °C (15 seconds) and extension at 72 °C (120 seconds). The six PCR reactions were combined and purified using a GeneJET PCR Purification Kit (Thermo Scientific). For the second PCR, 10 μ15x Phire Buffer (Thermo Scientific) was combined with 1 μ1 template DNA, 1 μ1 dNTPs (10 mM), 2 μ1 megaprimer (first PCR), 35 pl PCR grade water (Sigma-Aldrich) and 1 μ1 Phire II Hot Start DNA Polymerase (Thermo Scientific). The PCR protocol comprised an initial pre-denaturing step of 98 °C (30 seconds) followed by 25 cycles of denaturation at 98 °C (5 seconds) and extension at 72 °C (90 seconds). 5.7 μ15x FD green buffer (Thermo Scientific) and 1 μ1 Dpn1 enzyme (Thermo Scientific) was added to the PCR mix and let to digest at 37 °C for 1-3 hours.0.5 μ1 of the digested product was electrochemically transformed into 50 μ1 E. cloni 10G® (Lucigen) competent cells and grown on LB agar plates containing 100 mg/1 ampicillin and 1% glucose. Single colonies were enriched using a GeneJET Plasmid Miniprep Kit (Thermo Scientific) and the sequence was confirmed using the sequencing service of Macrogen Europe. [1038] The amino acid sequence of His6-tagged wild type Fragaceatoxin C is set forth as: MASADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIVLPHK VAHGKALLYNGQKNRGPVATGWGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRA DQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKAGSAHHHHHH (SEQ ID NO: 27). [1039] Unspecific lysozyme digestion. Lysozyme (Carl Roth, from chicken egg white, free from albumin) was dissolved in 8 M urea supplemented with 15 mM Tris (pH 9.5) to a final concentration of 20 mg/ml and left to denature at 95 °C for 5 minutes.200 μ1 denatured lysozyme solution was incubated for 30 minutes at 37 °C with 20 mM dithiothreitol (DTT), to reduce the cysteine residues. lodoacetamide (IAA) was added to the mixture, to react with reduced cysteines, with a final concentration of 45 mM and incubated in the dark for 30 minutes at room temperature. The mixture was diluted 5x with 100 mM Tris (pH 8.5) and trypsin (Alfa Aesar™ Trypsin, bovine pancreas) was added in a ratio of 1:50 (trypsin:protein). The mixture was left to digest overnight (~18 hours) at 37 °C. In order to denature and deactivate any remaining trypsin, the next day, the final mix was denatured at 95 °C for 10 minutes and HC1 was added to lower the pH (approximately pH 4). The mixture was then frozen at -20 °C until use. [1040] Planar lipid bilayer electrophysiological recordings. The electrophysiology chamber consisted of two compartments separated by a 25 μm thick Teflon (Goodfellow Cambridge Ltd) membrane. The Teflon membrane contained an aperture with a diameter of approximately 100-200 μm. Lipid membranes were formed by first applying 5 μl of 5% hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane, near the aperture. The pentane was left to dry and 400 μ1 of buffer (1 M KC1, 50 mM citric acid, titrated with WSGR Docket Number: 64828-710.601 bis-tris propane to pH 3.8) was added to both sides. 20 μ1 of a 6.25 mg/ml solution of DPhPC dissolved in pentane was added on top of the buffer on each side of the chamber. The chamber was left to dry for ~2 minutes to allow evaporation of pentane. Silver/silver chloride electrodes were attached to each compartment. The cis compartment was connected to the ground electrode and the trans was connected to the working electrode. Planar lipid bilayers were created using a Langmuir-Blodgett method. The orientation of FraC nanopores was determined by the asymmetry of the current-voltage relationship. A baseline of 2 minutes was recorded for each of the pores recorded. Analytes were added to the cis compartment of the chamber. [1041] Data recording. Recordings of ionic currents were obtained using an Axopatch 200B (Axon Instruments) combined with a Digidata 1550B A/D converter (Axon instruments). The sampling frequency was set at 50 kHz for analyte recordings, the analogue Bessel filter was set at 10 kHz. Data was recorded using Clampex 10 (Molecular Devices). [1042] Standard Data analysis and event detection. Various methods were employed when analyzing the stepwise current blockades measured from nanopore electrophysiology on the event types observed to extract useful data, which included but were not limited to blockade magnitude, blockade duration, blockade shape, blockade noise, other sub-features of the blockades (such as ministeps, or any combination thereof). [1043] A custom Python script was employed to analyze the raw electrical data. The open pore current (Io) and standard deviation of all traces was determined by calculating the mean current of 3 independent measurements, bootstrapped for 100 iterations of 10 second snippets for each measurement. For event detection, the baseline current and standard error of the recorded traces were determined from a full current histogram of the blank nanopore measurement containing no analytes. The value for the baseline was then used to determine the events when analyte was added. All data points above the baseline current and standard error that were separated by at least two times the sampling periods were detected as events. The excluded current (Iex%) of each event was calculated as the difference between the open-pore current Io and the blockade current Ib, over the open-pore current Io (Iex% = [Io-Ib]/Io). [1044] Impartial event detection. An impartial event detector method was employed to improve analyses. Some short lived events—with a dwell time near the sampling frequency—formed a spike or Gaussian profile under sampling and filtering effects, while some longer events followed a flat-top shape. A parameter describing the shape of current blockades was introduced in order to impartially compare the performance of mutant pores. It was assumed that the profile of ionic current blockades can be described by a generalized flat- top normal distribution function (gNDF). Each observed block was fit to the following equation using least- squares fitting, due to the non-polynomial nature of the function. WSGR Docket Number: 64828-710.601 [1045] Where μ is the events centre in the time domain with variance σ 2 and ΔIB is the current difference (pA) between the baseline (Io) and the event maximum. The variable β describes the shape of the function and can take any real number larger than zero. If β is less than one but larger than zero, the shape of the function is a spike. If β is equal to one, the function is equal to the normal distribution function. When β is larger than one, the function starts to follow a rectangular—flat-top—profile. The variable β can also be used to assess the quality of individual events in the following way. Events with a β < 1 may be mostly events that may be too short-lived to accurately measure the ionic current blockade. Those events with a β > 1 can be regarded as more accurate measurements of peptides. Events with a β > 10 were distinguished, since these events—having a flat- top shape— can permit an more accurate estimation of the blocked current. The gNDF fit also permits an estimation of the dwell time of an event by taking the full width at half maximum (FWHM) of the gNDF. Estimation of the dwell time using this equation can be advantageous, because it can allow the treatment of this parameters as continuous rather than discrete, which may be the case if the number of data points are counted within the event.
Figure imgf000300_0001
[1046] Where σ equals the square root of the variance (σ 2) and β describes the shape parameter. [1047] Spectral matching. Several of the residual current spectra obtained were expected to contain random events induced by factors other than the analyte (gating), so in order to reduce baseline sloping and to maintain high sensitivity, the squared first derivative Euclidean cosine correlation was utilized. This comparison is sensitive to the position of the peaks observed in the spectra, but not as sensitive to a shifting baseline.
Figure imgf000300_0002
[1048] Where Ai and A2 equal the vectors of excluded current counts and A1,i and A2,i represent the individual bins of the excluded current spectrum. In a more detailed description, A1 and A2 are set as the vector of counts observed for each residual current bin (e.g. An= counts (40-41%), counts(41- 42%), ..., counts (94-95%)). ΔAn, is the derivative of An (difference between bins). In the numerator, each element ΔAn is multiplied with the corresponding ΔAn of the comparing spectrum and take the squared sum of all items. In the denominator, the squared sum of each element in AAn is multiplied with that with the squared sum of each element in the spectrum to compare. So, if the two vectors A1 and A2 are equal, the correlation is 1, else it is less than 1, and because the derivative of A1 and A2 is taken, linear baseline sloping is less impactful. [1049] Hierarchal clustering was performed using the Ward distance as implemented in SciPy version 1.4.1. on the resulting correlation coefficients to determine which spectra were most similar. Example 2.1. Fragaceatoxin C mutant screening. WSGR Docket Number: 64828-710.601 [1050] Mutations of FraC. The sequence of WtFraC from the sea anemone Actinia fragacea was aligned with other actinoporins (FIG.16A) to identify sequence homology. A number of generally non-conserved positions were identified that would be more amenable to mutation, including D10, G13, G15, D17 and K20 (FIG.16B). These positions were engineered into different mutations to improve the ability of the pores to detect and discriminate different peptides. [1051] At position D10, mutations to arginine (R) and Glycine (G) were introduced to test changes in electro- osmotic capture of analytes. Each of the positions near the recognition site (G13), was modified to a basic residue (K, R or H) or acidic residue (D or E) as well as amino acids with neutral (G or Q) or aromatic (W,Y and F) groups. In FraC—a glycine residue is positioned at residue 15—while the most common amino acid in other actinoporins is a threonine. Mutation G15T was introduced to test whether increased hydrophobic mutations facing outwards into the membrane would stabilize and improve the behavior of FraC pores. [1052] Two mutants that have the same characteristics, T21D and the double-mutant K20D IT21K, were constructed based on sequence alignment (FIG. 16A) that showed a pair of opposite charges commonly at positions 20/21. A change of charge on position 20 by introducing a glutamic acid (K20D) was also tested. [1053] Oligomeric forms. Of the oligomeric forms of WtFraC, three forms were tested: octameric pores (or type I pores, T1), heptameric pores (or type II pores, T2), and hexameric pores (or type III pores, T3). [1054] Octameric oligomers were identified as the nanopores with the highest conductance. Several mutations significantly reduced the open pore current (I0) relative to WtFraC-Tl (95 ± 1 pA), some to an extent that the I0 resembled WtFraC-T2 (47 ± 3 pA). Decreased I0 were observed when residues with a larger volume were introduced, particularly for the aromatic residues (W/F/Y) introduced on position 13 (I0 = 64 ± 8 pA, 77 ± 4 pA and 82 ± 3 pA, respectively), which suggested that smaller recognition regions may be achieved. The introduction of a threonine residue on position 15 increased the open-pore current I0 flowing through the pore (100 ± 3 pA).The increased current was generally more sensitive to changes due to analyte binding. [1055] Peptide mixtures. In order to ensure a fair comparison between pores, a mixture of peptides was generated from the non-specific tryptic digestion of lysozyme (Gallus-Gallus) using trypsin or other proteases such as chymotrypsin or Lys-C protease. Trypsin cleaved preferentially after a K/R amino acid. All pores were tested with the same proteolytic mixture. [1056] Blockade event analysis. Events arising from nanopore current blockades were analyzed with a flat- top shape fitted using a least-squared Levenberg-Marquardt method and a generalized flat-top normal distribution function. The fit resulted in a β value that may classify the events as either a spike with β < 1, a normal distribution β = 1 or flat-top distribution β > 1. All events with β > 1 were used in subsequent analyses. For each blockade a number of characteristic metrics were extracted. These include but were not limited to the excluded current (Iex%), which is the percentage of the current that is blocked during a translocation event WSGR Docket Number: 64828-710.601 relative to the open pore current (Iex% =[Io-Ib]/Io)), the duration (termed dwell time) of the blockade, the shape of the blockade, and the noise in the blockade current. [1057] Experimental conditions. Peptide capture and discrimination in FraC nanopores was studied under a wide range of conditions. Peptide capture was observed over a wide range of voltages, for example from lower voltages of +-10 mV through to +-200 mV. The majority of sensing was carried out at +-50mV to +-100mV as this was generally found to be optimal for peptide capture and characterization. [1058] Peptide detection was observed over a wide range of salt types, concentrations and asymmetries across the membrane, all of which in combination with the pore type may alter the capture and detection properties of the system. Salt conditions for an experiment were about 1 M KC1 (or NaCl or LiCl) at pH <4.5 (e.g. pH 3.8). [1059] Results. Wild Type FraC-Tl and wild type FraC-T2 captured peptides at a frequency of about 10-13 events s-1 under pH 3.8 conditions. When the charge at position 10 or 17 was removed (D10G-FraC-T1 or D17Q-FraC-T1 mutation), the capture frequency was reduced by about 3.4 times and about 7.2 times relative to WtFraC-T. It was shown that the electro osmotic flow (EOF) can be used as a component for efficient capturing of peptides in the nanopore, and can act with or against to electrophoretic forces acting on analytes. It was also shown that the strength and direction of the EOF was dependent on charges in the constriction site (Table 4). Under low pH, which partially protonates water facing residues and generally increasing the net positive charge inside the pore (increasing anion selectivity), the native negative residues in wild type FraC result had almost zero net ion selectivity (Table 4) and thus almost zero net electro-osmotic flux across the nanopore (versus very high cation selectivity at pH 7.5). Removing the negative charge at position 10 further increased the anion selectivity at low pH, creating a stronger EOF component acting against the capture of mostly positively charged peptides, hence resulting in lower capture efficiency. Furthermore, pores with a positively charge constriction, such as D10R- FraC-T1, showed a destabilized baseline current under an applied bias of -50 mV, but stable under +50 mV, thereby behaving opposite to WtFraC. However, D10R mutations exhibited good capture of peptide analytes in the cis chamber at positive applied voltage (exhibiting similar capture to that of native D10 in WT under negative voltage), which indicated that the positive mutation created a strong net anion-selective electro-osmotic bias (flowing from cis to trans), which was dominant versus the weaker electrophoretic force acting against peptide capture at this polarity. [1060] FIGs.18A-18D show electrophysiology recordings of (mutant) Fragaceatoxin C with trypsin digested lysozyme. FIG. 18A shows representative electrical ionic current signals of (mutant) Fragaceatoxin C combined with equal units of trypsin digested lysozyme added to the cis side and under an applied potential of -50 mV, in accordance with some embodiments. The current signals show representative sections of ionic current data for various pores. The lowest current level is the open-pore current of the pore (Io), and the step- like upwards events may be the result of captured analytes occluding a portion of the ionic current flowing WSGR Docket Number: 64828-710.601 through the nanopore (event blockades, IB). FIGS. 18B-18D show representative signal of octameric Fragaceatoxin C (T1; FIG.18B), heptameric Fragaceatoxin C (T2; FIG.18C), Fragaceatoxin C mutant (G13F; FIG.18D). The raw current data in the signals are overlaid with a fit line from the application of edge-detecting event detection algorithms. The block above the signal aligns with the length of the events to indicate the duration of the pulses. Signals were collected in 1M KC1 and 50 mM citric acid titrated with bis-tris propane to pH 3.8 at a sampling frequency of 50 kHz, using a 10 kHz Bessel filter and 5 kHz Gaussian filter. [1061] Removing the charge on residue K20 by substitution to glutamine increased the capture frequency by 1.4 times relative to wild type Frac. Replacing the charge of K20 by introducing an aspartic acid reduced the capture frequency by 1.5 times relative to wild type FraC. The introduction of an aromatic residue (Y, F or W) increased the capture frequency by about 4 times relative to the wild-type FraC-T1 and FraC-T2 pores for all three mutations. The aromatic mutations also increased the duration of the peptide event blockades in the nanopores. Most of the blockades in pores with an aromatic residue on G13, were flat-top shaped with relatively long dwell times (e.g. FIG. 18D). The median dwell time of events in these aromatic pores was increased to 0.32 ± 0.06 ms, 0.18 ± 0.03 ms and 0.22 ± 0.06 ms for G13Y-FraC-T1, G13F-FraC-T1 and G13W- FraC-T1 respectively compared to 0.09 ± 0.06 ms for WtFraC-T1 and 0.10 ± 0.01 ms for WtFraC-T2. [1062] In order to compare the different mutants, the excluded current spectrum (shown for 4 pores in FIGS. 19A-19D) was constructed by creating a histogram of the excluded currents (Iex%) using all events with β > 1 (5 kHz Gaussian filter, see methods). The spectra were normalized and distinct patterns were observed for WtFraC-T1 and T2 (FIGS.19A-19B) with sharp gaussian shaped peaks for G13F-FraC-T1 (FIG. 19C). The majority of peaks of G13N-FraC-T1 were at low Iex% (FIG.19D), reflecting the faster translocation of peptides across the nanopore. The excluded current spectra were compared using a point-to-point spectral matching algorithm, using the excluded current spectrum where 40 % < Iex% < 95 %. Signals were collected in 1M KC1 and 50 mM citric acid titrated with bis-Tris propane to pH 3.8 at a sampling frequency of 50 kHz, using a 10 kHz Bessel filter and 5 kHz Gaussian filter. FIG. 19E shows squared first derivative Euclidean cosine correlation of residual current spectra of (mutant) Fragaceatoxin C combined with equal units of trypsin digested lysozyme. Similarity between the excluded current spectra of WtFraC and FraC mutants was calculated using a The darker shades of color indicate a higher similarity between the spectra of pore mutants. Several groups of pore mutants show highly similar nanopore spectra. Three groups are highlighted by black boxes (Boxes 1-3) in FIG. 19E. The external bias was -50 mV except for D10R# and G13H#, which were tested at +50 mV. Example 2.2. Fragaceatoxin C mutant characterization. [1063] Five mutants were selected for further characterization, namely: G15T-FraC- Tl, as it was comparable to WtFraC-Tl with a slightly increased Io, K20D- FraC-Tl as it had one of the higher SNRs and good capture WSGR Docket Number: 64828-710.601 frequency, and the aromatic mutations of at G13 (G13Y/F/W-FraC-Tl) for their increased dwell times compared to WtFraC-T2 and capture frequency. A mixture of well-defined peptides was used for the characterization of these pores. The peptide mixture was made by adding the individual peptides at equimolar concentrations. The mixture consisted of four peptides: Angiotensinogen (DRVYIHPFHLVIHN, 1758.9 Da, charge = +3.96), Angiotensin 1 (DRVYIHPFHL, 1296.5 Da, charge = +2.96), Angiotensin 3 (RVYIHPF, 931.1 Da, charge = +2.16) and Angiotensin 4 (VYIHPF, 774.9 Da, charge = + 1.16) abbreviated as Angiotensinogen, Ang-I, Ang- III and Ang-IV respectively. The resolution of the nanopores was quantified by measuring the separation between peptides using the difference between the peak centers and their mean standard deviation as shown in the following two equations. (^ + ^ ) ^ = ^ ^ 2
Figure imgf000304_0001
[1064] Where Rs is resolution, ^^ and ^^ are the peak centers with standard deviation
Figure imgf000304_0002
and ^^ espectively. If Rs < 2, the difference between the peak centers was less than twice the average standard deviation. Therefore, no baseline separation was achieved. To achieve an overlap of less than 5%, a Rs > 4 was required, that is, the difference between the peak centers was equal or bigger than twice the average standard deviation of the peaks, thus they may be considered separated. Larger values of Rs indicated a better separation (Table 3). Table 3: The differences between peptide peak centers (∆Iex%) and the observed baseline separation (Rs). MW: 1759 - 931 FraC-Tl FraC-T2 K20D- G15T- G13F- G13Y- G13W- FraC- Tl WtFraC- FraC-Tl FraC-Tl FraC-Tl T1 ∆Iex% 8.8±0.7% 18±3% 14±6%; 12±15% 9.2±0.3% 9.1±0.7% 5.0±0.3 % (Ang-IV — Ang- lll) ∆Iex% 17±2% 12.3±10.5% 15±1% 17±12% 24±1% 22±1% 19.9±0.2% (Ang-lll — Ang-I) ∆Iex% 19.0±0.2% 9.3±10.3% 16.2±0.4% 19.0±0.3% 10±1% 6.1±0.8% 6.4±0.2 % (Ang-I — Pre- Ang) Rs 2.1±0.7 4.1±1.2 2.6±1.4 2.0±0.5 4.6±0.5 4.4±1.1 3.6±0.4 (Ang-IV — Ang- lll) Rs 3.5±10.5 4.2±0.5 2.3±10.2 3.3±0.4 12.1±4.3 11.8±2.9 19.1±1.7 (Ang-lll — Ang-I) WSGR Docket Number: 64828-710.601 Rs 4.1±0.3 4.0±0.3 3.2±10.5 4.6±0.2 6.1±2.3 4.0±0.7 7.2±1.2 (Ang-I — Pre- Ang) [1065] FIGs. 20A-20C show the comparison between WtFraC-T2 and the selected engineered FraC pores. The aromatic pores G13F/Y/W showed marked improvement in the ability to discriminate between the peptides. The aromatic pores exhibited significantly longer blockade event durations versus WtFraC-T2. Longer duration events (with more raw data points at a given acquisition frequency) may allow the amplitude of the excluded current for the individual event blockades to be determined to a higher accuracy. [1066] FIG. 21 shows peptide recognition in further pore types, including heptameric and hexameric Fragaceatoxin C are shown. (Top panel) The fit of the residual current is shown for Leucine-enkephalin (YGGFL) [Leu-enk], Angiotensin II (4-8) (YIHPF) [AngII] and Kemptide (LRRASLG) [kemptide] each in 10 μM concentration, recorded under an applied potential of -70 mV. (Bottom panel) Excluded current % (IEX%) versus dwell time scatter plots of the single-molecule peptide event blockades for the different pore types. Signals were collected in 1M KC1 and 50 mM citric acid titrated with bis-Tris propane to pH 3.8 at a sampling frequency of 50 kHz, using a 10 kHz Bessel filter and 5 kHz Gaussian filter. FIG. 21 shows that aromatic nanopores can identify and discriminate between different peptides better than the wildtype Fragaceatoxin C. Example 2.3. Peptide analysis with T2 nanopores [1067] The resolution of aromatic heptameric (T2) nanopores was tested and compared to hexameric (T3) WtFraC-T3 and WtFraC-T2 nanopores using Leucine-enkephalin (Leu-enk, YGGFL, 555.6 Da), Angiotensin II (4-8) {Ang- II(4-8), YIHPF, 675.8 Da}, and Kemptide (LRRASLG, 771.9 Da). For WtFraC-T3, a FraC version was used with two altered membrane-interfacing modifications, W112S-W116S, which allowed forming hexameric nanopores. WtFraC-T2 showed no blockades (FIG. 20), suggesting that the majority of peptides may have translocated through the pore undetected. FraC-T3 and G13W- FraC-T2 showed leucine- enkephalin and angiotensin II (4-8) blockades, while kemptide blockades were not observed. Kemptide has a higher molecular weight than leucine-enkephalin and angiotensin II (4-8). [1068] Kemptide induced blockades to G13F-FraC-T2, indicating that this aromatic modification may be relevant to detection of this class of peptides. Without being bound to a particular theory, cation-II interactions between the ring of phenyl alanine residues and the two arginine residues may reduce the residence time of the peptide inside the nanopore. Table 4: The differences between peptide peak centers (∆Iex%) and the observed baseline separation (Rs). MW:772 -556
Figure imgf000305_0001
G13W- FraC-T2 WSGR Docket Number: 64828-710.601 ∆Iex% N.O. 27.6±0.8 % 19.1±0.1% 10.6±0.8 % (Leu-enk — Ang-ll (4-8)) ∆Iex% N.O. N.O. 6±2% N.O. (Ang-ll (4-8) - Kemptide) ∆Iex% N.O. 5±1 11±2 3±2 (Leu-enk — Ang-ll (4-8)) Rs N.O. N.O. 3±2 N.O. (Ang-ll (4-8) - Kemptide) Example 2.4. Analytical System comprising nanopores. [1069] Nanopores may be nanometer scale apertures in thin membranes that can detect analytes moving through the aperture. A non-limiting exemplary analytical system is schematically depicted in FIG. 22. It comprised two chambers filled with an electrolyte solution, separated by a membrane. The chambers are connected via a nanopore that is formed in the membrane. When a potential is applied across the membrane via the electrodes in either chamber, ions may move through the pore generating a small ionic current that is amplified and measured. When an analyte enters the nanopore, the ionic current flowing through the open-pore can be altered due to the displacement of ions by the analyte, which can result in a reduction in ionic current (blockade event). The characteristics of the current blockade (e.g. the magnitude, duration, shape, noise, or any combination thereof) can depend on the nature of the analyte captured and the conditions (e.g., applied potential, buffer conditions, temperature, or any combination thereof), and can be used to inform on the properties of the captured analyte. Example 2.5. FraC nanopore as a Next Generation Single-molecule Protein Analyzer. [1070] This example demonstrated that an engineered sub-nanometer biological nanopore of a mutant Fragaceatoxin C (FraC) was able to identify multiple trypsin digested proteins. By calibration through several synthetic peptides, a relation between the residual current spectrum and mass-spectrum may be found, allowing for protein identification, as illustrated conceptually in FIGs. 23A-23D. FIG. 23B shows an artistic representation of the experimental setup where a peptide fragment from a resulting peptide fragment mixture is captured and translocated through a FraC nanopore by applying an electric field across the membrane. FIG. 23D shows an artistic representation of a resulting residual current versus standard deviation spectrum obtained from analysis of the individual single-molecule event blockades, displaying distinct clusters for the different peptide populations. [1071] Protein digestion. 100 ^g of protein stock was taken and the volume was adjusted to 50 ^l using 20 mM Tris buffer (pH 7.5). A final concentration of 20 mM dithiothreitol (DTT) was added to reduce any disulphide bonds. The sample was incubated at 37°C for 15 minutes followed by a denaturing step at 95°C for 15 minutes. Afterwards, a 20 mM iodoacetamide (IAA) was added and the sample was left to incubate for 15 WSGR Docket Number: 64828-710.601 minutes at room temperature in the dark in order to alkylate the reduced cysteine residues. Finally, the total volume was adjusted to 100 ^l using 100 mM Tris Buffer (pH 8.5). [1072] For the tryptic digestion, a kit purchased from Sigma-Aldrich containing proteomics grade trypsin was used. 50 ^l of sample (containing 50 ^g of protein) was added to 1 ^g of mass-spec grade trypsin (1:50 enzyme:protein ratio) and the sample was subsequently incubated overnight at 37°C. Finally, large (> 2000 Da) peptides were removed using centrifugal filters with a molecular weight cut-off of 3000 Da (Amicon). Filtered samples were stored in -20°C prior to use. [1073] Expression of proteins for tryptic digestion. Five model proteins: DHFR (dihydrofolate reductase), BSA (Bovine serum albumin, Sigma-Aldrich), PAN (PAN unfoldase), ThpA (Thiamine binding protein) and HMWI_Act (C- terminal fragment of Haemophilus influenzae high-molecular weight adhesin protein, residues 1205-1536) were expressed and/or purified for the purpose of testing the nanopore sensors. [1074] Protein expression of DHFR/PAN/ThpA/HMWI_Act. All proteins were expressed via similar protocols. Briefly, plasmid containing the gene of interest, was electrochemically transformed into BL21(DE3) competent Escherichia coli cells. The cells were grown overnight at 37°C on LB agar plates supplemented with 100 mg/L ampicillin and 1% glucose. On the next day, grown LB plates were solubilized into 200 mL 2xYT medium, supplemented with 100 mg/L ampicillin. Cultures were grown under constant shaking at 37 °C until an optical density (OD600) of 0.6 was reached. Afterwards, 0.5 mM isopropyl ^-D-l-thiogalactopyranoside was added for induction and growth continued overnight at 21 °C. Bacterial cells were pelleted using centrifugation and stored for at least one hour at -80 °C. [1075] Protein Purification of DHFR/PAN/ThpA/HMWI_Act. Cell pellets were processed by first resuspending in lysis buffer and lysing by sonication (Branson Sonifier 450) in the presence of a protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation and supernatant was processed via Ni- affinity chromatography columns to recover the purified protein fractions. For PAN an additional purification was performed, purifying the protein via anion exchange using HiTrap Q HP anion exchange columns (GE Healthcare Life Sciences). Purity was confirmed by SDS-PAGE and the fractions with highest protein concentration were combined and concentrated using a 10 kDa MWCO spin filter (Amicon). For HMWIAct, fractions containing protein of interest were collected and dialyzed using SnakeSkin dialysis system (MWCO 10kDa, Thermo Fischer Scientific) against storage buffer (50 mM HEPES, 100 mM NaCl, 10% glycerol, pH 7.5). After dialysis protein was aliquoted and stored at -80 °C until further use. [1076] Protein purification of BSA. BSA was purchased from Acros Organics. The purity of BSA was increased using anion exchange chromatography (Akta pure) by processing 10 mg BSA (in 1 ml 50 mM Tris, pH 7.5) on a HiTrap Q HP anion exchange column (GE Healthcare Life Sciences). Eluted protein fractions WSGR Docket Number: 64828-710.601 were assessed by SDS-PAGE and the fractions with highest protein concentration were combined and concentrated using a 10 kDa MWCO spin filter (Amicon). [1077] Results [1078] Detection of a model protein digest. Detection and identification of proteins using mass spectrometry based techniques was conducted by fingerprinting of (tryptic) peptides. A properly digested protein was mimicked by employing a model peptide system containing 7 synthetic peptides with a mass between 700 and 1700 Da (Sigma Aldrich and Genscript) that would be predicted to result from complete trypsination of lysozyme (i.e. the protein is cleaved in-silico at all arginine (R) and lysine (K) residues unless they are followed by proline (P)). [1079] The 7 model peptides were individually added to separate nanopore experiments (G13F-FraC-Tl pores, IM KCI, pH 3.8, -50mV), generating a unique cluster of events when plotted by excluded current and dwell time. For each single experiment the average excluded current for the event blockades was calculated by fitting a gaussian to histograms of the clustered events. The average excluded current for each peptide type was calculated by averaging across n > 3 experiments performed on each peptide. In FIGs.24A-24B, G13F-FraC- T1 nanopores were characterized using synthetic model peptides that may be predicted to result from the complete tryptic digestion Gallus-gallus lysozyme. A strong correlation between the molecular weight of the peptides and their respective average excluded current blockade was observed (FIG.24A). The data were fitted with a logistic function (FIG. 24A), which could allow prediction of peptide mass from excluded current measurements. The dashed line represents a logistic function fit through the data and shows a clear correlation between excluded current and molecular weight, which can be used to characterize captured peptides and for predictive purposes when testing unknown peptides.
Figure imgf000308_0001
(1) [1080] Where a is the offset, k represent the width and ^ is the inflection point. The data of FIG. 24A were obtained from n>3 multiple separate experiments on separate pores for each model peptide. [1081] FIG. 24B shows a histogram of excluded current blockade events measured from a mixture of all 7 model peptides in the nanopore system (G13F-FraC- T1 pores, 1M KC1, pH 3.8, -50mV). The peaks are labelled according to the predictions from the logistic function, and match the same excluded current position observed in the individual experiments. The peaks are labelled according to the predictions determined from the experiments in FIG.24A, and match the same position observed in the separate experiments. WSGR Docket Number: 64828-710.601 [1082] Detection of digested Lysozyme protein and comparison with Mass Spectrometry. Lysozyme protein was digested via trypsination as described above. The resulting peptide fragment mixture was then analyzed both using nanopore sensing (G13F-FraC-Tl pores, IM KCI, pH 3.8, -50mV) and with Mass Spectrometry (LC ESI-MS). A histogram of the excluded current blockades measured from the mixture using the nanopores is plotted in FIG. 25A. For the purposes of comparison the mass data obtained from the Mass Spectrometry spectrum was transformed onto an pseudo excluded current axis using the predictions from the logistic fit parameters (FIG. 25B). Although the methods cannot be directly compared due to differences in detection efficiency for example, a significant correlation between the observed electrospray ionization (ESI) mass-spectrum and the nanopore mass spectrum was observed. [1083] Detection of trypsin digested proteins.9 proteins with highly divergent compositions were tested by nanopore spectrometry. The 9 proteins were: Bovine serum albumin (BSA), dihydrofolate reductase (DHFR), high molecular weight adhesin 1 (HMWlAct), PAN unfoldase, Thiamine binding protein (TbpA), beta casein, cytochrome C, lysozyme and trypsin. The proteins were digested via trypsination as described. The resulting peptide fragment mixtures were separately tested in multiple separate nanopore experiments (G13F-FraC- T1 pores, IM KC1, pH 3.8, -50mV). Similar to what was observed for the digested lysozyme peptide mixture, distinct clusters of blockade events were observed from the peptide mixtures for each of the digested proteins (FIGs.26A-26C and FIGs.27A-27B), with the clusters of event blockades separated by their excluded current Iex%. FIGs. 26A-26C shows that a high level of consistency for each unique spectra was observed between separate nanopore experiments for three representative protein samples. Each repeat was acquired from a separate nanopore experiment with a fresh nanopore, using the same digested sample in each repeat. All measurements were performed using G13F-FraC-T1 nanopores in 1M KC1 buffered to pH 3.8 using 50 mM citric acid titrated with bis-tris-propane under an applied potential of -70 mV. Recording was performed at 50 kHz using an analog Bessel-filter at 10 kHz and a digital Gaussian filter of 5 kHz. FIG. 26A shows the consistency between repeat 1 (2601), repeat 2 (2602), and repeat 3 (2603) for BSA. FIG. 26B shows the consistency between repeat 1 (2604), repeat 2 (2605), and repeat 3 (2606) for DHFR. FIG. 26C shows the consistency between repeat 1 (2607), repeat 2 (2608), and repeat 3 (2609) for EF-P. [1084] To account for pore to pore variations in the baseline current, the residual current spectra were aligned to a reference spectrum using a sliding window on Iex%. FIG. 27A plots the aggregated histogram “excluded current spectra” from fits to the individual peptide blockade event scatter plots of excluded current versus dwell time for each protein sample. The excluded current spectra for each protein display unique patterns of peaks that may be dependent on the unique composition of digested peptides in each system (with fragments varying by mass, length, and amino acid composition). The spectra of PAN and BSA showed distinct peptide clusters, WSGR Docket Number: 64828-710.601 despite the large amount of fragments predicted from the in-silico digestion, indicating that even large (50 kDa) proteins yield distinct spectra that can allow fingerprinting of the precursor protein. [1085] Protein fingerprinting and spectral matching. The unique excluded current spectra of the tryptic digests (FIG. 27A) was used to fingerprint proteins by spectral matching, wherein the measured spectra were compared to a previously measured database of known spectra. Different datasets showed a high level of reproducibility (as shown in FIGs. 26A-26C) after taking the baseline shift from pore-to-pore variation in separate repeat experiments into account. [1086] The uniqueness and reproducibility of the spectra were determined using spectral correlation, utilizing the squared first derivate Euclidean cosine correlation (DEuc).
Figure imgf000310_0001
(2) [1087] With ^^ and ^^ containing the vectors of the excluded current spectra and each element (i) in the vector. [1088] In order to ensure a representative example for spectral matching, a leave-one-out comparison was performed, where the comparison database was built from all spectra, excluding the one that was matched. The probability P(X) % was calculated from the DEuc score relative to the sum of all the DEuc scores (FIG.27B). 8 of the 9 tryptic digests were correctly assigned to the known protein (diagonal axis), except for DHFR, which was erroneously assigned to lysozyme. Visual inspection of the DHFR and lysozyme spectra (FIG. 27A) readily explained the erroneous assignment, as both digests shared some peak similarities for excluded current. This analysis employed only one “metric” of the events, their excluded current. The Iex% spectra were analyzed using a spectral matching algorithm incorporating the squared first derivate Euclidean cosine correlation (DEuc), which is advantageous, as it corrects for baseline sloping. The DEuc is a direct result of the dot product equation and allows the estimation of the angle between two vectors, which can be used as a measure of similarity. This was used to normalize counts of the excluded current spectra which are represented as vectors. The Iex% spectrum between 50 and 98 Iex% was considered, as noise below the limit-of-detection and fully blocked events may have skewed the comparison. Subsequently, a leave-one-out comparison was performed using the DEuc as a score, normalized to 100% for visualization. The leave-one-out comparison compared the Iex% spectrum of each measurement (sample), with the average Iex% spectrum of each protein (database). For each comparison, a database was constructed that contained all measurements except for the sample. Further analysis of spectra using other metrics, for example the standard deviation of the noise in each event, showed that clusters/peaks that may not easily be separated with one metric dimension were often possible to separate by another metric dimension. WSGR Docket Number: 64828-710.601 [1089] Detecting amino acid changes. To evaluate the resolution of the analytical detection system to discriminate between peptides that differ by only 4 Dalton, two different forms of the enkephalin peptide were tested: YGGFL, and YdAGFdL, wherein d represents a D-amino acid; all other amino acids being in the L- configuration. FIG.31A shows that two clear clusters were observed for the different peptides, illustrating that mass differences of at least 4 Da were differentiated along with differences in chirality using exemplary FraC G13F nanopore. Measurements were performed in 1 M KC1, 50 mM citric acid titrated with bis-tris-propane (pH 3.8) at -100 mV applied potential sampling at 50 kHz and filtered to 10 kHz using the G13F-FraC-T1 pore. FIG.31A plots the amplitude of the blockade versus the standard deviation of the noise in the blockade for the recorded event blockades, and illustrates that differences of at least 4 Da can be differentiated as two clear clusters. Detection of peptide chirality for peptides of the same mass was confirmed in FIG. 31B and 16C, showing a difference in nanopore signal due to the presence of D-amino acids. A mixture 10 ^M of [Ala2]- Leu Enkephalin and 10 ^M DADLE ([D-Ala2, D-Leu5]-Enkephalin) was added to either the cis compartment (FraC-G13F; FIG. 31B) or trans compartment (CytK-K128F; FIG. 31C). A mixture 10 μM of [Ala2]-Leu Enkephalin and 10 μM DADLE ([D-Ala2, D-Leu5]-Enkephalin) was added to the cis compartment (FraC- G13F; FIG.31B) or trans compartment (CytK-K128F; FIG.31C). Measurements were performed in 3M LiCl, 50mM citric acid, buffered to pH 3.8. Data recorded with 50 kHz sampling frequency and 10 kHz filter. Example 2.6. Detection of post-translationally modified peptides. [1090] An analytical system comprising a FraC-G13F nanopore as described herein above was used to detect post-translationally modified peptides, and was able to distinguish between a phosphorylated and non- phosphorylated peptide (see FIG. 28), an unmodified peptide, a peptide modified with a single or with two glycans (see FIG.29), and unmodified protein and rhamnosylated protein (FIG.30). For FIG. 28, 2.5 μM of kemptide (LRRASLG) and 2.5 μM of phosphorylated kemptide (LRRA{pS}LG) were added to the cis-chamber of a system comprising FraC_G13F nanopores. Measurements were taken in 1M KC1, 50mM citric acid buffered with bis-tris propane to pH 3.8 Recordings were performed at an applied potential of -70mV at 50kHz frequency with a 10kHz lowpass filter. Residual current (Ires = blockade current/open-pore current) was plotted against dwell time for both peptides. The peptides were detected in two distinct clusters (shown by the labels), demonstrating the ability of nanopore systems to differentiate a peptide from its phosphorylated form. For FIG. 29, nanopore systems distinguished between different numbers of glycan modifications on peptides when a mixture of peptides was used. 2.5 μM of unmodified peptide (ANVTLNTAG), 2.5 μM of peptide with one glycan (ANVT(Glc)LNTAG and 2.5 μM of peptide with two glycans (ANVT(Glc)LNTT(Glc)G) were added sequentially to the cis-chamber of a system comprising FraC_G13F-T1 nanopores (3M LiCl, 50mM citric acid buffered with bis-tris propane to pH 3.8, -50mV at 50kHz frequency with a 10kHz lowpass filter). FIG. 29 WSGR Docket Number: 64828-710.601 shows the residual current blockade histogram from all detected capture events when measuring a mixture containing all three glycosylated peptides. [1091] As shown in FIGs.30A-30B, 25 μg of unmodified Elongation Factor P (EF-P, FIG.30A) and 75μg of rhamnosylated EF-P (FIG.30B) were digested into peptide fragments using Lys-C. After digestion, in separate experiments 8 μg of digested protein was added to the cis-chamber of nanopore sensing systems comprising a FraC_G13F-T1 nanopore for peptide analysis (3M LiCl, 50mM citric acid at pH 3.8, -50mV at 50kHz frequency with a 10kHz lowpass filter). The rhamnosylation modification is on the SGRNAAVVK peptide fragment. The rhamnosylation modification was discriminated by the large shift in the residual current (Ires) between the modified peptide [SGR{rham}NAAVVK] and the unmodified peptide [SGRNAAVVK]. Example 2.7. Mutant proteinaceous nanopore comprising a beta-barrel pore forming toxin. [1092] Examples 2.1 to 2.6 relate to a mutant proteinaceous nanopore comprising an alpha-helical pore- forming toxin of the actinoporin family, and its application as single molecule sensor. To test whether these discoveries were more broadly applicable to different classes of nanopores, with similar dimensions in the sensing region but quite different structural makeup, similar mutations and conditions on beta-barrel pores were explored. [1093] In this example, beta-barrel pore-forming proteins, wherein the lumen-facing recognition region of the proteins comprises one or more mutations to an aromatic residue, were used to provide such nanopore-based sensors, including in combination with nearby acidic mutations. FIGs.32A-32D depict representative electrical ionic current signals from (mutant) Aerolysin nanopores with 4μg of trypsinated lysozyme added to the cis- chamber of a nanopore sensing system (+150 mV). Signals were acquired with 1M KC1 in cis and trans, with 50 mM citric acid buffered with bis-tris propane to about pH 3.8 or pH 3.0, or with 50 mM Tris buffered at pH 7.5, as indicated. [1094] It was found that lowering the pH of the buffer may increase the capture (FIG.32A versus FIG.32B) and resolution (FIG.33A versus FIG.33B) of a tryptic digested peptide mixture using the wild-type Aerolysin pore. For FIGs.33A-33H, the structure or schematic of the aerolysin nanopore, with indicated locations of, and spacing between, the modifications, and residual current versus dwell time scatter of individual peptide blockades provoked by 4μg of trypsinated lysozyme are shown. [1095] However, for the wild-type pore, even at low pH (e.g. pH 3.8) the observed events were extremely short (FIG.32A-32B) and peptide clusters resulting from different peptide populations had a wide distribution and were poorly resolved from each other, making the distinction of individual peptides from the mixture challenging (FIG.33B). [1096] It was found that replacing the Lysine at position 238 with a phenylalanine (Aer-K238F, FIG.32C and FIG.33C) did not significantly increase the dwell time of peptides (FIG.32C) and only marginally improved WSGR Docket Number: 64828-710.601 peptide cluster resolution under pH 3.8 (FIG.33C), and that replacing the Lysine residue at position 238 by the acidic amino acid aspartate (Aer-K238D) significantly increased the cluster resolution at low pH (FIG. 33D) over the wild-type pore. The improved peptide capture and resolution for the K238D mutation was partly due to reduced electrostatic repulsion between the recognition region of the nanopore and the mostly positively charged peptides at low pH, and partly due to the increased cation ion-selectivity. [1097] The K238D mutation was further combined with the introduction of the phenylalanine at either position Ala260, Ser264, Gln268 or S272 of Aerolysin, and observed a dramatic improvement in peptide resolution (FIG. 33E-33H). The improved resolution between different peptide clusters was the result of a combination of improvements, including 1) longer residence (dwell) times that can allow more accurate measurement of each single-molecule event (e.g. FIG. 32D), a lower spread of residual currents in each cluster that can allow closely separated clusters to be resolved from one another more easily, and 3) clusters spread out more widely over the full current range. The resolution of the analyte peptides was especially sharp when the distance between the aspartic acidic at position 238 and the introduced aromatic amino acid was less than 4 nm. Therefore, the combination of an increased negative pore and an aromatic substitution on the water-facing transmembrane was important for increasing the capture and resolution of unlabeled peptides. This appeared especially important when sampling at acidic pH values (< pH 4.5). This combination of mutations in the lumen of the beta-barrel pore created similar rings of sensing residues to those in the constriction of the FraC nanopore when engineered for improved peptide discrimination, showing that this combination of mutations was a general feature that may be engineered into the sensing constriction of a wide range of both alpha-helical and beta-barrel based nanopores with similar sensing constriction geometries (for example, engineering mutations into non-conserved inward facing residues). [1098] A tryptic digested peptide mixture was captured and resolved using the mutant Aer-K238W pore (FIG. 33I), demonstrating a significant improvement in peptide detection with the aromatic mutation versus the wild-type aerolysin. Measurements were performed in 1M KC1 cis and trans, with 50mM citric acid buffered with bis-tris propane to about pH 3.8 or pH 3.0 for low pH experiments, or with 50mM Tris buffered at pH 7.5 as indicated. Recordings were done at an applied potential of + 150mV at 50kHz frequency with a 10kHz lowpass filter. The figures show that aromatic mutations, especially in combination with modifications that increase the negative charge of the pore, improve the recognition of peptides especially at pH values lower than about 4. Measurements were in 1M KC1, 50mM citric acid, buffered to pH 3.8 at +150mV applied potential. Data was recorded with 50 kHz sampling frequency and 10 kHz filter. [1099] Expression and purification of pro-aerolysin. A plasmid containing a gene encoding for pro- aerolysin elongated by a hexa-histidine tag at the C-terminus was transformed into BL21(DE3) cells using electroporation. The transformed cells were grown overnight at 37°C on LB agar plates supplemented with 1% WSGR Docket Number: 64828-710.601 glucose and 100 ^g/ml ampicillin. On the next day, the colonies were resuspended and grown in 200 mL 2YT medium at 37°C until the OD6oo reached 0.6-0.8. At this point, the expression was induced by addition of 0.5 mM IPTG and the culture was incubated overnight at 25 °C. Afterwards, the cells were pelleted by centrifugation at 4000 rpm for 15 minutes and the cell pellets were stored at -80 °C for at least 30 minutes. For protein purification, cell pellets of 100 ml culture were resuspended in 20 ml lysis buffer, containing 150 mM NaCl, 20mM imidazole and 15 mM Tris buffered to pH 7.5, supplemented with 1 mM MgC12 , 0.2 units/ml DNasel and approximately 1 mg of lysozyme. The mixture is incubated for 30 minutes at RT and afterwards sonicated using a Branson Sonifier 450 (2 minutes, duty cycle 30%, output control 3) to ensure full disruption of the cells. Cell debris is pelleted by centrifugation at 6000 rpm for 20 minutes and the supernatant is carefully transferred to a fresh falcon tube. Meanwhile, 200 ^l of Ni-NTA bead solution is washed with wash buffer, containing 150mM NaCl, 20mM imidazole and 15mM Tris buffered to pH 7.5. The beads were added to the supernatant and incubated at RT for 5 minutes. Afterwards, the solution is loaded on a Micro Bio-Spin column (Bio-Rad) and subsequently washed with 5 ml of wash buffer. The bound protein is eluted in fractions of 200 ^l of elution buffer (150 mM NaCl, 300 mM imidazole, 15mM Tris buffered at pH 7.5. The pro-aerolysin fractions can be stored in at 4 °C for several weeks. [1100] Oligomerisation from pro-aerolysin using trypsin. Pro-aerolysin was incubated with trypsin in a 1:1000 mass ratio for 15 minutes at room temperature. The trypsin cleavage of the C-terminal peptide, resulted in aerolysin monomers that may assemble into heptameric pores which may be characterized in electrophysiology experiments. [1101] Tryptic digestion of lysozyme.100 ^g of lysozyme (Carl Roth, from chicken egg white, albumin free) was dissolved in 150mM NaCl, 15 mM Tris buffered at pH 7.5. Before digestion, free cysteines were alkylated to prevent formation of disulfide bridges after digestion. To that end, 3^L 200 mM DTT was added and the sample was incubated at 37 °C for 15 min, followed by 15 minutes of denaturation at 95°C. Subsequently, 7 ^L of 200 mM IAA was added and the sample was incubated for 15 min at RT in the dark. After alkylation, the lysozyme was digested overnight at 37°C in a 50:1 lysozyme:trypsin mass ratio using the Trypsin Singles, Proteomics Grade-kit (Sigma Aldrich, Catalog #T7575- 1KT). [1102] Detection of lysozyme digest using Aerolysin pores. Aerolysin was added to the cis-chamber and the bilayer was broken and reformed until a single channel inserted into the bilayer. The orientation of the pore can be detected by a small asymmetry in the IV curve of the pore. First, a 2 minute blank was recorded at +150mV applied potential and afterwards 4 ^l of trypsin-digested lysozyme was added to the cis compartment of the chamber. The analyte was measured for at least 10 minutes at an applied potential of+ 150mV. [1103] Data recording. Recordings of ionic currents were obtained using an Axopatch 200B (Axon Instruments) combined with a Digidata 1550B A/D converter (Axon instruments), similar to preceding work WSGR Docket Number: 64828-710.601 (Huang et al. Nat. Commun. 2019). The sampling frequency was set at 50 kHz for analyte recordings, the analogue Bessel filter was set at 10 kHz. Data was recorded using Clampex 10 (Molecular Devices). Example 2.8. Mutant proteinaceous nanopore comprising a cytolysin k beta-barrel pore forming protein. [1104] Example 2.7 relates to single molecule analysis using a modified beta-barrel pore-forming protein Aerolysin. In this example, functionally similar mutations were introduced into the cytolysin k (CytK) nanopore to demonstrate that aromatic mutations, in combination with nearby acidic mutations, and when used under low pH conditions (<pH 4), improve the ability to capture and resolve unlabeled peptides for other beta- barrel pores. [1105] To identify the beta-barrel region, and the putative analyte recognition region of CytK, a homology model was built by mapping the CytK sequence to the sequence and structure of the alpha-hemolysin nanopore from Staphylococcus aureus (FIG. 35A). The beta-barrel region was identified as comprising the stretch running from amino acid E112 to amino acid S134, and from amino acid S137 to amino acid K155, with the even residues in the range E112-S130 and odd residues in the range S137-K155 being the inward lumen water- facing residues (FIG.35A). [1106] Expression and purification of (mutant) CytK. Plasmid containing a gene encoding for CytK elongated by six histidine residues at the C-terminus was transformed into BL21(DE3) cells by electroporation. Transformed cells were grown overnight at 37°C on LB agar plates (1% glucose, 100 ^g/ml ampicillin). Colonies were resuspended and grown in 200 mL 2YT medium at 37 °C until OD6oo 0.6-0.8, then expression was induced by addition of 0.5 mM IPTG and the culture was incubated overnight at 25 °C. Cells were pelleted by centrifugation and stored at -80 °C for at least 30 minutes. Cell pellets were lysed by resuspension in lysis buffer (150 mM NaCl, 20mM imidazole, 15 mM Tris pH 7.5, 1 mM MgCl2, 0.2 units/ml DNasel, ~1 mg of lysozyme), incubated for 30 minutes at RT, then sonicated (Branson Sonifier 450, 2 minutes). Cellular debris was pelleted by centrifugation and the supernatant containing CytK was recovered. CytK was extracted from the supernatant and purified using Ni- NTA beads, with final elution in 200 ^l aliquots (150 mM NaCl, 300 mM imidazole, 15mM Tris buffered at pH 7.5) before storage at 4 °C. [1107] Planar lipid bilayer electrophysiological recordings. Electrophysiology measurements were performed as described in Example 2.7. CytK was added to the cis-chamber and the DPhPC bilayer in the nanopore system was broken and reformed until a single nanopore inserted into the bilayer. The orientation of the pore can be detected by the asymmetry in the IV curve of the pore. All recordings were performed with 1 M KC1 in both the cis and trans compartments at either pH 3.8 (50 mM citric acid, titrated with bis-tris propane to pH 3.8) or pH 7.5 (50mM Tris buffered at pH 7.5). First, 2 minutes of blank open-pore current was recorded at +100mV applied potential, and afterwards 4 ^l of trypsin- digested lysozyme was added to either this cis or WSGR Docket Number: 64828-710.601 trans compartment of the chamber. The analyte was measured for at least 10 minutes at an applied potentials of -l00mV to +100mV as indicated. The ionic current was recorded using a Digidata 1440A (Molecular Devices) connected to an Axopatch 200B amplifier (Molecular Devices). The sampling frequency was set at 50 kHz for analyte recordings, the analogue Bessel filter was set at 10 kHz. Data was recorded using Clampex 10 (Molecular Devices). Event blockade data was analyzed as described herein, measuring the event blockades resulting from peptide capture and extracting metrics including average open-pore current, average blockade current, blockade duration (dwell time), standard deviation of blockade current, or any combination thereof [1108] Results. Similar to Example 2.7, nanopore sensing systems containing CytK nanopores were tested using a digested peptide mixture resulting from trypsinated lysozyme. Wild Type CytK exhibited little to no capture of the peptides from a trypsinated lysozyme sample, including when the sample was added to either the cis or trans compartments, under either positive or negative applied potentials over a wide range of voltages, at either pH 7.5 or pH 3.8. For example, FIG.34A and FIG.35B shows the low number of detected events using wildtype CytK nanopores when trypsinated lysozyme sample was added to the trans compartment, with a positive applied potential at the trans electrode to drive electrophoretic capture of the mostly positively charged peptides (+100 mV, IM KC1, pH 3.8). Representative electrical ionic current signals from (mutant) Cytolysin K nanopores with 4μg of trypsinated lysozyme added to the trans-chamber of a nanopore sensing system (+100 mV). Signals were acquired with 1M KC1 in chambers and 50mM citric acid buffered to pH 3.8. [1109] According to the predicted structure, a Lysine residue at position 128 and a Glutamate residue at position 139 were predicted to be inward facing residues in the recognition region. In accordance with previous findings described herein, a phenylalanine was substituted into the K128 position of the CytK monomers adjacent to the acidic E139, thus serving both to reduce the net positive charge in the nanopore and introduce an aromatic for improved peptide detection. The K128F mutation produced a dramatic improvement in the ability to both capture (FIG. 34B) and resolve (FIG.35C) different peptides at low pH versus the wild-type nanopore. Very good results were also obtained with the K128W mutation (FIG.35H). [1110] In another implementation, similar to the strategy employed in Example 2.7, an aromatic amino acid was introduced adjacent to an additional negative mutation by substituting the lysine at 238 with an aspartic acid and substituting the serine at 126 with a phenylalanine (CytK-S126F-K128D). Similar to what was observed for the Aerolysin nanopore system, this combination of an aromatic amino acid substitution adjacent to an acidic amino acid substitution further improved the resolution of different peptides through a combination of improved metrics, including: better capture (FIG.34C), longer residence (dwell time) of peptide blockades (FIG.34C), tighter clusters with less residual current spread (FIG.35D), and clusters spread widely over the full min-max current range (FIG.35D). WSGR Docket Number: 64828-710.601 [1111] Aromatic mutations placed higher up in the barrel of aerolysin (position S120, Q122 or G124) combined with K128D also yielded a good resolution of peptides of a trypsinated lysozyme sample. See FIG. 35E-35G. For FIGs. 35B-35H, measurements were in 1M KC1, 50mM Tris, buffered to pH 7.5 at + 100mV applied potential. Data were recorded with 50 kHz sampling frequency and 10 kHz filter. To the left of each panel is indicated the schematic position of the substituted amino acid. Recordings were done at an applied potential of + 100 mV at 50kHz frequency with a 10kHz lowpass filter. The figures show that aromatic mutations in combination with modifications that increase the negative charge of the pore allowed the recognition of peptides especially at pH values lower than about 4. [1112] Accordingly, the data demonstrated that aromatic mutations, preferably adjacent to acidic amino-acid substitutions, created a sensing region that improved the ability to capture and discriminate unlabeled peptides, in particular at low pH conditions. [1113] In examples 2.7 and 2.8 two different dominant mechanisms were demonstrated for controlling peptide capture in CytK and aerolysin nanopores. For example, it was demonstrated that Aerolysin nanopores may capture and discriminate peptides effectively at positive applied potential when analytes are in the cis compartment. The analytes, being mostly positively charged at pH 3.8 or pH 3.0, were captured against the electrophoretic direction due to dominant electro-osmotic capture conditions. In contrast, CytK was demonstrate to capture and discriminate peptides effectively at positive applied potential when analytes were in the trans compartment. The analytes were captured primarily by electrophoretic forces under the pH 3.8 conditions, and the electro-osmotic component was tuned to be close to zero by substitution of additional acidic residues (see Table 4). These results indicated that the introduction of aromatic residues in beta-barrel pore- forming toxins works regardless of the capture mechanism of the analyte, and that the introduction of acidic residues under low pH conditions can be an important tool for tuning and controlling cation selectivity and electro-osmotic capture. Table 5: Ion selectivity of FraC, Aerolysin and CytK nanopores. [1114] The reversal potential was measured from IV curves between -100 mV and +100 mV under asymmetric salt conditions (2M KC1 in trans and 0.5 M KC1 in cis), buffered to indicated pH using 50 mM Tris for pH 7.5 or 50 mM citric acid titrated to pH 3.8 using bis-tris propane. The reversal potential (the applied voltage at which there is zero net current flow) was determined by linear regression of the IV curve between -20mV and +20mV. Reversal potential (mV) P(K+)/P(Cl) Wt Aerolysin pH 7.5 -3.5±0.4 0.78 pH 3.8 -13.2±0.4 0.37 WSGR Docket Number: 64828-710.601 Aerolysin K238D pH 3.8 -8.8±0.9 0.52 Aerolysin_K238W pH 3.8 -10.0±0.8 0.48 Wt CytK pH 7.5 -0.3±0.3 0.98 pH 3.8 -7.8±0.8 0.57 CytK_K128F pH 7.5 12.8±0.2 2.61 pH 3.8 0.5±0.3 1.03 CytK_128D pH 7.5 10.5±0.2 2.17 pH 3.8 1.7±0.7 1.12 WtFraC* pH 7.5 17.2± 1.2 3.6±0.4 pH 3.8 1.0±1.7 1.03±0.04 FraC_G13F pH 7.5 17.0±0.7 3.7±0.2 pH 3.8 0.0±0.4 1.00±0.03 *replicated from Huang et al., Nat. Commun.2019, 10 (1), 835. Example 2.9. Mutant proteinaceous nanopore comprising a Lysenin beta-barrel pore forming protein. [1115] In this example, Lysenin, a further exemplary beta-barrel pore forming protein, was successfully mutated to demonstrate that an aromatic mutation of a non-aromatic lumen facing residue improves the ability to capture and resolve unlabeled peptides. [1116] Plasmids containing the Lysenin gene from Eisenia fetida were transformed into BL21(DE3) E.coli competent cell by electroporation. Next, the cells were grown on lysogeny broth (LB) agar plate containing 100 ^L/mL ampicillin overnight at 37 °C. The LB plate was harvested and inoculated into 400 mL 2xYT media. Then, the culture was grown at 37 °C while shaking at 200rpm until the optical density at 600 nm of the cell culture reached 0.8. This was followed by addition of 0.5 mM isopropyl-D-thiogalactoside (IPTG) to the media and the culture was grown overnight at 25 °C while shaking at 200 rpm. The next day, cells were harvested by centrifugation (4000rpm, 15 min) and the resulting pellets were frozen at -80 °C for 30 min. [1117] The cells were resuspended and mixed for 30 min in 40 mL of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.02% DDM supplemented with, 10 mM imidazole, 1 mM MgC12) together with 0.2 mg/mL lysozyme, and 10 ^L DNasel. The lysate was sonicated for 2 min (40% output power) and centrifuged down at 4 °C for 15 min (4000 rpm). Next, the supernatant was incubated with 150 ^L washed Ni-NTA beads for 15 min at 20 rpm. The Ni-NTA beads were loaded on a gravity-flow column and washed with wash* buffer: ([50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM imidazole, and 0.02% DDM)]. The proteins were eluted in 3 elution steps with 150 ^L elution buffer:* ([50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 300 mM imidazole and 0.02% DDM)]. Lysenin monomers were stored at 4°C. WSGR Docket Number: 64828-710.601 [1118] Lysenin was oligomerized by incubation with liposomes (with a 1:1 sphingomyelin:DPHPC lipid composition) in a 1:10 protein:liposome ratio at 37°C for 1 hour. The liposomes were then disrupted by addition of 0.6% LDAO. The solution was diluted 20x using wash buffer and mixed with 150^l washed Ni-NTA beads. The solution was subsequently loaded on a gravity-flow column and washed with wash buffer. Oligomers were eluted by an elution buffer containing IM Imidazole, 150 mM NaCl and 15 mM Tris buffered to pH 7.5 in fractions of 150^l. Oligomers were stored at 4°C. [1119] In this example, 0.5 ^g Lys-C digested lysozyme were added to the trans compartment (final concentration 1.25 ng/^l) of an analytical system comprising either wildtype Lys (FIG. 36A) or Lys-E76F (FIG. 36B). Introduction of the aromatic residue in the lumen resulted in a clear peptide cluster for larger peptides, as seen in FIG.36. Measurement of 0.5 μg Lys-C digested lysozyme added to the trans compartment (final concentration 1.25 ng/μl) of a system comprising either (A) wild type (WT-Lys) or (B) mutant Lys-E76F nanopores. Measurements were performed in 1M KC1, 50mM Citric acid, buffered to pH 3.8 at -70mV applied potential. Data were recorded with 50 kHz sampling frequency and 10 kHz filter. [1120] Non-proteinaceous analytes were also detected by nanopore systems described herein. Analytes were added to the cis-chamber (Thioflavin 2.0 μM) or to the cis and trans chambers (Vitamin B12, 10.0 μM) of a nanopore system. The system comprised heptameric wild-type FraC (FIG.37A), mutant FraC_G13F nanopores with Thioflavin (FIG. 37B and FIG. 37C), octameric wild-type FraC (FIG. 37D), or mutant FraC_G13F nanopores with Vitamin B12 (FIG.37E and FIG. 37F). Measurement in 1 M KC1, 50mM Tris.HC1 pH 7.5 Recordings were done at an applied potential of -70mV (Vitamin B12) or -50 mV (Thioflavin) at 50kHz frequency with a 10kHz lowpass filter. The graph demonstrated that the molecules can be detected as a distinctive cluster, plotting residual current (Ires = blockade current/open-pore current) versus dwell time. Example 3. Pore with a Proteasome. [1121] Examples 3.1-3.7 used the following materials and methods. [1122] General materials. Oligonucleotides and gBlock gene fragments were obtained from Integrated DNA Technologies (IDT). Phire Hot Start II DNA Polymerase, restriction enzymes, T4 DNA ligase, and Dpn I were purchased from Fisher Scientific. Angiotensin I, dynorphin A, pentane, hexadecane, and Trizma base were obtained from Sigma-Aldrich. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was purchased from Avanti Polar Lipids. Sodium chloride and Triton X-100 was bought from Carl Roth. [1123] Plasmid Construction for proteins. gBlock gene fragments were ordered for synthesis by IDT, and cloned into pT7-SCl plasmid using Nco I and Hind III restriction digestion sites. Plasmid and gene were ligated together using T4 ligase (Fermentas).0.5 μL of the ligation mixture was incorporated into 50 μL E. cloni® 10G (Lucigen) competent cells by electroporation. Transformants were grown overnight at 37°C on LB agar plates WSGR Docket Number: 64828-710.601 supplemented with ampicillin (100 μg/mL). Ampicillin-resistant colonies were picked and inoculated into 5 mL LB medium supplemented with of ampicillin (100 μg/mL) for plasmid DNA preparation. The plasmid was extracted with GeneJET Extraction Kit (Fisher Scientific). The identity of the clones was confirmed by sequencing at Macrogen. [1124] Plasmid Construction for building a sequencing proteasome machine. gBlock gene fragments of Thermoplasma acidophilum α and β were ordered for synthesis by IDT. The gene encoding for the α subunit was cloned upstream of pETDuet-1 vector (Novagen) between the Nco I and Hind III sites with the gene of Strep-tag at the C-terminus. Subsequently, the gene encoding for an untagged β subunit was cloned downstream between the Nde I and Kpn I sites. PA-nanopore was fused to α subunit gene through PCR splicing by overlap extension, and cloned into pET-28a vector (Novagen) using Nco I and Hind III restriction digestion sites with His tag at the N terminus. [1125] Construction of mutants. All mutants were constructed using the QuickChange protocol for site-directed mutagenesis on a circular plasmid template DNA with Phire Hot Start II Polymerase. Partially overlapping primers were used to avoid primer self-extension. PCR amplification was as follows: denaturation at 98°C for 3 min, followed by 30 cycles of 98°C for 30 s, 55°C for 30 s, and 72°C for 3 min, and a final extension cycle of 72°C for 5 min. After the PCR reaction, the parental DNA template was digested with Dpn I enzyme for 1 h at 37°C. The PCR amplified plasmid was separated on 1% agarose gel, extracted with GeneJET Gel Extraction Kit (Fisher Scientific), and incorporated into 50 μL E. cloni® 10G (Lucigen) competent cells by electroporation. Transformants containing the plasmid were grown overnight at 37°C on LB agar plates supplemented with ampicillin (100 μg/mL). Ampicillin-resistant colonies were picked and inoculated into 5 mL LB medium supplemented with of ampicillin (100 μg/mL) for plasmid DNA preparation. The plasmid was extracted with GeneJET Extraction Kit (Fisher Scientific), and sequenced at Macrogen for confirmation of the mutation. [1126] Expression and purification. The gene of the PA nanopore was transformed into E. coli. BL21 (DE3) pLysS chemically competent cells. Transformants were selected after overnight growth at 37°C on lysogeny broth (LB) agar plates supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin. The cells were grown at 37°C (180 rpm shaking). After the optical density reached an absorbance of 0.6 at 600 nm, the expression was induced by addition of 0.5 mM isopropyl β-D-l-thiogalactopyranoside (IPTG). The temperature was lowered to 25°C, and the cell cultures were further grown overnight. The cells were harvested by centrifugation for 20 min (4000 x g) at 4°C and the pellets were stored at-80°C. About 100 mL of cell culture pellet was thawed and solubilized with ~20 mL lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgC12, 0.1 units/mL DNase I, 10 pg/mL lysozyme, 1% v/v Triton X-100) and stirred with a vortex shaker for 1 hour at 22°C. The bacteria were then lysed by WSGR Docket Number: 64828-710.601 sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000 x g at 4°C for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Strep-Tactin resin (IBA) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (1% v/v Triton X-100, 150 mM NaCl, 15 mM Tris-HCl, pH 7.5). After 1 hour, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). In total, 10 mL of wash buffer (1% v/v Triton X-100, 150 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole) was used to wash the beads. The protein was eluted with approximately 100 pL elution buffer (2.5 mM desthiobiotin, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X-100). [1127] The genes encoding for test peptides S1 and S2 were separately transformed into E. coli. BL21 (DE3) electrocompetent cells. Transformants were selected after overnight growth at 37°C on lysogeny broth (LB) agar plates supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin. The cells were grown at 37°C (180 rpm shaking). After the optical density reached an absorbance of 0.6 at 600 nm, the expression was induced by addition of 0.5 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) at 37°C. And the cell cultures were further grown 4 hours. The cells were harvested by centrifugation for 20 min (4000 x g) at 4°C and the pellets were stored at-80°C. About 100 mL of cell culture pellet was thawed and solubilized with ~20 mL lysis buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgC12, 0.1 units/mL DNase I, 10 pg/mL lysozyme, 0.2% v/v Triton X-100) and stirred with a vortex shaker for 1 hour at 4°C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000 x g at 4°C for 20 min and the cellular debris discarded. The supernatant was mixed with 100 pL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X- 100). After 1 hour at 4°C, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre- washed with 5 mL wash buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X-100). In total, 10 mL of wash buffer (300 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole) was used to wash the beads. The protein was eluted with approximately 200 pL elution buffer (500 mM imidazole, 300 mM NaCl, 50 mM Tris- HCl, pH 7.5). [1128] The genes encoding for VAT and GFP were separately transformed into E. coli. BL21 (DE3) electrocompetent cells. Transformants were selected after overnight growth at 37°C on lysogeny broth (LB) agar plates supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin. The cells were grown at 37°C (180 rpm shaking). After the optical density reached an absorbance of 0.6 at 600 nm, the expression was induced by addition of 0.5 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) at 25°C. And the cell cultures were further grown overnight. The cells were harvested by centrifugation for 20 min (4000 x g) at 4°C and the pellets were stored at-80°C. About 100 WSGR Docket Number: 64828-710.601 mL of cell culture pellet was thawed and solubilized with ~20 mL lysis buffer (150 mM NaCl, 50 mM Tris- HCl, pH 7.5, 1 mM MgC12, 0.1 units/mL DNase I, 10 pg/mL lysozyme) and stirred with a vortex shaker for 1 hour at 4°C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000 x g at 4°C for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre- equilibrated with wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). After 1 hour at 4°C, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5). In total, 10 mL of wash buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole) was used to wash the beads. The protein was eluted with approximately 200 μL elution buffer (500 mM imidazole, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5). [1129] Proteasome co-expression and purification. For the assembly of the proteasome-nanopore, the pETDuet-1 containing the gene encoding for the α and β subunits of the proteasome and pET28a containing the gene encoding for the PA28-α∆20 nanopore plasmids were co-transformed into E. coli BL21 (DE3) electrocompetent cells. Transformants were selected after overnight growth at 37°C on LB agar plates supplemented with ampicillin (100 mg/L) and kanamycin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin and kanamycin. Protein expression was induced by 0.5 mM β-d-thiogalactopyranoside (IPTG) when the A600 reached about 0.6. The temperature was lowered to 25°C. After 12 h induction, the cells were collected, and the pellets were stored at-80°C. [1130] About 100 mL of cell culture pellet was thawed and solubilized with ~20 mL lysis buffer (150-1000 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM MgC12, 20 mM imidazole, 0.1 units/mL DNase I, 10 μg/mL lysozyme, 1% v/v Triton X-100) and stirred with a vortex shaker for 1 hour at 22°C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000 x g at 4°C for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (1% v/v Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.5). After 1 hour, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 15 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). The protein was eluted with approximately 200 μL elution buffer (500 mM imidazole, 150-1000 mM NaCl, 15 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). Subsequently, the eluted protein was mixed with 50 pL of Strep-Tactin resin (IBA) to a 2 mL tube, which was pre-equilibrated with wash buffer (1% v/v Triton X-100, 150 mM NaCl, 15 mM Tris-HCl, pH 7.5). After 30 minutes, the resin was loaded into a column (Micro Bio Spin, Bio-Rad), which was pre-washed with 5 mL wash buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% v/v Triton X-100). In total, 10 mL of wash buffer (150-1000 mM NaCl, 50 mM Tris, pH 7.5, 20 mM imidazole, 0.2% v/v Triton X-100) was used to wash the beads. The protein was eluted WSGR Docket Number: 64828-710.601 with approximately 100 pL elution buffer (2.5 mM desthiobiotin, 150-1000 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.2% v/v Triton X-100). [1131] Proteolytic activity of artificial proteasome-nanopore (complex 3). To determine the proteolytic activity of artificial proteasome-nanopore, β-casein was incubated with purified complex 3 under a variety of incubating time, temperature, and salt concentration (FIGs.42A-42C). Firstly, an aliquot of 0.1 mL β-casein (1 mg/mL) was incubated with complex 3 at 53°C in buffer A (50 mM Tris, pH 7.5, 150 mM NaCl). The final β-casein/complex 3 concentration ratio was 42 (FIG.42A). In the absence of the protease, no degradation of β-casein was observed. After 15 min of incubation at 53°C with complex 3, almost all β-casein was digested, with about three quarters of the initially observed proteins no longer detectable on SDS-PAGE. After 30 minutes of incubation, all detectable β-casein was digested. Then, a variety of temperature and salt concentration for degradation of β-casein were tested. As shown in FIG. 42B and FIG. 42C, the proteolytic activity increased with the temperature and decreased with increasing the salt concentration. [1132] Electrical recordings in planar lipid bilayers. The setup consisted of two chambers separated by a 25 pm thick polytetrafluoroethylene film (Goodfellow Cambridge Limited), which contain an aperture of approximately 100 pm in diameter, which was formed by applying a high voltage spark. To form a lipid bilayer, the aperture was pre-treated with a drop of 5% hexadecane/pentane solution. After waiting about 1-5 minutes in order to allow pentane to evaporate, 500 μL of a buffered solution (150 mM NaCl, 15 mM Tris-HCl, pH 7.5) was added to each compartment. Then a drop of l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in pentane (~10 mg/mL) was added to each compartment. After evaporation of the pentane, a lipid monolayer formed spontaneously by pipetting the solution up and down over the aperture. Silver/silver-chloride electrodes were submerged into the solution of each compartment. Nanopores were added to the trans side. All experiments were performed at ~23°C. [1133] Data recordings and analysis. Electronic signals were recorded by using an Axopatch 200B (Axon Instruments) with digitization performed with a Digidata 1440 (Axon Instruments). Clampex 10.7 software and Clampfit 10.7 software (Molecular Devices) were used for electronic signal recording and subsequent data analysis, respectively. Events were collected using the single-channel search feature in Clampfit and events shorter than 0.05 ms were ignored. [1134] Ion selectivity. The current—voltage (I-V) current signals were recorded with an automated voltage protocol that applied each potential for 0.4 s from-30 to +30 mV with 1 mV steps. Ag/AgCl electrodes were surrounded with 2.5% agarose bridges containing 2.5 M NaCl. Reversal potential was measured from extrapolation from I-V curves collected under asymmetric salt concentration condition. The experiment proceeded as follow: First an individual nanopore was reconstituted using the same buffer in both chambers (1 M NaCl, 15 mM Tris, pH 7.5, 500 μL). This allowed assessing the orientation of the nanopore and allowed WSGR Docket Number: 64828-710.601 balancing the electrodes. Then 500 μL solution containing 4 M NaCl, 15 mM Tris, pH 7.5 was slowly added to cis side and 500 μL of a buffered solution containing no NaCl (15 mM Tris, pH 7.5) was added to trans side (trans:cis, 2.0 M NaCl: 0.5 M NaCl). Example 3.1. Design of an Artificial Nanopore. [1135] A transmembrane protein device for single-molecule protein analysis is shown in FIG.38. The structure of mouse PA28α (PDB ID: 5MSJ) is denoted by 3801. The sticks diagram of the structure of serine-serine-glycine linker is represented by 3802. A ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C) can be shown by 3803. The transmembrane region of the protective antigen is shown embedded in the lipid bilayer. The lipid molecules are indicated schematically by a circular polar head region and two flexible acyl chains, 3804 shows the structure of artificial nanopore generated by molecular dynamics simulations, in accordance with some embodiments. PA28 was genetically fused to the transmembrane region of the protective antigen via a short linker. 3805 shows the structure of T. acidophilum proteasome α and β subunit (PDB ID: 1YA7). 3806 shows the structure of the designed proteasome nanopore. 3807 shows the structure of the Thermoplasma VCP-like ATPase from Thermoplasma acidophilum(VAT) (PDB ID: 5G4G), 3808 and 3809, VAT bound to the artificial nanopore. Then the translocated protein can be degraded to peptides or released. [1136] The 20S proteasome from Thermoplasma acidophilum has a cylindrical structure made of four stacked rings composed of 14 α-and 14 β-subunits (FIG.38E). The two flanking outer α-rings allow for the association of the 20S proteasome with several regulatory complexes, among which is proteasome activator PA28 (FIG. 38A) that controls the translocation of substrates into the catalytic cavity. A PA28 nanopore was designed by replacing the disorder region in a subunit of PA28 (from 163 to P100) with the transmembrane region (VHGNAEVHASFFDIGGSVSAGF; SEQ ID NO: 28) of anthrax protective antigen flanked by a short flexible linker (SSG) on each side (FIG. 38A-38D, FIG. 39A). The 22 residues of this transmembrane (TM) region were sufficient to span the hydrophobic core of a lipid bilayer. The full sequence of the transmembrane region with the linkers was set forth as: GSS VHGNAEVHASFFDIGGSVSAGFSSG (SEQ ID NO: 29). [1137] The amino acid sequence of a subunit of the artificial PA28-nanopore comprising the transmembrane region is set forth as: MATLRVHPEAQAKVDVFREDLCSKTENLLGSYFPKKISELDAFLKEPALNEANLSNLKAPLDIGSSVH GNAEVHASFFDIGGSVSAGESSGCGPVNCNEKIVVLLQRLKPEIKDVTEQLNLVTTWLQLQIPRIEDG NNFGVAVQEKVFELMTNLHTKLEGFHTQISKYFSERGDAVAKAAKQPHVGDYRQLVHELDEAEYQ EIRLMVMEIRNAYAVLYDIILKNFEKLKKPRGETKGMIYGSSWSHPQFEK (SEQ ID NO: 30). [1138] The amino acid sequence of a subunit of the artificial PA28-nanopore can be the following amino acid sequence (SEQ ID NO: 31): WSGR Docket Number: 64828-710.601
Figure imgf000325_0001
[1139] The transmembrane region of protective antigen flanked by 2 short linkers (SSG) (indicated in bold) was inserted in the polypeptide sequence of PA28α, which insertion also involved deletion of the stretch of amino acids of PA28 that is indicated in italics. [1140] In order to optimize the fusion nanopore, the length of the linker was varied by adding or removing residues on each side of the transmembrane region. The transmembrane region was inserted in the middle of PA28 via a short linker (SSG, red). Three phenylalanine and one valine residue define the lipid-water boundary and are highlighted with green squares. The side chains that point towards the outside and inside of the barrel are highlighted with gray and black lines, respectively. Each of the seven subunits contributes two β-strands separated by a turn (black line). The firstly designed nanopore is highlighted with wider arrow. One deletion mutant ( ^2) and five insertion mutants (∇2, ∇4, ∇8, ∇12, and ∇16) were prepared based on the sequence of protective antigen nanopore (FIG. 39A). With the exception of ^2, all variants could insert into the lipid bilayer. However, the insertion efficiency and subsequent bilayer stability differed amongst the mutants. ∇8, ∇12, and ∇16 showed large current fluctuations, which prevented nanopore analysis, suggesting the linker may have introduced a large conformational flexibility to the nanopore. ∇4 showed low-noise conductance with occasional full current blocks at positive applied potentials.∇4 nanopores also showed a heterogeneous unitary conductance and often closed at negative applied potentials (FIG. 39B). Among all the constructs, ∇2, which was efficiently expressed and purified, produced the most uniform pores in lipid bilayers (mean unitary conductance of 1.17 ± 0.14 nS at-35 mV, 1 M NaCl, 15 mM Tris, pH 7.5, n = 59, FIG.39C). [1141] ∇2 inserted as efficiently and as uniformly as other nanopores found in nature (e.g. alpha hemolysin). The individual peptides corresponding to the TM region of anthrax protective antigen could not form nanopores, indicating that a soluble scaffold stabilized the nanopore in lipid bilayers. Data were collected at ±35 mV in 1 M NaCl, 15 mM Tris, pH 7.5 using 10 kHz sampling rate and a 2 kHz low-pass Bessel filter. WSGR Docket Number: 64828-710.601 [1142] Molecular dynamics (MD) simulations were performed on the ∇2 PA-nanopore (hereafter PA- nanopore) to better understand the electrostatic and hydrophobic interactions between the nanopore and the lipid bilayer. As shown in FIG.39D, the MD simulations showed two rings of hydrophobic residues anchoring the TM region to the hydrophobic edges of the bilayer, while alternated residues with aliphatic side-chains interfaced the core of the bilayer. The MD simulations also showed lumen of the pore as being kept hydrated by hydrophilic residues and the hydrophilic side-chain of the linker residues as interacting with the charged head groups of membrane lipids. Example 3.2. Electrical and Functional Properties of the Optimized Artificial Pore. [1143] FIG.40A shows the schematic of an ion-current measurement setup. The artificial pore is added to the cis side and inserted into a suspended lipid membrane. An electrical potential is applied via two Ag/AgCl electrodes, which induces a current of Na+ and Cl-ions through the nanopore (1 M NaCl, 15 mM Tris, pH 7.5). Darker gray (positive) and lighter gray (negative) regions of the pore are colored according to the vacuum electrostatic potential as calculated by PyMOL. FIG.40B shows a current signal recorded through an efficient single pore after optimization at ±35 mV. The average current value is 41.24 ± 0.02 pA at-35 mV and 45.43 ± 0.06 pA at +35 mV. Similar to other β-barrel nanopores such as αHL and aerolysin nanopore, the artificial PA-nanopore showed an asymmetric current-voltage (I-V) relationship (FIG.40C), which allowed identifying the orientation of the pore in the lipid bilayer. Ion-selectivity measurements using asymmetric NaCl concentrations (0.5 M/cis and 2 NL/trans) revealed a cation selective nanopore (PK+/PCF = 1.76 ± 0.20, FIG. 40D). The current signals were filtered at 2 kHz and sampled at 10 kHz. Throughout the example, errors indicate the standard deviations obtained from three experiments. The correct folding of the PA-nanopore was characterized using cyclodextrins (CDs), circular molecules that bind to β-barrel nanopores. α-CD, β-CD and γ-CD were added to the cis side of the artificial nanopore and the magnitude of the ionic current associated with a blockade (IB) was measured. The percentage of excluded current (Ires%), defined as [(Io-IB)/IO] X 100, where Io represents the open pore current, was used to characterize the blockade. α-CD may have translocated across the nanopore too quickly, as no current blockades were observed. By contrast, β-CD and γ-CD showed characteristic blockades (FIG.40E and FIG.40F). Finally, the ability of the nanopore to identify peptides was tested using angiotensin I and dynorphin A. The two peptides were found to induce blockades which may be easily distinguished using several parameters, including the residual current and the duration of the current blockades (FIG.40G and FIG.40H). Example 3.3. Design of an Artificial Transmembrane Proteasome. [1144] In cells, PA28 docks onto the 20S proteasome and controls the translocation of substrates into the catalytic cavity. When the proteasome was added to the cis side of individual PA28-nanopores in 1 M NaCl solutions, no clear interaction was observed. It may be that the high ionic strength used did not allow such WSGR Docket Number: 64828-710.601 interaction. The crystal structure of the Thermoplasma acidophilum proteasome in complex with PA26 from Trypanosoma brucei, a homolog of PA28, showed that the carboxy-terminal tails of PA26 slide into a pocket on the 20S proteasome, near the amino-terminus of the α subunit (FIG.41A). Hence, the C-terminal of PA28 (S231) was fused with L21 of the proteasome α subunit. In the designed protein complex the first 20 residues of the α subunit were removed, leaving the proteasome gate open towards the PA28 nanopore. The proper assembly of the proteasome required co-assembly of the α and β subunits. Thus, PA28 fused to proteasome ∆20-α subunit (PA28-α∆20 nanopore) containing an N-terminal His-tag was cloned into pET-28a vector, carrying a gene for kanamycin resistance. The proteasomal α∆12, containing a C-terminal Strep-tag, and β subunit were both cloned into a pETDuet-1 vector, carrying a gene for kanamycin resistance (FIG. 41B). PA pore was fused to the proteasome α subunit (α∆A20) with the N-terminal His-tag and cloned into pET-28a vector. Untagged B subunits and a second α subunit (α∆12) with the C-terminal Strep-tag were cloned into pETDuet-1 vector. First a His-tag affinity chromatography co-purified complex 1 and 3. Then a Strep-Tag affinity chromatography purified 3. In α∆12 the first 12 residues of the α subunit were removed allowing fast degradation of unfolded substrates without the need for a proteasome activator. The co-assembled proteasome- nanopore was then purified in two steps by affinity chromatography using 1 M NaCl, 50 mM Tris, pH 7.5 solutions (FIG. 41B). SDS-PAGE and native PAGE confirmed the successful assembly of the multi-protein complex (FIG.41C). SDS-PAGE revealed the presence of three unique bands of PAuA20 (top), α∆12 (middle), and β (bottom) with molecular weights of 52.7, 25.8, and 22.3 kDa, respectively. These results suggest that PA α∆20, β, and α∆12 can form a stable subcomplex 3. Activity assays revealed that the proteasome nanopore was active, with the proteolytic activity increasing with the temperature and decreasing with the salt concentration. The transmembrane proteasome inserted efficiently in lipid bilayers and showed low-noise current recordings; some extent of fast gating at positive potentials was observed (FIG.41D). The native PAGE showed only one band indicating that the complex is stable, shown in FIG. 41D, behavior of a single pore at ±35 mV in 1 M NaCl, 15 mM Tris, pH 7.5. The I-V curve of the proteasome-nanopore in 1 M NaCl solutions was similar to that of PA-nanopore (data not shown), suggesting that the transmembrane region was unchanged and the gate of the α-subunit was open. These results suggested that co-expression and two-step purification procedure may be used for the effective isolation of stable subcomplex 3 (PAα∆20-ββ-α∆12 nanopore) formed in E. coli, in solutions containing 1 M NaCl. Subcomplex 3 displayed some fast gating behavior at positive potential, shown in FIG.41E, a cut-through of a surface representation of artificial transmembrane proteasome shaded according to the vacuum electrostatic potential as calculated by PyMOL. FIGs. 42A-42C shows SDS- PAGE analysis the hydrolyzing activity of subcomplex 3. β-casein was incubated with subcomplex 3 at different conditions to assess hydrolyzing capability. Example 3.4. Real-Time Protein Processing. WSGR Docket Number: 64828-710.601 [1145] The activity of the transmembrane proteasome was tested using substrates containing a C-terminal ssrA tag, which mediates the interaction with VAT (Valosin-containing protein-like ATPase of Thermoplasma acidophilum), an unfoldase that threads substrate proteins through the proteasome chamber. The first substrate, named S1, was 123 amino acid long and was designed to be unstructured and to contain four stretches of 15 serine residues flanked by a group of 10 arginines and three hydrophobic residues. The second substrate was S2, a longer polypeptide of 210 amino acids. The third substrate was green fluorescent protein (GFP) carrying 10 arginines and a ssrA tag (AANDENYALAA) at the C-terminus. Table 6. Sequences for Analytes and GFP Description Sequence SEQ ID NO. S1 MGHHHHHHSSRRRRRRRRRRSSSSSSSSSSSSSSSFGYGWSSSSSSSSSSSSSSSRRR 5 RRRRRRRSSSSSSSSSSSSSSSFGYGWSSSSSSSSSSSSSSSRRRRRRRRRRSSAAND ENYALAA S2 MGHHHHHHSSRRRRRPVPLPIPVPLPIPVPLPIPRRRRRSSSSSSSSSSSSSSSSSSS 32 SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSRRRRRPVPLPIPVPLPIPVPLPIPRRR RRSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSEEEEEP VPLPIPVPLPIPVPLPIPEEEEESSAANDENYALAA GFP MGHHHHHHSSSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT 33 TFKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYK TRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANF KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF VTAAGITHEFVTAAGITHGMDELYKSSAANDENYALAA [1146] Initial tests were performed using a transmembrane proteasome, in which the proteolytic activity was removed by substituting the amino-terminal threonine 1 in the active site with alanine. Reactions were performed in 1 M NaCl, 15 mM Tris-HCl, pH 7.5, 20 mM MgC12 solutions. The addition of 20.0 µM of S1 to the cis compartment of an inactive proteasome-nanopore induced both short (average dwell time is 0.62 ±0.11 ms) and second-long current blockades (FIG.43A). Most likely, the short events represent the substrate either translocating across the nanopore, and the long events the substrate remaining blocked within the proteasome chamber. Both blockades showed a residual current close to zero (Ires% = 11.56 ± 0.13), suggesting that during translocation the unstructured substrates occluded most of the nanopore. When VAT (20.0 µM) was added in solution in the presence of 2.0 mM ATP, the second-long blockades were no longer observed (FIG.43B). More ionic current was observed during the VAT-assisted translocation events compared to un-assisted translocation events (Ires %= 83.81 ± 0.11), suggesting that the substrate may have been stretched while VAT unfolded the substrate. Several recurring current signatures were observed during translocation (average dwell time is 5.8 ± 3.9 ms), suggesting that the different features of the substrate may be reflected in the ionic signal (FIG.43B). [1147] When a GFP was used instead of S1, the current blockades became longer (average dwell time was 22.1 ± 20.2 ms) and the current signature was strikingly different compared with S1 (FIG.43B-43C ), indicating WSGR Docket Number: 64828-710.601 that the two substrates can be differentiated based on their ionic current signal. When the ATP concentration was increased to 6.0 mM, the average dwell time of GFP blockades decreased 10-fold to 2.4 ± 1.7 ms (data not shown). Hence, it was demonstrated that VAT may feed the polypeptide through the nanopore at a speed that may be tuned by the concentration of ATP. [1148] When the active proteasome was used in the presence of S1 but in the absence of VAT and ATP, uniform and short blockades were observed (FIG.43D). Their average dwell time (0.51 ± 0.03 ms) was shorter than that observed for the analogous events recorded with the inactive proteasome, suggesting that the proteasome processed at least in part the substrate during translocation. When a longer unfolded substrate was tested (S2), the average dwell time of the observed events was longer (2.26 ± 0.26 ms) and deeper residual currents were observed compared to S1, indicating that larger polypeptide fragments may have been formed. Mixtures of S1 and S2 may be readily distinguished by ionic current blockades. When S1 was tested with VAT (20.0 µM) and ATP (2.0 mM), more spaced and shorter blockades were observed (FIG.43E), suggesting that the reduced speed of polypeptide threading across the proteasomal chamber may allow more efficient degradation of the polypeptide into small peptides that could be quickly transported across the nanopore. Accordingly, when GFP was tested under the same conditions no blockades were observed, suggesting that the slower unfolding of GFP compared to the unstructured S1 may have allowed for a yet more efficient proteolysis of the substrate into yet smaller peptides. These smaller peptides may have transported across the nanopore too quickly to be observed. FIGs. 44A-44B show discrimination of substrates with proteasomal nanopore. The sequences of substrate 1 (S1) and the longer substrate 2 (S2) are shown in FIG.44A. FIG.44B shows scatter plots of fraction blockade versus time and representative blockades induced by cleaved S1 and S2 at 40°C and- 30 mV in 1 M NaCl, 15 mM Tris, pH 7.5 Example 3.5. PA26-Artificial Nanopore. [1149] In this example an artificial nanopore was designed and characterized comprising the ring-forming multimeric proteasome activator protein PA26, which is a homolog of PA28. [1150] The transmembrane sequence (bold) of anthrax protective antigen (PDB ID: 3J9C) was fused in the middle of a subunit of PA26 (PDB ID: 1YA7), from which the 12-amino acid sequence shown in italics was deleted, via 2 linkers (GSSSE ---- SNSSG). Table 7 shows the sequences of the artificial pore and its components. Table 7. Sequences of artificial PA26 pore and components Pore Component Sequence SEQ ID NO. PA26 Full sequence MGWSHPQFEKSSGPPKRAALIQNLRDSYTETSSFAVIEEWAAGTLQEIEGIAKA 34 Pore AAEAHGVIRNSTYGRAQAEKSPEQLLGVLQRYQDLCHNVYCQAETIRTVIAIRI PEHKEEDNLGVAVQHAVLKIIDELEIKTLGSGEKSGSGGAPTPIGMYALREYLS WSGR Docket Number: 64828-710.601 ARSTVEDKLLGGSSSEVHGNAEVHASFFDIGGSVSAGFSNSSGSQSPSLLLELR QIDADFMLKVELATTHLSTMVRAVINAYLLNWKKLIQPRTGSDHMVS Transmembrane VHGNAEVHASFFDIGGSVSAGF 28 sequence Linker GSSSE 35 Linker SNSSG 36 Transmembrane GSSSEVHGNAEVHASFFDIGGSVSAGFSNSSG 37 with linkers [1151] The complete sequence of an N-terminally Strep-tagged subunit of the artificial PA26-nanopore is as follows:
Figure imgf000330_0001
[1152] [1153] FIG. 45 shows the structure of the resulting artificial PA26-nanopore, and typical current signal demonstrating insertion of an individual pore. FIG. 45A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C), with the transmembrane region shown as 4501. FIG.45B shows a structure of PA26 (PDB ID: 1YA7), with FIG. 45C depicting the structure of the artificial PA26-nanopore. FIG.45D shows a current signal shows insertion of individual pore. Data were collected at ±35 mV in 1 M NaCl, 15 mM Tris, 20 mM MgCl2, pH 7.5. Example 3.6. ATPase-Artificial Nanopore. [1154] In this example an artificial nanopore was designed and characterized comprising the ring-forming multimeric Aquifex aeolicus ATPase (PDB ID: 3M0E), as an example of a protein capable of transporting a polynucleotide. [1155] The transmembrane sequence (bold) of anthrax protective antigen (PDB ID: 3J9C) was inserted in the middle of a subunit of the ATPase, from which the amino acid sequence indicated in italics was deleted (insertional replacement). The inserted TM sequence was flanked on both sides with a linker (SSSSS; SEQ ID NO.: 43) as indicated in bold. The complete sequence of a designed N-terminally Strep-tagged a subunit of the artificial ATPase-nanopore is as follows: WSGR Docket Number: 64828-710.601 [1156] [1157] FIGs. 46A-46C show the structure of the assembled subunits to provide an artificial ATPase transmembrane nanopore. FIG. 46A shows a ribbon diagram of the structure of anthrax protective antigen (PDB ID: 3J9C).4601 shows the transmembrane region of the anthrax protective antigen. FIG.46B shows the structure of Aquifex aeolicus ATPase (PDB ID: 3M0E). FIG. 46C shows the structure of artificial ATPase transmembrane pore. FIG.46D shows a current signal shows insertion and ATP hydrolysis of individual pore. The ATPase nanopore displayed gating at positive potentials. The current signals became noisy and bigger when ATP (2 mM) was added in solution. Data were collected at ±35 mV in 1 M NaCl, 15 mM Tris, 20 mM MgC12, pH 7.5. The artificial ATPase nanopore may be efficiently expressed and reconstituted into lipid bilayers to form nanopores. Addition of ATP to the solution increased the noise of the baseline nanopore, indicating that the protein was active. [1158] Herewith, another example of an artificial nanopore is provided that is based on the fusion of a beta barrel to a toroidal protein. Example 3.7. ClpP-Artificial Nanopore. [1159] In this example an artificial nanopore was designed for single-molecule protein analysis. It was based on an artificial PA28-nanopore as described in Example 3.1, fused at its N-terminus to a subunit of ClpP. ClpP (PDB ID: 1TYF) is the caseinolytic Clp protease (ClpP) from E. coll. Wang et al. (1997) Cell 91: 447-456) determined a structure of ClpP at 2.3 A resolution. The active protease resembles a hollow, solid-walled cylinder composed of two 7-fold symmetric rings stacked back-to-back. Its 14 putative proteolytic active sites may be located within a central, roughly spherical chamber approximately 51 Å in diameter. Access to the proteolytic chamber may be controlled by two axial pores, each having a minimum diameter of approximately 10 Å. WSGR Docket Number: 64828-710.601 [1160] The sequence of a designed C-terminally Strep-tagged subunit of the artificial ClpP-nanopore is shown in Table 8. Table 8. Sequences of the components of the artificial ClpP nanopore. Description Sequence SEQ ID NO. Full sequence of MGSYSGERDNFAPHMALVPMVIEQTSRGERSFDIYSRLLKERVIFLTGQVEDHMANLI 44 artificial ClpP VAQMLFLEAENPEKDIYLYINSPGGVITAGMSIYDTMQFIKPDVSTISMGQAASMGAF nanopore LLTAGAKGKRFSLPNSRVMIHQPLGGYQGQATDIEIHAREILKVKGRMNELMALHTGQ SLEQIERDTERDRFLSAPEAVEYGLVDSILTHRNATLRVHPEAQAKVDVFREDLCSKT ENLLGSYFPKKISELDAFLKEPALNEANLSNLKAPLDIGSSSEVHGNAEVHASFFDIG GSVSAGFSNSSGCGPVNCNEKIVVLLQRLKPEIKDVTEQLNLVTTWLQLQIPRIEDGN NFGVAVQEKVFELMTNLHTKLEGFHTQISKYFSERGDAVAKAAKQPHVGDYRQLVHEL DEAEYQEIRLMVMEIRNAYAVLYDIILKNFEKLKKPRGETKGMIYGSSWSHPQFEK Primary MGSYSGERDNFAPHMALVPMVIEQTSRGERSFDIYSRLLKERVIFLTGQVEDHMANLI 45 sequence of VAQMLFLEAENPEKDIYLYINSPGGVITAGMSIYDTMQFIKPDVSTISMGQAASMGAF ClpP from E. LLTAGAKGKRFSLPNSRVMIHQPLGGYQGQATDIEIHAREILKVKGRMNELMALHTGQ coli SLEQIERDTERDRFLSAPEAVEYGLVDSILTHRN PA-nanopore ATLRVHPEAQAKVDVFREDLCSKTENLLGSYFPKKISELDAFLKEPALNEANLSNLKA 46 with Strep-tag PLDIGSSSEVHGNAEVHASFFDIGGSVSAGFSNSSGCGPVNCNEKIVVLLQRLKPEIK DVTEQLNLVTTWLQLQIPRIEDGNNFGVAVQEKVFELMTNLHTKLEGFHTQISKYFSE RGDAVAKAAKQPHVGDYRQLVHELDEAEYQEIRLMVMEIRNAYAVLYDIILKNFEKLK KPRGETKGMIYGSSWSHPQFEK Strep-tag peptide WSHPQFEK 47 Linker GSS 48 Linker SSG 49 Transmembrane SEVHGNAEVHASFFDIGGSVSAGFSN 50 region [1161] Residues 1-208 (italics) represent the primary sequence of ClpP from E. coli; residues 209-462 is the PA-nanopore including the C-terminal Strep-tag peptide WSHPQFEK; underlined residues 271-273 and 300- 302 are linkers; and residues 274-299 (bold) represent the TM region. [1162] FIG.47 depicts the schematic design of the artificial ClpP-nanopore. 4701 shows the structure of PA- nanopore. 4702 show a ribbon diagram of the structure of ClpP (PDB ID: 1TYF). A PA-nanopore was genetically fused to ClpP (4703). 4704 shows a structure of the designed ClpP-nanopore. 4705 shows the structure of unfoldase ClpX (PDB ID: 3HWS). SDS-PAGE analyses of the purified ClpP-nanopore the presence of two unique bands corresponding well the molecular weights of active ClpP-PApore, active ClpP, inactive ClpP-PApore, and inactive ClpPPAα ^20 (data not shown). [1163] FIG. 48 shows current—voltage (I-V) characteristics of three different nanopores. The artificial opened and closed ClpP-nanopore did not alter the conductance of the nanopore. The current signals were recorded in 0.5 M KC1, 20 mM HEPES, pH 7.5, filtered at 2 kHz, and sampled at 10 kHz. [1164] FIG. 49 shows the controlled translocation of a protein (GFP) through the ClpP-nanopore. ClpX- assisted transport of GFP across opened ClpP-nanopore in the presence of 2.0 mM ATP. The ClpP-nanopore, WSGR Docket Number: 64828-710.601 ClpX and GFP were added to the cis side. Data were collected at 22 °C and-50 mV in 0.1 M KC1, 0.3 M NaCl, 10% glycerol, 15 mM Tris, pH 7.5, using a 10 kHz low-pass Bessel filter with a 50 kHz sampling rate. The signals were then filtered digitally with a Gaussian low-pass filter with a 5 kHz cut-off. Example 4. Tunable pore diameters. [1165] Examples 4.1-4.4 used the following materials and methods. [1166] Chemicals. Endothelin 1 (≥97%, CAS# 117399-94-7), endothelin 2 (≥97%, CAS# 123562 20-9), dynorphin A porcine (≥95%, CAS# 80448-90-4), angiotensin I (≥90%, CAS# 70937-97-2), angiotensin II (≥93%, CAS# 4474-91-3), c-Myc 410-419 (≥97%, # M2435), Asnl-Val5-Angiotensin II (≥97%, CAS# 20071- 00-5), He7 Angiotensin III (≥95%, #A0911), leucine enkephalin (≥95%, #L9133), 5 methionine enkephalin (≥95%, CAS# 82362-17-2), endomorphin I (≥95%, CAS# 189388-22-5), pentane (≥99%, CAS# 109-66-0), hexadecane (99%, CAS# 544-76-3), Trizma®hydrochloride (≥99%, CAS# 1185-53-1), Trizma®base (≥99%, CAS# 77-86-1), Potassium chloride (>99%, CAS# 744740-7), NN-Dimethyldodecylamine N-oxide (LADO, >99%, CAS# 1643-20-5) were obtained from Sigma-Aldrich. Pre angiotensin 1-14 (≥97%, # 002-45), angiotensin 1-9 (≥95%, # 002-02), angiotensin A (≥95%, # 002-36), angiotensin III (≥95%, # 002-31), angiotensin IV (≥95%, # 002-28) were purchased from Pheonix Pharmaceuticals. Angiotensin 4-8 (≥95%) was synthesized by BIOMATIK. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, #850356P) and sphingomyelin (Porcine brain, # 860062) were purchased from Avanti Polar Lipids. Citric acid (99.6%, CAS# 77-92-9) was obtained from ACROS. n-Dodecyl β-D-maltoside (DDM, ≥99.5%, CAS# 69227-93-6) was bought from Glycon Biochemical EmbH. DNA primers were synthesized from Integrated DNA Technologies (IDT), enzymes from Thermo scientific. All peptides were dissolved with Milli-Q water without further purification and stored in -20°C freezer. pH 7.5 buffer containing 15 mM Tris in this study was prepared by dissolving 1.902 g Trizma®HCl and 0.354 g Trizma@ base in 1 litre Milli-Q water (Millipore, Inc). [1167] FraC monomer expression and purification. FraC gene containing NcoI and HindIl restriction site at the 5’ and 3’ ends, respectively, and a sequence encoding for a poly histidine tag at the 3’ terminus was cloned to a pT7-SC1 plasmid. Plasmids were transformed into BL21(DE3) Ecloni competent cell by electroporation. Cells were grown on LB agar plate containing 100 µg/mL ampicillin overnight at 37C. The entire plate was then harvested and inoculated into 200 mL fresh 2YT media and the culture was grown with 220 rpm shaking at 37 °C until the optical density at 600 nm of the cell culture reached .0.8. Then 0.5 mM IPTG was added to the media and the culture was transferred to 25 °C for overnight growth with 220 rpm shaking. The next day the cells were centrifuged (2000 x g, 30 minutes) and the pellet stored at -80 °C. Cell pellets harvested from 100 mL culture media were used to purify FraC monomer.30 mL of cell lysis buffer (150 mM NaCl, 15 mM Tris, 1 mM MgCl2, 4 M urea, 0.2 mg/mL lysozyme and 0.05 unit/mL DNase) was added to WSGR Docket Number: 64828-710.601 resuspend the pellet and vigorously mixed for 1 hour. Cell lysate was then sonicated with Branson Sonifier 450 for 2 minutes (duty cycle 10%, output control 3). Then the crude lysate was centrifuged down at 4 °C for 30 minutes (5400 x g), and the supernatant incubated with 100 pL Ni-NTA beads (Qiagen) for 1 hour with gentle shaking. Beads were spun down and loaded to a Micro Bio-spin column (Biorad).10 mL of SDEX buffer (150 mM NaCl, 15 mM Tris, pH 7.5) containing 20 mM imidazole was used to wash the beads, and proteins were eluded with 150 pL elution buffer (SDEX buffer, 300 mM imidazole). The concentration of protein was measured by measuring the absorption at 280 nm with Nano-drop 2000 (Thermo scientific) using the elution buffer as blank. To further confirm the purity of monomer, monomeric FraC was diluted to 0.5 mg/mL using the elution buffer and 9 pL of the diluted sample was mixed with 3 µL of 4x loading buffer (250 mM Tris HCl, pH 6.8. 8% SDS, 0.01% bromophenol blue and 40% glycerol) and then loaded to 12% SDS-PAGE gel. Gels were run for 30 min with 35 mA constant applied current, and stained with coomassie dye (InstantBlueTM, Expdedeon) for more than 1 hour before viewing using a gel imager (Gel DocTM, Bio-rad). Table 9. Sequences for FraC nanopores. Description Sequence SEQ ID NO. Wildtype (WT) FraC nanopore SADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWT 51 (Uniprot number: B9W5G6) AMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVIAY SMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEELYY HRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILE1HVTKA 6x His-Tag with WT FraC MASADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKT 52 Bold residues indicate residues of WTAMNTYFRSGTSDIVLPHKVAHGKALLYNGQKNRGPVATGVVGVI the N- and C-terminal end that AYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVYKGQKRADQRMYEEL were added to the original YYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKAGSA sequence HHHHHH 6x His-Tag with WT FraC ATGGCGAGCGCCGATGTCGCGGGTGCGGTAATCGACGGTGCGGGTC 53 (nucleotide sequence) TGGGCTTTGACGTACTGAAAACCGTGCTGGAGGCCCTGGGCAACGT TAAACGCAAAATTGCGGTAGGGATTGATAACGAATCGGGCAAGACC TGGACAGCGATGAATACCTATTTCCGTTCTGGTACGAGTGATATTG TGCTCCCACATAAGGTGGCGCATGGTAAGGCGCTGCTGTATAACGG TCAAAAAAATCGCGGTCCTGTCGCGACCGGCGTAGTGGGTGTGATT GCCTATAGTATGTCTGATGGGAACACACTGGCGGTACTGTTCTCCG TGCCGTACGATTATAATTGGTATAGCAATTGGTGGAACGTGCGTGT CTACAAAGGCCAGAAGCGTGCCGATCAGCGCATGTACGAGGAGCTG TACTATCATCGCTCGCCGTTTCGCGGCGACAACGGTTGGCATTCCC GGGGCTTAGGTTATGGACTCAAAAGTCGCGGCTTTATGAATAGTTC GGGCCACGCAATCCTGGAGATTCACGTTACCAAAGCAGGCTCTGCG CATCATCACCACCATCACTGATAAGCTT [1168] FraC mutation preparation. FraC mutants were prepared according to MEGAWHOP method.25 µL REDTaq® ReadyMixTM was mixed with 4 µM primer (see Table 5) containing the desired mutation with 50 ng WSGR Docket Number: 64828-710.601 plasmid (pT7-SC1 with wild type FraC gene) as template and the final volume was brought to 50 µL with MilliQ water. Table 10. Primer sequences used in this Example for preparing FraC mutants. Primer Name Nucleotide Sequence (in 5’ to 3’) SEQ ID NO. T7 promoter TAATACGACTCACTATAGGG 54 T7 terminator GCTAGTTATTGCTCAGCGG 55 W112S Fw ACGATTATAATAGCTATAGCAATTGGTGG 56 W116S Fw ATTGGTATAGCAATAGCTGGAACGTG 57 W112/116S Fw GTACGATTATAATAGCTATAGCAATAGCTGGAACGTGC 58 D109S ReV TGCTATACCAATTATAGCTGTACGGCA 59 [1169] The PCR protocol was initiated by 150 seconds denature step at 95 °C, followed by 30 cycles of denaturing (95 °C, 15 s), annealing (55 °C, 15 s), and extension (72 °C, 60 s). The PCR products (MEGA primer) were combined and purified using a QlAquick PCR purification kit with final DNA concentration around 200 µg/L. The second PCR was performed for whole plasmid amplification.2 µL of MEGA primer, 1 µL Phire II enzyme, 10 µL 5x Phire buffer, 1 µL 10 mM dNTPs, were mixed with PCR water to 50 µL final volume. PCR started with pre-incubated at 98 °C (30 s) and then 25 cycles of denaturing (98 °C, 5 s), annealing (72 °C, 90 s), extension (72 °C, 150 s). When the PCR was completed, 1 µL Dpn I enzyme was added and the mixture kept at 37 °C for 1 hour. Then the temperature was raised to 65 °C for 1 minute to inactivate the enzyme. Products were then transformed into E. clonil® 1OG cells (Lucigen) competent cell by electroporation. Cells were plated on LB agar plates containing 100 µg/mL ampicillin and grew at 37 °C overnight. Single clones were enriched and sent for sequencing. [1170] Sphingomyelin-DPhPC liposome preparation. 20 mg sphingomyelin and 20 mg DPhPC (1,2- diphytanoyl-sn-glycero-3 phosphocholine) were dissolved in 4 mL pentane with 0.5% v/v ethanol and brought to a 50 mL round flask. The solvent was then evaporated by rotation and using a hair-dryer to warm-up the flask. After evaporation, the flask was kept at ambient temperature for an additional 30 minutes. The lipid film was resuspended with 4 mL SDEX buffer (150 mM NaCl, 15 mM Tris, pH 7.5) and the solution immersed in a sonication bath for 5 minutes. Liposome suspensions were stored at -20°C. [1171] FraC oligomerization. FraC oligomerization was triggered by incubation of FraC monomers with sphingomelyin-DPhPC liposomes. Frozen liposome were thawed and sonicated in a water bath for one minute. FraC monomers were diluted to one mg/mL using SDEX buffer, and then 50 µL of FraC monomers were added to 50 µl of a 10 mg/mL liposome solution to obtain a mass ratio of 10:1 (liposome : protein). The lipoprotein solution was incubated at 37 °C for 30 min to allow oligomerization. Then 10 pl of 5% (w/v, 0.5% final) LADO was added to the lipoprotein solution to solubilize the liposomes. After clarification (typically 1 minute) the WSGR Docket Number: 64828-710.601 solution was transferred to a 50 mL Falcon tube. Then 10 mL of SDEX buffer containing 0.02% DDM and 100 µL of pre-washed Ni-NTA beads were added to the Falcon tube and mixed gently in shaker for 1 hour at room temperature. The beads were then spun down and loaded to a Micro Bio-spin column.10 mL wash buffer (150 mM NaCl, 15 mM Tris, 20 mM imidazole, 0.02% DDM, pH 7.5) was used to wash the beads and oligomers eluded with 100 µL elution buffer (typically 200 mM EDTA, 75 mM NaCl, 7.5 mM Tris pH 7.5,0.02% DDM). The FraC oligomers were stored at 4 °C. Under these conditions the nanopores were stable for several months. [1172] W112S-W116S-FraC oligomer separation with His-Trap chromatography. 200 µL of W112S- W116S-FraC monomers (3 mg/mL) were incubated with 300 µL of Sphingomyelin-DPhPC liposome (10 mg/mL) and kept at 4 °C for 48 hours after which 0.5% LADO (final concentration) was added to solubilize the lipoprotein. Then the buffer was exchanged to the 500 mM NaCl, 15 mM Tris, 0.01% DDM, 30 mM imidazole, pH 7.5 (binding buffer) using a PD SpinTrap G-25 column. W112S-W116S-FraC oligomers were then loaded to Histrap HP 1 mL column (General Electric) using an AKTA pure FPLC system (General Electric). The loaded oligomers were washed with 10 column volumes of 500 mM NaCl, 15 mM Tris, 0.01% DDM, 30 mM imidazole, pH 7.5, prior applying an imidazole gradient (from 30 mM to 1 M imidazole, 500 mM NaCl, 15 mM Tris, 0.01% DDM, pH 7.5) over 30 column volumes. The signal was monitored with the absorbance at 280 nm and fractions were collected when the absorbance was higher than 5 mAu. [1173] Electrophysiology measurement and data analysis. Electrical recordings were performed as explained in details previously 27,37. Io, referring to open pore current, were measured by fitting Gaussian functions to event amplitude histograms. Residual current values (Ires%) were calculated by dividing the blockade current (IB) by open pore current (IB/1O X 100%). Dwell times and inter-event times were measured by fitting single exponentials to histograms of cumulative distribution. [1174] Ion permeability measurement. In order to measure reversal potentials, a single channel was obtained under symmetric conditions (840 mM KCl, 500 µL in each electrophysiology chamber) and the electrodes were balanced. The 400 µL of a buffered stock solution of 3.36 M KCl was slowly added to cis chamber, while 400 µL of salt free buffered solution was added to the trans chamber to obtain a total volume of 900 µL (trans:cis, 467 mM KCl:1960 mM KCl). After the equilibrium was reached, IV curves were collected from -30 to + 30 mV. The resulting voltage at zero current is the reversal potential (Vr). The ion selectivity (^^ ^/^^^ ^) was then calculated using the Goldman-Hodgkin-Katz equation where ^^ + - ^^ /^^ ^ ^Cis/trans is the activity of the K or Cl in the cis or trans compartment, R the gas constant, T the temperature and F the Faraday’s constant. WSGR Docket Number: 64828-710.601 [1175] The activity of ions was calculated by multiplying the molar concentration of the ion for the mean ion mobility (0.649 for 500 mM KCl, and 0.573 for 2000 mM). Ag/AgC1 electrodes were surrounded by 2.5% agarose bridge in 2.5 M NaCl. [1176] Molecular models of Type I, II and III FraC nanopores. The 3D models with different multimeric order, ranging from five to nine monomers, were constructed with the symmetrical docking function of Rosetta 3 . A monomer without lipids was extracted from the crystal structure of FraC with lipids (PDBID 4tsy). Symmetrical docking arranged this monomer around a central rotational axis ranging in order form 5 to 9. In total Rosetta generated and scored 10000 copies for each symmetry. In all cases, a multimeric organization with a symmetry similar to the crystal structure may be identified as a top scoring solution. However, in the pentameric assembly the multimer interface was not fully satisfied as compared to the crystal structure, with large portions left exposed. The 9-fold symmetric model however exhibited a significant drop in Rosetta score compared to the 6- 7- and 8-fold symmetric models indicating an unfavored assembly of the nonameric assembly with the 6- 7- and 8-fold assemblies as the most plausible. To create lipid bound models, the crystal structure with lipids was superimposed on each monomer of the generated models, allowing the lipid coordinates to be transferred. The residues within 4.5 angstrom of the lipids were minimized with the Amber10 forcefield. Example 4.1. Engineering the size of FraC Nanopores. [1177] In this example different nanopore sizes were obtained by engineering the protein oligomeric composition. FIG. 50A shows a cut-through representation of wildtype (WT) FraC pore. One protomer is shown as a carton presentation with tryptophans 112 and 116 displayed as spheres. At pH 7.5 a small fraction of Wild Type FraC (WT-FraC) nanopores showed a lower conductance (1.26 ±0.08 nS, -50 mV, type II WT- FraC, FIG.50B) compared to the dominant fraction (2.26 ±0.08 nS, -50 mV, type I WT-FraC), suggesting that FraC might be able spontaneously to assemble into nanopores with smaller size. At pH 4.5 yet a smaller nanopore conductance was observed (0.42 ±0.03 nS, type III WT FraC, -50 mV, FIG. 50B). It was observed that the reconstitution of lower conductance nanopores depended to several purification conditions. It was observed that the occurrence of type II and type III nanopores increased when the oligomers were stored in solution for several weeks or when the concentration of monomeric WT-FraC was reduced during oligomerisation. [1178] In an attempt to enrich for type II and type III FraC nanopores, the interaction between the nanopore and the lipid interface was weakened by substituting W112 and W116 at the lipid interface of FraC (FIG.50A) with serine. without being bound to a particular theory, it was proposed that a lower concentration of monomers during liposome-triggered oligomerisation may increase the population of lower molecular mass oligomers. It was found that at pH 4.5 using W116S-FraC and W112S-W116S-FraC oligomers, type II and type III FraC WSGR Docket Number: 64828-710.601 nanopores were the dominant species, respectively (FIG.50B, FIG. 51).The different nanopore types may be separated by Ni NTA affinity chromatography using an imidazole gradient. It was found that enrichment of type II and type III FraC nanopores may also be obtained at pH 7.5 by replacing aspartate 109 at the lipid interface with serine (see FIG.50E, Table 11). Table 11: relative amounts of Type I, Type II and Type III for each of the FraC nanopores investigated at neutral and acidic pH. Type 1(%) SD Type 11(%) SD Type SD III(%) pH 7.5 Wild type 85.7 3.8 14.3 3.8 0.0 0.0 W112S 61.9 4.3 38.1 4.3 0.0 0.0 W116S 61.1 5.7 38.9 5.7 0.0 0.0 W112116S 27.1 3.9 72.9 3.9 0.0 0.0 D109S 50.3 3.8 48.0 3.6 1.7 1.5 D109SW116S 29.3 9.5 66.7 8.4 4.0 4.0 pH 4.5 Wild type 42.5 10.6 51.9 7.7 5.6 7.9 W116S 29.0 4.0 47.0 4.6 24.0 2.3 W112SW116S 21.7 4.7 38.0 8.5 40.3 9.3 D109S 35.7 2.1 56.3 9.1 8.0 7.0 D109SW116S 19.3 8.4 64.3 6.7 16.3 3.2 [1179] Among FraC nanopores of the same type, the lipid interface modifications caused by W112S and W116S substitutions did not alter the conductance and ion selectivity as compared to that of wild type (FIG. 50C, FIGs. 51A-51F, Table 7) suggesting that the modifications may not have altered the overall fold of the nanopores. When characterized in lipid bilayers, type I, type II and type III nanopores showed a well-defined single conductance distribution and a steady open pore current (FIG.50D-50E). Type I, Type II and Type III nanopores showed increasing cation selectivity (from 2.0 for type I to 4.2 for type III W116S-FraC nanopores at pH 4.5 (FIG. 50F, Table 12), possibly reflecting a larger overlap of the electrical double layer in the nanopores with a narrower constriction. For the conductance measures of FIGs.51B-51F, single channels were collected under -50 mV applied potential. pH 7.5 and 4.5 were obtained using 1 M NaCl, 15 mM Tris, or 1 M KCl, 0.1 M citric acid, 180 mM Tris base respectively. Table 12: Ion selectivity of different FraC pores at pH 7.5 and 4.5. pH 7.5 pH 4.5 Reversal PK+ Pcl- Reversal PK+ Pcl- potential (mV) potential (mV) Type 1 17.2±1.2 3.6±0.4 10.5±1.4 2.1 ±0.2 WT-FraC Type II 20.8±1.6 5.2±0.9 12.3±1.2 2.4±0.2 Type III / / 20.6±1.1 5.0±0.6 Type 1 / / 10.1 ±0.9 2.0±0.1 W116S-FraC Type II / / 12.8±0.7 2.5±0.2 WSGR Docket Number: 64828-710.601 Type III / / 18.8±0.5 4.2±0.2 Type I / / 8.8±1.2 1.9±0.2 W112S-W116S-FraC Type II / / 14.0±0.1 2.8±0.1 Type III / / 20.1 ±0.6 4.8±0.3 [1180] The ion selectivity (PK+/PC1-) was calculated from the reversal potential according to the Goldman- Hodgkin-Katz equation:
Figure imgf000339_0001
[1181] Where Vr is the reversal potential, P - K+/PCl the ion selectivity, a the activity of ions and F the Farady constant. Electrophysiology recordings were carried out with 1960 mM KC1 in the cis solution and 467 mM KCl in the trans solution. The activity of ions was calculated by multiplying the molar concentration of the ion for the mean ion mobility (0.649 for 500 mM KCl, and 0.573 for 2000 mM). Errors are given as standard deviations calculated from 3 experiments at least. [1182] Without being bound to a particular theory, these findings suggested that the three types of FraC nanopores may represent nanopores with different protomeric compositions. Molecular modelling allowed predicting the diameter of type 11 (1.1 nm) and type III (0.8 nm) nanopores (FIG.50G); and revealed that type III FraC was potentially the biological nanopore with the smallest constriction known at the time of experimentation. Example 4.2. Identification of peptides containing single amino acid substitutions using type II or type III FraC nanopores as sensor. [1183] Type II FraC nanopores were used to sample a series of angiotensin peptides, which in blood regulate blood pressure and fluid balance. The peptides were added to the cis side of type II W116S-FraC nanopores and the induced ionic current blockades (IB) was measured. Residual currents percent (Ireso%, defined as IB / 1O x 100) were used instead of current blockades because they provided more reliable values when comparing different nanopores. Results are shown in FIG.52A and Table 8. [1184] Angiotensin I (DRVY1HPFHL, 1296.5 Da), showed the deepest blockade (Ires%= 8.8±0.2) and angiotensin IV (VYIHPF, 774.9 Da) the shallowest blockade (Ireso= 38.9±4.0). The residual current of angiotensin II (DRVYIHPF, 1046.2 Da, Ires%= 17.9±1.3) and angiotensin III (RVYIHPF, 931.1 Da, Ires%= 22.1±0.5) fell at intermediate values. When the four peptides were tested simultaneously, individual peptides may be readily discriminated (FIG. 52A). All measurements and recordings were performed in pH 4.5 buffer containing 1 M KCl, 0.1 M citric acid, 180 mM Tris base with a 50 kHz sampling and 10 kHz filter. Standard deviations were calculated from minimum three repeats. Density plot was created with Origin. Table 13: Peptide analysis using different types of FraC nanopores at pH 4.5 WSGR Docket Number: 64828-710.601 [1185] The electrophysiology solution contained 1 M KCl, 0.1 M citric acid, 180 mM Tris base at pH 4.5. Recordings were performed using a 50 kHz sampling and applying 10 kHz Bassel filter. Standard deviations were obtained for at least three measurements. The charges of the peptides were calculated according to the pKa for individual amino acid.
WSGR Docket Number: 64828-710.601 Charge Peptide Sequence Molecular PH 7.5 pH 4.5 Ires% (I /I )% Dwell time weight (g/mol) pH4.5 (ms) WT-FraC type I pore, -30 mV Endothelin 2 CSCSSWLDKECVYFCHLDIIW (SEQ 2546.9 -2.15 0.36 6.1±1.8 104.0±29.9 ID NO.: 60) Endothelin 1 CSCSSLMDKECVYFCHLDIIW (SEQ 2491.9 -2.15 0.36 7.5±0.5 19.73±1.95 ID NO.: 61) Dynorphin A YGGFLRRIRPKLKWDNQ (SEQ ID 2147.5 3.76 4.48 15.1±2.6 3.68±0.76 NO.: 62) Pre angiotensinogen DRVYIHPFHLVIHN (SEQ ID NO.: 1758.9 0.03 3.45 24.6±2.3 0.29±0.04 63) W116S-FraC type II pore, -30 mV Angiotensin I DRVYIHPFHL (SEQ ID NO.: 64) 1296.5 -0.06 2.46 43.4±0.9 0.15±0.04 Angiotensin I DRVYIHPFHL (SEQ ID NO.: 64) 1296.5 -0.06 2.46 8.8±0.2 0.54±0.01 c-Myc 410-419 EQKLISEEDL (SEQ ID NO.: 65) 1203.3 -3.24 -1.19 30.0±3.4 0.12±0.01 Angiotensin 1-9 DRVYIHPFH (SEQ ID NO.: 66) 1183.3 -0.06 2.46 14.0±0.2 0.37±0.04 Angiotensin II DRVYIHPF (SEQ ID NO.: 67) 1046.2 -0.15 1.47 17.9±1.3 0.37±0.04 AsnlVal5 NRVYVHPF (SEQ ID NO.: 68) 1031.2 0.85 2.03 19.6±0.2 0.34±0.06 Angiotensin II Angiotensin A ARVYIHPF (SEQ ID NO.: 69) 1002.2 0.85 2.03 21.0±0.6 0.34±0.02 Angiotensin III RVYIHPF (SEQ ID NO.: 70) 931.1 0.85 2.03 22.1±0.5 0.35±0.04 Ile7 Angiotensin III RVYIHPI (SEQ ID NO.: 71) 897.1 0.85 2.03 24.3±0.4 0.19±0.05 Angiotensin IV VYIHPF (SEQ ID NO.: 72) 774.9 -0.15 1.02 38.9±4.0 0.15±0.06
WSGR Docket Number: 64828-710.601 W112S-W116S-FraC type III pore, -50 mV Angiotensin IV VYIHPF (SEQ ID NO.: 73) 774.9 -0.15 1.02 1.1+0.8 0.61±0.07 Angiotensin 4-8 YIHPF (SEQ ID NO.: 74) 675.8 -0.15 1.02 8.2+0.4 0.40+0.04 Endomorphin I YPWF (SEQ ID NO.: 75) 610.7 -0.24 0.04 19.2+0.5 0.32+0.04 Met5 Enkephalin YGGFM (SEQ ID NO.: 76) 573.7 -0.24 0.04 33.5±0.7 0.16+0.02 Leucine Enkephalin YGGFL (SEQ ID NO.: 77) 555.6 -0.24 0.04 34.5±2.4 0.20+0.05
WSGR Docket No.64828-710.601 [1186] The resolution limit of the nanopore sensor was challenged by sampling a mixture of peptides. Angiotensin II and angiotensin A, having an identical composition except for the initial amino acid (aspartate in angiotensin II vs. alanine in angiotensin A), appeared as distinctive peaks in Ires% plots (FIG.52B). Smaller differences in peptide mass (e.g. the 34 Da difference between phenylalanine and leucine in angiotensin III and He7 angiotensin III) were observed but not easily detected (data not shown), indicating the resolution of the system at ~40 Da. Smaller peptides such as angiotensin II4-8 (Y1HPF, 675.8 Da), endomorphin I (YPWF, 610.7 Da) or leucine enkephalin (YGGFL, 555.6 Da) translocated too quickly across type II W116S-FraC nanopores to be sampled. However, they could be readily measured using type III W112S-W116S-FraC nanopores (Table 8; FIG.55). Example 4.3. A nanopore mass spectrometer for peptides. [1187] In an effort to assess FraC nanopores as peptide mass analyzer, additional peptides were tested at pH 4.5 and 1 M KCl using type I, type II and type III FraC nanopores (FIGS. 53A-53C, Table 8). Crucially, analytes with largely different charge compositions were included. Current blockades were measured at -30 mV for type I and II pore, and at -50 mV for type III pore. Error bars are standard deviations obtained from at least three measurements. [1188] It was found that for most of peptides there was a direct correlation between the size and the residual current (FIGS. 53A-53C). An exception was c-Myc 410-419 (1203.3 Da), an intentionally selected peptide because it includes a long stretch of negatively charged residues (FIG. 54A). The overall negative charge of the peptide at pH 4.5 (see Table 8) was expected to have an effect on both peptide capture and recognition. c Myc 410-419 could be readily captured at negative applied potentials (trans), indicating that the cis to trans electroosmotic flow across the nanopore can overcome the electrostatic energy barrier opposing peptide capture. The Ires% of c-Myc 410-419 (30.0 ±3.4) was higher than the expected value (FIG.53B). [1189] Without being bound to a particular theory, such anomaly might be due to the interaction between the acidic amino acids of the peptide and the negatively charged constriction of FraC nanopores. To test this, the pH solution to values where the aspartate and glutamate side chains in the peptides were expected to be protonated was lowered, hence become neutral (FIG.54A).At pH 3.8, the signal corresponding to c-Myc 410- 419 (1203.3 Da) fell between the signal of angiotensin 1 (1296.5 Da), and angiotensin 11 (1046.2 Da, FIG. 54B, c). This indicates that, after losing its negative charges, the peptide blockades may have scaled with the expected mass of the peptides. [1190] The voltage dependence of the average dwell time (Toff) can report on the translocation of a molecule across a nanopore. Under a negative bias (trans) for positively charged peptides (added in cis) both electrophoretic and electroosmotic forces (from cis to trans) can promote the entry and translocation across the nanopore. For negatively charged peptides, such as c-Myc 410-419 at pH 4.5 (FIG.54A), the electroosmotic driving force may need to be stronger than the opposing electrophoretic force. The voltage dependence of Toff WSGR Docket Number: 64828-710.601 was examined for c-Myc 410-419 at different pH values (FIG. 54D). At pH 4.5 the peptide exhibited a maximum in Toff at -50 mV, suggesting that at low potentials c-Myc 410-419 can return to the cis chamber (<50 mV), and at higher potentials (>50 mV) c-Myc 410-419 can exit to the trans chamber. At pH 3.8 and lower, a decrease in Toff was observed at higher potentials, indicating that c-Myc 410419 crosses the membrane to the trans chamber. All electrophysiology measurements were carried out in 1 M KCl, 0.1 M citric acid, and pH was adjusted with 1 M Tris base to desired values. 50 kHz sampling rate and 10 kHz filter was used for collecting the current events. Error bars are standard deviations obtained from at least three measurements. The charges of the peptides were calculated according to the pKa for individual amino acids. [1191] As shown in FIGs.55A-55C, type III FraC nanopore can detect differences in peptide length down to 4 amino acids (mass around 500 Dalton) in a peptide mixture. FIG. 55B shows current blockades provoked by the different peptides. It was also found that the residual current signal correlated well with the mass of peptides, suggesting that Type III can be used as a detector for a peptide having a mass down to ~500 Da. All measurements and recordings were performed in pH 4.5 buffer containing 1 M KCl, 0.1 M citric acid, 180 mM Tris base with a 50 kHz sampling and 10 kHz filter. Standard deviations were calculated from three repeats at least. Example 4.4. Peptide mass identifier at pH 3. [1192] This example shows that mutation D1OC can be used as additional mutation to obtain a FraC pore showing a quiet signal in electrophysiology recordings. [1193] Using mutant W116S as exemplary mutant, the aspartic acid at position 10 of FraC was converted to cysteine by site-directed mutagenesis. The thiol group of cysteine was then oxidized to sulfonic acid by incubation of FraC monomers with 10% hydrogen peroxide (v/v), which was dissolved in regular buffer (e.g. 10 mM Tris buffer pH 7.5, 150 mM NaCl). As a control, the double mutant was left without oxidation. [1194] D10C/W116S FraC was oligomerized, and the oligomers tested in electrical recordings. FIGs. 56A- 56B show the signal comparison between the D1OC/ W116S pore and oxidized DIOC/ W116S pore to demonstrate the difference after oxidization. Recordings were performed in a buffer containing 1 M NaCl, pH 7.5, ±50 mV. Oligomerized pores from oxidized DIOC / W116S FraC monomers showed a quiet signal in electrophysiology recordings, as compared to a more noisy signal observed for nanopores that had not been subjected to oxidation. Example 5. FraC actinoporin. [1195] Examples 5.1-5.8 used the following methods and materials -342- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1196] Materials. Unless otherwise specified, all chemicals were bought from Sigma-Aldrich. DNA was purchased from Integrated DNA Technologies (IDT). Enzymes were acquired from Fermentas and lipids from Avanti Polar Lipids. All errors in this example are given as standard deviations. [1197] FraC cloning. To allow cloning, a Neo I restriction site (CCATGG) was introduced at the beginning of the DNA sequence (5’ end) corresponding to mature WtFraC from A. fragacea. To maintain the reading frame an additional two bases were inserted after the Neo I site, resulting in an additional alanine residue after the starting methionine. For purification purposes, at the C-terminus of FraC, a His9 affinity tag was attached via a flexible glycine-serine-alanine linker and the open reading frame was terminated by two consecutive stop codons, followed by a Hind III restriction site (3’ end). 50 ng of the synthetic gene with optimized codon composition (IDT) served as a template for the following PCR reaction: the gene was amplified by Phire Hot Start II DNA polymerase (Finnzymes) using 6 µM of primers Frf and Frr (Table 9) in 300 µL volume. The PCR protocol was as follows: a pre-incubation step at 98°C for 30 s was followed by 30 cycles of denaturation at 98°C for 5 s and extension at 72°C for 1 min. The resulting PCR product containing the Hi9- tagged WtFraC gene was purified with QIAquick PCR Purification Kit (Qiagen) and digested with Neo I and Hind HI (FastDigest, Fermentas). The gel purified insert (QIAquick Gel Extraction Kit, Qiagen) was cloned under control of the T7 promoter into the pT7-SCl expression plasmid using sticky-end ligation (T4 ligase, Fermentas) via Neo I (5’) and Hind III (3’) sites. Of the ligation mixture 0.6 µL was transformed into 50 µL of E. cloni® 10G cells (Lucigen) by electroporation. The transformed bacteria were grown overnight at 37°C on ampicillin (100 µg/ml) LB agar plates. The identity of the clones was confirmed by sequencing. [1198] Construction of 10R FraC. Of the pT7-SCl plasmid containing the WtFraC gene, 100 ng served as a template for PCR reaction: the gene was amplified by Phire Hot Start II DNA polymerase (Finnzymes) using 6 µM of primers 10Rf (encoding for D10R) and T7 terminator (Table 9) in a 300 µL volume. The PCR reaction cycling protocol was as follows: a pre-incubation step at 98°C for 30 s was followed by 30 cycles of denaturation at 98°C for 5 s and extension at 72°C for 1 min. The PCR product was gel purified (QIAquick Gel Extraction Kit, Qiagen) and cloned into a pT7 expression plasmid (pT7-SCl) by MEGAWHOP procedure: about 500 ng of the purified PCR product was mixed with about 300 ng of the pT7-SCl plasmid containing WtFraC gene and the amplification was carried out with Phire Hot Start II DNA polymerase (Finnzymes) in 50 µL final volume (pre-incubation at 98°C for 30s, then 30 cycles of: denaturation at 98°C for 5 s, extension at 72°C for 1.5 min). The circular template was eliminated by incubation with Dpn I (1 FDU) for 2 hr at 37°C. Of the resulting mixture 0.6 µL was transformed into E. cloni® 10G cells (Lucigen) by electroporation. The transformed bacteria were grown overnight at 37°C on ampicillin (100 µg/ml) containing LB agar plates. The identity of the clones was confirmed by sequencing. [1199] Construction of 10R FraC libraries by error-prone PCR. -343- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1200] Libraries were constructed by amplifying the D10R FraC gene from plasmid DNA using T7 promoter and T7 terminator primers (Table 14). Table 14: Oligonucleotides employed in this study. [1201] “5Biosg” stands for biotin group conjugated to 5’ end of DNA via C6 linker (IDT). Name DNA sequence SEQ ID NO. Frf atatatatatccATGGCGAGCGCCGATGTCGCGGGTGCGG 78 Frr atatatatatAAGCTTATCAGTGATGGTGGTGATGATGCGCAG 79 10Rf GCCGATGTCGCGGGTGCGGTAATCcgtGGTGCGGGTCTGGGCI1IGACGIAC 80 Oligonucleot /5Biosg/AAAAAAAAAAAAAAAAAAAAGTGCTACGACTCTCTGTGTGCCCCCC 81 ide I CCCCCCCCCCCCCC Oligonucleot CACACAGAGAGTCGTAGCAC 82 ide II A20 /5Biosg/ATATATAAAAAAAAAAAAAAAAAAAA 83 C20 /5Biosg/ATATATCCCCCCCCCCCCCCCCCCCC 84 T20 /5Biosg/ATATATTTTTTTTTTTTTTTTTTTTT 85 T7- GCTAGTTATTGCTCAGCGG 86 terminator T7-promoter TAATACGACTCACTATAGGG 87 [1202] In the first round of mutagenesis a plasmid containing the synthetic gene encoding for 10R FraC was used as a template. In the second mutagenesis round the pool of DNA plasmids from the clones with highest activity identified in the first round of screening was used as a template. DNA amplification was performed by error prone PCR: 400 µL of PCR mix (200 µl of REDTaq ReadyMix, 6 µM T7 promoter and T7 terminator primers, ~400 ng of plasmid template) was split into 8 reaction volumes containing from 0 to 0.2 mM of MnCl2 and cycled for 27 times (pre-incubation at 95°C for 3 min, then cycling: denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 3 min). These conditions typically yielded 1-4 mutations per gene in the final library. The PCR products were pooled together, gel purified (QIAquick Gel Extraction Kit, Qiagen) and cloned into a pT7 expression plasmid (pT7-SCl) by MEGA WHOP procedure: ~500 ng of the purified PCR product was mixed with ~300 ng of pT7-SCl plasmid containing 10R FraC gene and the amplification was carried out with Phire Hot Start II DNA polymerase (Finnzymes) in 50 µL final volume (pre-incubation at 98°C for 30s followed by 30 cycles: denaturation at 98°C for 5 s, extension at 72°C for 1.5 min). The circular template was eliminated by incubation with Dpn I (1 FDU) for 2 hr at 37°C. Of the resulting mixture 0.6 µL was transformed into E. cloni® 10G cells (Lucigen) by electroporation. The transformed bacteria were grown overnight at 37°C on ampicillin (100 µ/ml) LB agar plates typically resulting in >105 colonies which were harvested for plasmid DNA library preparation. -344- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1203] Screening for hemolytic activity in crude lysates after FraC overexpression. Overnight starter cultures from 600 clones (see above) were inoculated into 450 µL of fresh medium in new 96-deep-well plates and cultures were grown at 37°C until OD600~0.8. Then, IPTG (0.5 mM) was added to induce overexpression and the temperature was reduced to 25°C for an overnight incubation. Bacteria were harvested the following day by centrifugation at 3000 x g for 15 min at 4°C. The supernatant was discarded and pellets were frozen at -80°C for two hours to facilitate cell disruption. Cell pellets were then resuspended in 0.4 mL of lysis buffer (15 mM Tris-HCl pH 7.5, 1 mM MgC12, 10 µg/ml lysozyme, 0.2 units/mL DNAse I) and lysed by shaking at 1300 RPM for 30 min at 37 °C. Of the crude lysate 0.5-5 µL were then added to 100 µL of ~1% horse erythrocytes suspension. The latter was prepared by centrifuging horse blood (bioMérieux Benelux) at 6000 x g for 5 min at 4°C and pellet resuspension in 15 mM Tris-HCl pH 7.5, 150 mM NaCl. If the supernatant had a red color, the solution was centrifuged again and the pellet resuspended in the same buffer. The hemolytic activity was monitored by the decrease in OD at 650 nm over time (Multiskan GO Microplate Spectrophotometer, Thermo Scientific). [1204] Screening for mutations that compensate for deleterious effects of D10R amino acid substitution in FraC. D10R amino acid substitution resulted in ~5 fold decrease in hemolytic activity of FraC (FIG.61). Although D10R FraC still may be oligomerized and reconstituted in planar bilayers made from 1,2- diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) a search was conducted for compensatory mutations that recovered the hemolytic activity of D10R FraC back to the WtFraC level. Maintaining the hemolytic activity of FraC may be important for two reasons: firstly, it may indicate the ability to assemble into oligomeric pores on targeted lipid bilayers and, therefore, may translate into more efficient preparation of oligomeric nanopores. Hemolytic activity offers a convenient way to screen the functionality of variants with arbitrary amino acid sequences changes and thus may facilitate future engineering efforts on the FraC nanopore. In order to identify compensatory mutations, a random mutagenesis library was constructed based on D10R FraC gene, then transformed into B121 DE3 E. coli and individual variants were screened for hemolytic activity against horse erythrocytes in crude lysates after overexpression, using WtFraC as reference. In the first round, 600 variants were screened and 12 clones selected as a template for second round of random mutagenesis combined with hemolytic activity screening. Then, 7 clones with the highest level of hemolytic activity were selected for further characterization. Sequence changes that occurred in the corresponding genes are summarized in Table 15. Table 15: Sequence changes that compensate for deleterious effects of D10R mutation in FraC. FraC variant Amino acid sequence changes relative to WtFraC (residue numbering as in crystal structure PDB ID 4TSY) 1 D10R, T150I, W112L -345- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 2 I9T, D10R 3 A2S, D10R, G153D 4 D10R, A34V, A159E ReFraC D10R, K159E 5 D10R, I171T 6 I9T, D10R, F52Y, K159E [1205] Purified variants were oligomerized in sphingomyelin:DPhPC (1:1) liposomes and solubilized in 0.6% LDAO. After exchanging the detergent to 0.02% DDM by Ni-NTA chromatography, oligomeric proteins were tested for pore-forming activity in planar lipid bilayers composed of DPhPC. Initially, variants named 3, 4 and ReFraC (Table 10) were identified as the most promising pore-formers. Nanopores made by variant 3 were heterogeneous (less than 50% yielded octameric pores), while pore-forming activity of variant 4 was decaying within days when stored at 4°C. [1206] Oligomeric ReFraC maintained pore–forming activity for months when stored at 4°C and formed nanopores nearly as uniform as a WtFraC while being able to capture ssDNA. Therefore, ReFraC was selected for further DNA analysis in this example. Aspartate 10 was replaced with asparagine in ReFraC yielding a D10N K159E variant, but ssDNA entry was not detected. [1207] >WtFraC (protein sequence) [1208] MASADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSD IVLPHKVAHGKALLYNGQKNRGPVATGWGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVRVY KGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKAGSAHHHH HH** (SEQ ID NO.: 130) [1209] >WtFraC (DNA sequence) [1210] ATGGCGAGCGCCGATGTCGCGGGTGCGGTAATCGACGGTGCGGGTCTGGGCTTTGACGT ACTGAAAACCGTGCTGGAGGCCCTGGGCAACGTTAAACGCAAAATTGCGGTAGGGATTGATAA CGAATCGGGCAAGACCTGGACAGCGATGAATACCTATTTCCGTTCTGGTACGAGTGATATTGTG CTCCCACATAAGGTGGCGCTGGTAAGGCGCTGCTGTATAACGGTCAAAAAAATCGCGGTCCTGT CGCGACCGGCGTAGTGGGTGTGATTGCCTATAGTATGTCTGATGGGAACACACTGGCGGTACTG TTCTCCGTGCCGTACGATTATAATTGGTATAGCAATTGGTGGAACGTGCGTGTCTACAAAGGCC AGAAGCGTGCCGATCAGCGCATGTACGAGGAGCTGTACTATCATCGCTCGCCGTTTCGCGGCGA CAACGGTTGGCATTCCCGGGGCTTAGGTTATGGACTCAAAAGTCGCGGCTTTATGAATAGTTCG GGCCACGCAATCCTGGAGATTCACGTTACCAAAGCAGGCTCTGCGCATCATCACCACCATCACT GATAAGCTT (SEQ ID NO.: 131) [1211] FraC overexpression and purification. E. cloni® EXPRESS BL21 (DE3) cells were transformed with the pT7-SCl plasmid containing the FraC gene. Transformants were selected after overnight growth at -346- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 37°C on LB agar plates supplemented with 100 mg/L ampicillin. The resulting colonies were inoculated into 2xYT medium (Sigma) containing 100 mg/L of ampicillin. The culture was grown at 37°C, with shaking at 200 rpm, until it reached an OD600 of ~ 0.8. The expression of FraC was then induced by the addition of 0.5 mM IPTG and the growth was continued at 25°C. The next day the bacteria were harvested by centrifugation at 6000 x g for 25 min and pellets were stored at -80°C. The pellets (derived from 50-100 ml of bacterial culture) containing monomeric FraC were thawed and resuspended in 40 ml of 15 mM Tris-HCl pH 7.5, 1 mM MgC12 and 0.05 units/mL of DNase I (Fermentas). Then, to initiate cell disruption, bacteria suspension was supplemented with 0.2 mg/ml lysozyme and 2 M urea (to prevent debris formation) and was subjected to vigorous shaking at ambient temperature for 40 min. The remaining bacteria were disrupted by probe sonication. The crude lysates were clarified by centrifugation at 6000 x g for 20 min and supernatant mixed with 200 µL (bead volume) of Ni-NTA resin (Qiagen) that was pre-equilibrated with wash buffer (10 mM Imidazole 150 mM NaCl, 15 mM Tris-HCl pH 7.5 ). [1212] After 1 hour of gentle mixing at ambient temperature, the resin was loaded onto a column (Micro Bio Spin, Bio-Rad) and washed with ~5 ml of wash buffer. FraC was eluted with approximately ~0.5 mL of wash buffer containing 300 mM imidazole. Protein concentration was determined by the Bradford assay. FraC monomers were stored at 4°C until further use. [1213] Hemolytic activity assay. Defibrinated horse blood (bioMérieux Benelux) was washed with 150 mM NaCl, 15 mM Tris-HCl pH 7.5 until the supernatant was clear. The erythrocytes were then resuspended with the same buffer to ~1% concentration (OD 650nm 0.6 – 0.8). The suspension was then mixed with 200 nM of FraC. Hemolytic activity was measured by monitoring the decrease in OD650 using a MultiskanTM GO Microplate spectrophotometer (Thermoscientific). The rate of hemolysis was determined as one over the time elapsed till 50% decrease in turbidity. [1214] Preparation of Sphingomyelin:DPhPC liposomes. 20 mg of the sphingomyelin (Brain, Porcine, Avanti Polar lipids) and DPhPC (1:1) mixture was dissolved in 4 ml of pentane supplemented with 0.5 % ethanol (to help dissolving sphingomyelin) and placed in a round bottom flask. The solvent was evaporated while slowly rotating the flask in order to deposit lipid film on the walls. After deposition of the lipid film, the flask was kept open for 30 min to allow the complete evaporation of the solvent. The lipid film was then resuspended in 150 mM NaCl, 15 mM Tris-HCl pH 7.5 (final concentration of the total lipid 10 mg/ml) using a sonication bath (5-10 minutes at ambient temperature). Obtained liposomes were stored at - 20°C. [1215] Oligomerization of FraC. Monomeric FraC was mixed with liposomes (lipid/protein mass ratio 10:1) in 150 mM NaCl, 15 mM Tris-HCl pH 7.5 buffer. The mixture was briefly sonicated (sonication bath) and incubated for 30 min at 37°C. -347- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1216] Proteoliposomes were then solubilized with 0.6% LDAO and incubated for 5 min and the mixture was diluted 20-fold with DDM-containing wash buffer (0.02% DDM 150 mM NaCl, 15 mM Tris-HCl pH 7.5) and mixed with ~ 100 µl (bead volume) of Ni-NTA agarose resin (Qiagen) that was pre-equilibrated with DDM- containing wash buffer. After gentle mixing for 1 hour, the resin was loaded onto a column (Micro Bio Spin, Bio-Rad) and washed with ~ 2 ml of DDM wash buffer. FraC was eluted from the column with 50 pl of elution buffer (200 mM EDTA, 0.02 % DDM, pH 8 - alternatively 1M imidazole 0.02% DDM could have been used, however, EDTA proved more efficient). Purified FraC oligomers were stored at 4°C. [1217] Alternatively, FraC oligomers can be formed by mixing FraC monomers with liposomes formed by sphingomyelin alone (1hr at 37°C and then 4°C overnight). Next day, 5 mM EDTA and 1% DDM (final) is added to the proteoliposomes and incubated for 15 minutes at room temperature. The solution is then diluted to 1 ml volume containing 5mM EDTA, 0.05% DDM 15 mM Tris HCl 7.5150 mM NaCl. The solution is then concentrated to ~100ul with 100 kDa cutoff ultrafiltration device. [1218] Electrical recordings in planar lipid bilayers. The applied potential refers to the potential of the trans electrode. FraC nanopores were inserted into lipid bilayers from the cis compartment, which was connected to the ground electrode. The two compartments were separated by a 25 µm thick polytetrafluoroethylene film (Goodfellow Cambridge Limited) containing an orifice of ~ 100 µm in diameter. The aperture was pretreated with ~5 µl of 10% hexadecane in pentane and a bilayer was formed by the addition of ~10 µL of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) in pentane (10 mg/mL) to both electrophysiology chambers. Typically, the addition of 0.01-10 ng of oligomeric FraC to the cis compartment (0.5 mL) was sufficient to obtain a single channel. WtFraC nanopores displayed a higher open pore current at positive than at negative applied potentials, which provided a useful tool to determine the orientation of the pore. Electrical recordings were carried out in 1M (initial characterization of the FraC nanopores) and in 3M NaCl (for polynucleotide analysis to increase amplitudes), 15 mM Tris-HCl pH 7.5. [1219] Data recording and analysis. Electrical signals from planar bilayer recordings were amplified using an Axopatch 200B patch clamp amplifier (Axon Instruments) and digitized with a Digidata 1440 A/D converter (Axon Instruments). Data were recorded by using Clampex 10.4 software (Molecular Devices) and the subsequent analysis was carried out with Clamp fit software (Molecular Devices). [1220] Electrical recordings were acquired by applying a 2 kHz low-pass Bessel filter and a 10 kHz sampling rate. Current transitions from level 0 to level 1 were analyzed with the “single-channel search” function in Clampfit. [1221] Residual current values (Ires) were calculated from blocked pore current values (IB) and open pore current values (Io) as Ịres = 100* IB/IO. IB and Io were determined from Gaussian fits to amplitude histograms of events. In case of events showing stepwise current enhancements, residual current levels were calculated from -348- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Gaussian fits to whole point current histograms. To determine event lifetimes, event dwell times (toff) were binned together as cumulative distributions and fitted to a single exponential. Frequency of events that show stepwise current enhancements (FIG.59A) and rotaxane forming blockades were calculated manually. Graphs were made with Origin (OriginLab Corporation) or Clampfit software (Molecular Devices). [1222] Graphic representation of FraC nanopore. Molecular graphics were generated with Chimera (http://www.cgl.ucsf.edu/chimera). Example 5.1: Reconstitution of wild type FraC pores in planar lipid bilayers. [1223] Recombinant wild type FraC (WtFraC, FIGS.57A-57B) protein monomers, genetically fused to a Hi9 tag at the C-terminus, were expressed in a BL21(DE3) E. cob strain. Pore assembly of actinoporins may be triggered by the presence of SM in lipid bilayers. Water-soluble monomers of WtFraC purified by Ni-NTA chromatography did not form pores in DPhPC planar lipid bilayers. Monomers were pre-oligomerized with DPhPC:SM (1:1) liposomes. After solubilization of the liposomes in 0.6 % N,N-Dimethyldodecylamine N- oxide (LDAO), to prevent the dissociation of the oligomers, LDAO was exchanged to 0.02% β-Dodecyl maltoside (DDM) by a second round of Ni-NTA chromatography (SI). The addition of purified sub-microgram quantities of oligomeric WtFraC in 0.02% DDM to the cis side of the DPhPC planar lipid bilayer yielded pores readily. Distribution of unitary channel conductance for WtFraC pores at 1 M NaCl, 15 mM Tris-HCl pH 7.5 buffer revealed chiefly a single conductance type (FIG. 60A, top), which may correspond to the octamer observed in the determined crystal structure. The conductance was measured at -50 mV applied potential. The orientation of each individual channel was verified according to the asymmetry in conductance. WtFraC channels showed asymmetric current-voltage (I-V) relationship (FIG. 60B) allowing the determination of orientation of the pore. Experiments were repeated 3 times, and error bars indicate the standard deviations between experimental values. Recordings were carried out in 15 mM Tris-HCl pH 7.5 and IM NaCl. An example signal, obtained in 3 M NaCl, 15 mM Tris-HCl pH 7.5 buffer, is shown in FIG.57D, top. Example 5.2: Engineering of WtFraC for nucleic acid analysis. [1224] The crystal structure of octameric WtFraC (FIG. 57A) suggests that this nanopore may be large enough to allow the threading of ssDNA (1.2 nm constriction diameter). In the initial experiments, ssDNA blockades were not observed, possibly because of the negatively charged constriction region of the WtFraC pore prevented DNA translocation. To induce the threading of ssDNA through FraC, aspartate 10 was substituted with arginine, producing a nanopore with a positively charged constriction (FIG. 57B, bottom). Because D10R FraC showed a low pore-forming activity (FIG. 61), random mutagenesis was performed on the background of the D10R FraC gene and screened hemolytic activity of obtained variants (SI). As a result, the compensatory mutation lysine 159 to glutamic acid (K159E) was identified, which is located on the outer rim of the wide vestibule (FIG. 57A). The double mutant D10R, K159E of FraC (ReFraC) displayed near -349- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 wild type-levels of hemolytic activity (FIG. 61) and yielded uniform pores (FIG.57C), albeit with altered I- V relationship compared to WtFraC (FIG. 60B and FIG.60D, bottom). The lower conductance of ReFraC pores at ± 50 mV in 1 M NaCl, 15 mM Tris-HCl pH 7.5 may be be attributed to a narrower constriction as arginine has bulkier side chain than aspartate (FIG.57B). In assessing the hemolysis rate of pores in FIG.61, hemolysis rate was calculated as inverse of the time elapsed till 50% decrease in turbidity (measured as optical density at 650 nm wavelength) observed in 1% of horse erythrocytes suspension in 15 mM Tris-HCl pH 7.5 150 mM NaCl. Proteins were added in 200 nM concentration, hemolysis rates are presented as percentage of WtFraC. Experiment was repeated 3 times, and error bars indicate the standard deviation between experimental values. Example 5.3: Polynucleotide discrimination with ReFraC [1225] DNA was immobilized with neutravidin (NA) with αHL and MspA. 5’- end biotinylated A20/C20/T20 ssDNA homopolymers were complexed with tetrameric NA to assess the ability of ReFraC to translocate and discriminate DNA strands. Pre-mixed DNA (1 µM) and NA (0.25 µM) were added to the cis compartment of the planar lipid bilayer setup and DNA discrimination experiments were performed in 3 M NaCl, 15 mM Tris-HCl, pH 7.5 buffer and +70 mV applied potential (referring to the trans electrode). Permanent current blockades were observed, which may have been provoked by pseudorotaxanes where ssDNA is stably threaded through the pore until the applied potential is reversed (FIG. 58A and FIG. 65A). The residual currents, Ires, which are the percentage ratios of the amplitudes of blocked and open pore currents multiplied by 100 ((IB/IO) x 100) were: 13.1±0.4 % for NA:A20 (N=5, n=364, where N is a number of independent single pore experiments and n the analyzed blockades), 10.8 ± 0.3 % (N=4, n=920) for NA:C20 and 14.0 ± 0.3 %(N=5, n=780) for NA:T20 (FIG.58B). To exclude effects of pore-to-pore variation, mixtures of homopolymers (FIG. 58C-58F) were also resolved. The relatively low residual currents suggest a tight closure of the pore around threaded ssDNA. Signals were recorded in 3 M NaCl, 15 mM Tris-HCl, pH 7.5, using 2 kHz low-pass Bessel filter and 10 kHz sampling rate. Signals C and E were subjected to additional 100 Hz Gaussian digital filtering. Example 5.4: DNA unzipping and double strand DNA translocation by ReFraC nanopores [1226] The constriction of ReFraC (1.2 nm) is smaller than the B-form of double stranded DNA (dsDNA, ~2 nm). To evaluate dsDNA as a stopper for DNA analysis, two oligonucleotides were designed: oligo I with a biotin group attached at the 5’-terminus with the sequence bio-5’-A20- GTGCTACGACTCTCTGTGTG-C20- 3’ and a short oligo II with reverse complement sequence to the underlined part of oligo I. Annealing yielded an A(dsDNA)C substrate: a 20 base pair long central segment of dsDNA, flanked by A20 and C20 ssDNA segments. Addition of 1 µM of A(dsDNA)C to the cis compartment at +50 mV caused transient blockades to the ReFraC pore (blockade lifetime 2±5 s, Ires = 10.0±0.2%, N=3, n=290, FIG. 62A, left). Increasing the -350- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 applied potential to +70 mV shortened the blockades lifetime to 2.9±0.4 ms (the residual currents showed two current levels: 12.8±0.6 % and 3.5+0.5 % N=3 n=2700 FIG. 62B, left). The decrease of blockade lifetime, with the potential suggested the translocation of A(dsDNA)C through ReFraC. To prove DNA translocation, NA was added to the cis chamber. NA:A(dsDNA)C blockades became permanent both at | +50 mV, (FIG. 62A, right) and at +70 mV (FIG.62B, right), suggesting the transient blockades in the absence of NA may not be provoked by the retraction of A(dsDNA)C to the cis compartment. Two levels of the residual current detected for free A(dsDNA)C blockades indicated with pale dashed line (6201). Current levels corresponding to the blocked and open pores are shown as pale violet and grey dashed lines respectively. Voltage stepping protocols are shown with the red lines below the signals. Recordings were carried out in 15 mM Tris-HCl pH 7.5 and 3M NaCl, sampling frequency was 10 kHz, and data were smoothed by 2 kHz low-pass Bessel filter upon acquisition. [1227] At +50 mV, 31±4% of the NA:A(dsDNA)C blockades (N=3, n=468) showed a stepwise enhancement of the residual current from a transient level (FIG.59A, state “2”, Ires = 8.8±0.7%) to a stable level (FIG.59A, state “3”, with Ires= 12.5±0.7%, N=4, n=46; more examples in FIG. 63). The current level of state “2” was slightly lower than that of NA:C20 (Ires=10.5±0.7; N=3, n=206 at +50 mV). The current level of state “3” matched that of NA:A20. Without being bound to a particular theory, a potential explanation for above current enhancements is that at +50 mV the C20 segment of NA: A(dsDNA)C was dwelling in the constriction of the nanopore (FIG. 59A, state “2”), with the duplex segment preventing the further translocation. After the unzipping of the duplex, A20 occupies the constriction of ReFraC, with NA arresting the translocation (FIG. 59A, state “3”). At +50 mV, NA:A(dsDNA)C blockades were immediately relieved when the potential was reversed to -30 mV, indicating that at +50 mV translocation of A(dsDNA)C may have been mediated by unzipping (FIG.59A, brackets). Two levels can be observed in the block: firstly, a lower level (“2”), likely corresponding to homopolymeric cytosine which converts via an intermediate level (unzipping, brackets) into a higher level (“3”), most likely corresponding to homopolymeric adenine. [1228] In FIG. 63, within the blockades residual current has switched from 8.8±0.7% (initial level) to 12.5±0.7% (final level). Voltage stepping protocol is shown with the red lines at the bottom. Recordings were carried out in 15 mM Tris-HCl pH 7.5 and 3M NaCl, sampling frequency was 10 kHz, and data were smoothed by 2 kHz low-pass Bessel filter upon acquisition. [1229] At +70 mV, a significant fraction of blockades were not immediately released at -30 mV (FIG. 59B, inset), indicating the formation of an interlocked state (FIG.59B, states “2” and “3”). These interlocked states were generated more frequently with increasing the potential (e.g. from 7±4% of all blockades at +70 mV to 54±14 % at + 100 mV, N=3, n=739; FIG.59B, insert). Signals were recorded in 3 M NaCl, 15 mM Tris-HCl, pH 7.5, using 2 kHz low-pass Bessel filter and 10 kHz sampling rate. Blockades of oligo I alone in complex -351- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 with NA were released immediately at -30 mV (FIG.65A). Without being bound to a particular theory, such an interlocked state may be attributed to a rotaxane where NA and the duplex DNA segment of A(dsDNA)C serve as cis and trans stoppers, respectively (FIG. 59B, right). Such rotaxanes may also be formed from NA:oligo I cis blockades by adding oligo II in trans (FIG. 65B). Switching potential to -40 mV dismantled rotaxanes quickly, presumably via unzipping of the dsDNA stopper in trans (FIG.64 and FIG.65B). Transient state describing unzipping of dsDNA is shown in brackets. Voltage stepping protocol is shown with the red lines at the bottom. Recordings were carried out in 15 mM Tris-HCl pH 7.5 and 3M NaCl, sampling frequency was 10 kHz, and data were smoothed by 2 kHz low-pass Bessel filter upon acquisition. Formation of a rotaxane from NA:A(dsDNA)C present in cis may require the deformation of the ReFraC pore in order to allow the translocation of the duplex segment of the A(dsDNA)C substrate (FIG.59B, brackets). [1230] This structural flexibility may be a general feature of α-helical pores. Previously, it was observed that the blockades of human thrombin (~4.2 nm diameter) to type I ClyA-CS nanopores (~3.3 nm constriction diameter) were followed by a transient increase in the open pore current. This phenomenon may be interpreted as translocation of the protein via the deformed constriction of ClyA. [1231] Materials. Chymotrypsin(from bovine pancreas, ≥85%, C4129), β2-microglobulin (from human urine, ≥98%, M4890), endothelin I(≥97%, E7764), endothelin 2(≥97%,E9012), angiotensinI(≥90%, A9650), pentane(≥99%, 236705) and hexadecane(99%, H6703), Trizma® hydrochloride (Lot#SLBG8541V) and Trizma® base(Lot#SLBK4455V), N,N -Dimethyldodecylamine N-oxide (LADO, ≥99%, 40234) and n- Dodecyl β-D-maltoside (DDM, ≥98%, D4641) were obtained from Sigma-Aldrich. Human EGF(≥98%, CYT- 217) was obtained from PROSPEC. 1,2-diphytanoyl-sn-glycero-3-phosphocholine(DPhPC, 850356P) and sphingomyelin (Brain, Porcine, 860062) were purchased from Avanti Polar lipids. Potassium chloride(>99%, Lot#BCBL9989V) was bought from Fluka. Citric acid(≥99%, Lot#A0365028) was obtained from ACROS. All polypeptide biomarkers and chemicals were used directly without further purification.15 mM Tris, pH 7.5 buffer below used was prepared with the formula from Trizma® protocol: 1.902 g Trizma® HC1 and 0.354 g Trizma® Base dissolved in 1 liter H2o to be 15 mM Tris, pH 7.5. [1232] FraC monomer expression and purification. A gene encoding FraC with a C-terminal His6 tag was cloned into a pT7- SCl expression plasmid1 using Ncol and HindIII restriction digestion sites. For expression, the plasmid was transferred into E.cloni® EXPRESS BL21(DE3) competent cell by electroporation. Transformants were harvested from the LB agar plate containing 100 mg/l ampicillin after overnight incubation at 37°C, and inoculated into 200 ml fresh liquid 2-YT media with 100 mg/l ampicillin. The cell culture was grown at 37°C, with 220 rpm shaking to an optical density at 600 nm of 0.8, then 0.5 mM IPTG was added to the cell culture. The temperature was lowered to 25°C to induce the expression of FraC protein for 12 hours. Cells were recovered by 4,000 RPM centrifugation for 30 minutes at 4°C and the cell pellets were kept at - -352- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 80°C. 50-100 ml of cell culture pellet was thawed at room temperature, resuspended with 30 ml lysis buffer (15 mM Tris pH 7.5, 1 mM MgCb, 4 M Urea, 0.2 mg/ml lysozyme and 0.05 units/ml DNase) and mixed vigorously with a vertex shaker for 1 hour. In order to fully disrupt the cells, the suspension was sonicated for 2 minutes (duty cycle 10%, output control 3 of a Branson Sonifier 450). The crude lysate was then centrifuged at 6,500 RPM, 20 minutes at 4°C. The supernatant (containing FraC monomers) was transferred to a 50 ml falcon tube containing a 100 µl of Ni-NTA resin (Qiagen, stored at 4°C, and suspended before pipetting out 100 µl), which was pre-washed with 3 ml of washing buffer (10 mM imidazole, 150 mM NaCl, 15 mM Tris, pH 7.5), and incubated at room temperature for 1 hour with gentle mixing. The resin was spun down at 4,000 RPM for 5 minutes at 4°C. Most of the supernatant was discarded and the pellet containing the Ni-NTA resin within ~5 ml of buffer was transferred to a Micro Bio Spin column (Bio-Rad) at RT. The Ni-NTA beads were washed with 10 ml wash buffer and the protein was eluded with 500 µl of 300 mM imidazole. Protein concentration was determined with NanoDrop 2000 (Thermo Scientific). The monomers were stored at 4°C. [1233] Preparation of sphingomyelin-DPhPC liposomes. 20 mg sphingomyelin (Brain, Porcine, Avanti Polar lipids) was mixed with 20 mg of l,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar hpids) and dissolved in 4 ml pentane (Sigma) containing 0.5% v/v ethanol. This lipid mixture was placed to a round flask and rotated slowly near a hair dryer to disperse the lipid well around the wall to evaporate the solvent. The flask was kept open at room temperature for another 30 minutes to let the solvent to evaporate completely. Then 4 ml of buffer (150 mM NaCl, 15 mM Tris, pH 7.5) was added to the dried lipids and the flask was added to a sonication bath for 5 minutes. Liposomes solution was kept at -20°C. [1234] Oligomerization of FraC. Frozen liposomes were sonicated after thawing and mixed with monomeric FraC in a mass ratio 1:1. The FraC and liposome mixture was sonicated in a water bath for ~30 seconds and then kept at 37°C for 30 minutes. The proteo-liposome was solubilized with 0.6% LADO(N(N- Dimethyldodecylamine N-oxide, 5% w/v stock solution in water), then transferred to a 50 ml falcon tube and diluted 20 times with buffer (150 mM NaCl, 15 mM Tris, pH 7.5, 0.02% DDM). 100 µl of pre-washed Ni- NTA resin (Qiagen) was added to the diluted protein/liposome mixture. After incubation with gentle shaking for 1 hour, the beads were loaded to column (Micro Bio Spin, Bio-Rad) and washed with 10 ml buffer (150 mM NaCl, 15 mM Tris, pH 7.5). FraC oligomers were eluted with 300 gl elution buffer (200 mM EDTA, 75 mM NaCl, 7.5 mM Tris, pH 8, 0.02% DDM). Oligomers can be stable for several weeks at 4°C. [1235] Electrical recording in planar lipid bilayers. Electrical recordings were performed as described follows. In short, two chambers were separated by a 25 µm polytetrafluoroethylene film (Goodfellow Cambridge Limited) containing an aperture with diameter of around 100 pm. Two silver/silver-chloride electrodes were submerged into each compartment of the electrophysiology chamber, which was filled with 0.5 ml of buffer. The ground electrode was connected to the cis compartment, the working electrode to trans -353- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 side. To form a lipid bilayer, ~5 µl of hexadecane solution (10% v/v hexadecane in pentane) was added to the polytetrafluoroethylene film. After ~2 minutes, 10 µl of a 10 mg/ml solution of l,2-diphytanoyl-sn-glycero-3- phosphocholine (DPhPC) in pentane was added directly to the buffer in both compartments. A lipid bilayer then spontaneously formed by lowering the buffer above and below the aperture in the Teflon film. FraC oligomers were added to the cis side. Under an applied potential, the ionic current of FraC is asymmetric, which helps assessing the orientation of FraC nanopores in the lipid bilayer. FraC nanopores showed the orientation as shown in FIGs. 66A-66C when a higher conductance was measured at negative applied potential. Analytes were then added to cis chamber. Two kinds of buffer solutions were used for electrophysiology recording in this study depending on the pH. At pH 7.5 recordings were performed using 1 M KCl and 15 mM Tris. When the pH was varied from 7.5 to 4.5, the buffer used contained 1 M KCl, 0.1 M citric acid, and 180 mM Tris-Base. FraC and ReFraC oligomers could insert into lipid bilayer from pH 4.5 to 7.5. Endothelin 1 and chymotrypsin enter WtFraC under negative applied potentials, while they enter ReFraC under positive applied potentials. Chymotrypsin blockades to WtFraC were also observed under -50 mV at pH 7.5 and 4.5, however, the applied potential was increased to -100 mV to obtain a sufficient number of blockades. At pH 7.5, blockades to ReFraC by chymotrypsin under positive applied bias required higher potential than to WtFraC under negative applied bias. The buffer at pH 7.5 included 1 M KC1, 15 mM Tris, and the buffer at pH 4.5 contained 1 M KCl, 0.1 M citric acid, 180 mM Tris-Base. Endothelin 1 and chymotrypsin were added into cis compartment. All signals were recorded using 50 kHz sampling rate and a 10 kHz low-pass Bessel filter. Structures were rendered using PyMOL. [1236] Data recording and analysis. Planar bilayer recordings were collected using a patch clamp amplifier (Axopatch 200B, Axon Instruments) and the data digitized with a Digidata 1440 A/D converter (Axon Instruments). Data were acquired by using Clampex 10.4 software (Molecular Devices) and the subsequent analysis was carried out with Clampfit software (Molecular Devices). Events duration (dwell time), time between two events (inter-event time), blocked current levels and open pore levels were detected by “single channel search” function. The current levels of blockades were referred as Is, while the open pore current was referred as Io. Ires%, defined as (IB/IO)*100, was used to describe the extent of blockade caused by different biomarkers. Average Inter-event times were calculated by binning the inter-event times and applying a single exponential fit to cumulative distributions. [1237] Ion selectivity measurement. Ion permeability ratio (K+/CF-) was calculated using the Goldman– Hodgkin–Katz equation, which uses the reverse potential (Vr) as variable input. The Vr was measured from extrapolation from I-V curves using asymmetric salt concentration condition as follow: Individual FraC nanopores were reconstituted using the same buffer in both chambers (symmetric conditions, 840 mM KCl, 15 mM Tris, pH 7.5, 500 µl) to assess the orientation of the nanopore.400 µl solution containing 3.36 M KCl, -354- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 15 mM Tris, pH 7.5 was slowly added to cis chamber and 400 µl of a buffered solution containing no KCl (15 mM Tris, pH 7.5) was added to trans solution (trans:cis, 467 mM KCl: 1960 mM KCl). The solutions were mixed and I-V curves collected from -30 mV to 30 mV with 1 mV steps. Experiments at pH 4.5 were carried out using the same method but using 0.1 M citric acid buffered solutions. Initially, 500 µl buffer of 840 mM KCl, 0.1 M citric acid, 180 mM Tris-Base was added into both chamber and a single FraC channel obtained. Then, 400 µl of pH 4.5 solution containing 3.36 M KCl, 0.1 M citric acid, 180 mM Tris-Base was slowly added to cis chamber and 400 µl of a buffered solution containing no KCl (0.1 M citric acid, 180 mM Tris-Base, pH 4.5) was added to trans solution (thus yielding a trans:cis ration of 467 mM KCl: 1960 mM KCl). The solutions were mixed and I-V curves collected from -30 mV to 30 mV with 1 mV steps. The directionality of the ion selectivity was also tested by using high KCl concentration in trans chamber and low KCl concentration in the cis chamber. Ag/AgCl electrodes were surrounded with 2.5% agarose bridges containing 2.5 M NaCl. Example 5.5: Polypeptides and protein capture with FraC nanopores [1238] To assess FraC nanopores as a sensor for oligopeptides biomarkers endothelin 1 was initially selected. Endothelin 1 is a 2.5 kD oligopeptide of 21 amino acids and α-II-chymotrypsin (henceforth chymotrypsin), a 25 kD globular protein of 245 amino acids (FIG.66A). Analytes were added to the cis side of wild type FraC (WtFraC) nanopores (FIG.66A) using 1 M KCl, 15 mM Tris, pH 7.5 solutions and an external potential was applied to the “working” electrode located in the trans compartment. WtFraC shows gating above ~+50 mV, but is stable at potentials as high as -300 mV, so potentials were applied between those limits. Addition of 1 pM of endothelin 1 to the cis compartment did not provoke blockades at ±50 mV (FIG. 66B) and up to - 300 mV. The constriction of ClyA is lined with aspartic acid residues (FIG. 66A), so without being bound to a particular theory, the protonation of these residues at more acidic conditions may diminish the energy barrier for the translocation of endothelin 1 (carrying a net charge of -2) through the WtFraC constriction. Simultaneously, a less negative endothelin 1 may also migrate more easily towards the trans electrode under negative applied potentials. Endothelin 1 blockades started to be appear at pH 6.4, and their capture frequency increased linearly with decreasing the pH (from 0.6±0.2 events S-1 µM-1 at pH 6.4 to 10.8±2.3 events s-1 µM-1 at pH 4.4). At pH 4.5 (1 M KCl, 0.1 M citric acid, 180 mM Tris-Base), endothelin 1 blockades to WtFraC were observed at -50 mV (Ires%: 9.1±0.1%, dwell time: 5.6±2.0 ms, inter-event time: 5.8±0.7 ms), but not at +50 mV (FIG.66B). [1239] Next, the capture of endothelin 1 was investigated with the D10R, K159E FraC (ReFraC) nanopore, a pore with arginine residues at the constriction engineered above for purposes of DNA analysis. ReFraC is stable under positive applied potentials but displays gating at potentials of ~-50 mV. Thus, only voltages between -50 mV to +200 mV were applied to ReFraC. Addition of 1 µM endothelin 1 to the cis compartment elicited blockades at pH 7.5 at +50 mV (dwell time: 3.3±2.2 ms, inter-event time: 1413±223 ms) but not -50 -355- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 mV (FIG. 66B). Decreasing to pH 4.5 (1 M KCl, 0.1 M citric acid, 180 mM Tris-Base) led to an increase in capture frequency at +50 mV (FIG. 66B, dwell time: 8.5±1.8 ms, inter-event time: 402±79 ms), despite the reduced electrophoretic mobility towards the trans electrode. [1240] Next, the protein chymotrypsin (pI 8.75, Sigma) was tested as an example of a relatively large protein analyte. Protein blockades were observed at -50 mV in pH 7.5 buffer (1 M KCl, 15 mM Tris), although they became homogeneous when the potential was increased to -100 mV (45.2±19.1 events s-1 µM-1, dwell time: 12.0±5.7 ms), and no capture was observed at positive applied potentials (FIG.66C). The capture frequency of chymotrypsin remained constant between pH 7.5 and 5.5 (45.2±19.1 events S-1 µM-1 at pH 7.5, 50.5±22.6 events s-1 µM-1 at pH 6.4, 45.2±20.6 events s-1 µM-1 at pH 5.5,), and decreased when the pH was lowered to 4.4 (20.8±5.3 events s-1 µM-1 at pH 4.4). Using ReFraC at pH 7.5, only few blockades were observed at high positive applied potentials (dwell time: 0.2±0.1 ms, inter-event time: 174.3±22.9 ms at +200 mV) but not at -50 mV (FIG. 66C). Decreasing the pH to 4.5 led to an increase in capture frequency (dwell time: 1.3±0.7 ms, 112.5±9.5 events s-1 µM-1, FIG. 65B). ReFraC showed often shallow gating events at negative applied potentials under acidic conditions as shown in FIG. 66C bottom right. Taken together, both nanopores can capture analytes differing 10-fold in molecular weight (2.5 kD versus 25 kDa). Example 5.6: Ion selectivity and electrostatic potential of FraC nanopores [1241] To gain a better insight into the influence of pH on the electrostatic environment and electro-osmotic flow on the entry of polypeptides inside FraC nanopores, the Adaptive Poission-Boltzmann Solver (APBS) and a modified version of the PDB2PQR software were used to estimate the electrostatic potential inside homology models of WtFraC and ReFraC at pH 7.5 and 4.5 in IM KCl. The simulations showed that the constriction regions of WtFraC and ReFraC at the center of the nanopore exhibited highly negative and positive potentials, respectively (FIG. 67A). While for WtFraC the lowering of the pH from 7.5 to 4.5 caused a reduction potential at the center of the constriction from -1.2 to - 0.7 kBT/ec (1 kBT/ec= 25.6 mV at 298 K) respectively, no such effect was observed for ReFraC. All simulations were performed using APBS(13) at 1M KCl, with the partial charge of each titratable residue adjusted according to their average protonation states with a modified version of the PDB2PQR software. Residue pKa values were estimated using PROPKA. [1242] The contribution of the electro-osmotic flow to the capture of analytes with WtFraC and ReFraC pores was estimated by measuring the ion-selectivity of both pores using asymmetric KCl concentrations on either side of the nanopore (1960 mM and 467 mM). The reversal potential (Vr), i.e. the potential at which the current is zero (FIG. 67B), was then used, together with the Goldman-Hodgkin-Katz equation, to calculate the ion selectivity (PK+/PCl-) of both nanopores:
Figure imgf000358_0001
-356- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1243] where [αx]comp is the activity of ion X in the cis/trans compartments, R the gas constant, T the temperature and F the Faraday constant. It was found that the ion selectivity of FraC nanopores is dominated by the charge at the constriction, with WtFrac being strongly cation-selective (PK+/PCl- = 3.55±0.30, pH 7.5) and ReFraC anion-selective (PK+/PCl- = 0.57±0.04, pH 7.5). Lowering of the pH to 4.5 decreased the cation- selectivity of WtFraC (PK+/PCl-= 2.02±0.15, pH 7.5) while it increased the anion-selectivity of ReFraC (PK+/PCl- = 0.36±0.08, pH 4.5, FIG. 67B). Lowering the pH from 7.5 to 4.5, reduced the ion selectivity of WtFraC (P_(K^+)P_(〖Cl〗^-) ) by ~43%, in accordance with the reduced magnitude of the simulated electrostatic potential. By contrast, the ~37% increase in ion selectivity of ReFraC at pH 4.5 was not predicted by the simulations. All reversal potentials were measured under asymmetric salt conditions (467 mM KCl in trans and 1960 mM KCl in cis) and the ion selectivity determined using the Goldman-Hodgkin-Katz equation. The envelopes behind every current-voltage curve represent their respective standard deviations. Example 5.7: Biomarker detection with the WtFraC nanopore [1244] After assessing the capture of chymotrypsin (25 kD, 245 amino acids) and endothelin 1 (12.5 kD, 21 amino acids), which can be protein biomarkers for pancreatic cysts and bronchiolitis obliterans, respectively, the WtFraC nanopores were used to detect a larger range of protein biomarkers including β2-microglobulin, a 11.6 kDa (99 amino acids) biomarker for peripheral arterial disease, human EGF, a 6.2 kDa (53 amino acids) biomarker for chronic kidney disease, and angiotensin I, a 1.3kD (10 amino acids) biomarker for hypertensive crisis. [1245] All biomarkers were assessed under negative applied potentials and, with the exception of chymotrypsin, at pH 4.5. The capture frequency of all biomarkers increased with the applied potential. All other parameter tested showed a non-uniform voltage dependency. The residence time of the biomarkers inside WtFraC increased (chymotrypsin), decreased (β2- microglobulin and angiotensin 1) or showed a bi-phasic behavior with the applied potential (EGF and endothelin 1), see FIGs.68A-68E. The voltage dependence of the residual current percentage (Ires%) of chymotrypsin decreased with the potential, the Ires% of endothelin 1 increased with the potential, while the Ires% of β2-microglobulin, EGF and angiotensin 1 remained constant. Despite the complex voltage dependency of the current blockades, the results showed that the WtFraC nanopore was capable of distinguishing differently sized oligopeptide and protein biomarkers by virtue of the Ires% of their current blockades alone (FIGs. 68A-68E). The concentrations of the biomarkers were: 200 nM for chymotrypsin, 200 nM for β2-microglobin, 2 µM for human EGF, 1 µM for endothelin 1, and 2 µM for angiotensin I, respectively. Isoelectric points of biomarkers were obtained from literatures or with the online calculation tool Pepcalc. Error bars represent the standard deviation obtained from at least 3 repeats and at least 300 events for each repeat. Data were fitted using a B-spline function (Origin 8.1). All recordings were collected with 50 kHz sampling rate and 10 kHz low-pass Bessel filter. -357- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Example 5.8: Near-isoform oligopeptide discrimination [1246] In order to challenge the experimental system, identification of highly similar analytes was tested. Endothelin 1 (ET-1) and endothelin 2 (ET-2) were selected, as these near-isomeric oligopeptides differing in only one out of twenty-one amino acids (FIG. 69A and FIG. 69B). At -50 mV, distinguishable blockades were observed with unique Ires% and dwell time (FIG.68B) for ET-1 (Ires% 8.9±0.1%, dwell time 5.6±2.0 ms, N=3, n=600) and ET-2 (6.1±1.4%, dwell time 19.0±5.3 ms, N=3, n=384). This made it such that their identification on an individual blockade level could be accomplished (FIG.69C). [1247] When first 2 µM ET-1 (FIG.69D) followed by 8 µM ET-2 were consecutively added to the same pore (FIG.69E), a mixture of both two distinct populations may be separated by plotting the standard deviation of the amplitude of events over their corresponding Ires%. This observation indicated that highly similar (oligo)peptides or other analytes can be discriminated with a FraC nanopore. Graphs were created with custom R scripts. All recordings were conducted with 50 kHz sampling rate and 10 kHz Bessel low-pass filter. Example 6: Alternate EOF-EPF Arrangements [1248] Examples 6.1-6.2 used the following methods and materials [1249] Preparation of ClpX Translocase. E. coli ClpX was employed as exemplary translocase to control the movement of the polypeptide through the nanopore (FIG.74). ClpX was selected as it a well-studied AAA+ translocase systems, and can unfold and translocate a variety of proteins, and can generating a high force through NTP hydrolysis. The monomer and covalently linked trimer of N-terminal truncated ClpX variants (residues 61–423) were purified with minor modifications and used for ClpX nanopore experiments. The gene encoding for monomer and trimer of ClpX-ΔN were separately transformed into E. coli BL21 (DE3) electrocompetent cells. Transformants were selected after overnight growth at 37 °C on lysogeny broth (LB) agar plates supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 100 mg/L of ampicillin. The ClpX protein expression was induced at an A600 of ∼0.6 by addition of 0.5 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) and incubated at 25 °C overnight. The cells were harvested by centrifugation for 20 min (4000 x g) at 4°C and the pellets were stored at -80°C. About 100 mL of cell culture pellet was thawed and solubilized with ~20 mL lysis buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 20 mM imidazole, 1 mM dithiothreitol (DTT), 0.1 units/mL DNase I, 10 µg/mL lysozyme) and stirred with a vortex shaker for 1 hour at 4°C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000 x g at 4 °C for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 20 mM imidazole, 1 mM dithiothreitol (DTT), ). Proteins were purified from the supernatant via Ni-NTA resin -358- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 (Qiagen) using standard procedures and eluted with approximately 600 μL elution buffer (600 mM imidazole, 1 mM dithiothreitol (DTT), 100 uM EDTA, 200 mM KCl, 25 mM MgCl2, 50 mM Tris, pH 7.5). The proteins were further purified using a Superose 6 column Increase 10/600 GL and eluted in 200 ul fractions in elution buffer 2 (1 mM dithiothreitol (DTT), 100 uM EDTA, 200 mM KCl, 25 mM MgCl2, 50 mM Tris, pH 7.5). The fractions containing pure protein were concentrated using Amicon Ultra Centrifugal Filters. Purified proteins were then flash frozen in small aliquots supplemented with 30 % glycerol and stored at –80 °C. Protein concentrations were determined by Bradford assay with bovine serum albumin as a standard. [1250] Preparation of Protein Analytes. Maltose Binding Protein (MBP) and Green Fluorescent protein (GFP) were used to test protein translocation through the nanopores. The model proteins were provided with a His-affinity tag and further modified via genetic fusions to express full length substrates with C- terminal “leader construct” extensions of design similar to that shown in FIG. 77B. The leader constructs contained elements to promote binding to ClpX (ssrA recognition motif), stalling of the ClpX (polyglycine stall motif) and EPF capture motifs to promote nanopore capture (polyanion or polycation stretches). The gene encoding for target proteins GFP-0, GFP-1, GFP-2, GFP-3, MBP-1, MBP-2, or MBP-4 (see Table 11 below) was separately transformed into E. coli. BL21 (DE3) electrocompetent cells. Transformants were selected after overnight growth at 37 °C on lysogeny broth (LB) agar plates supplemented with kanamycin (50 mg/L). The resulting colonies were inoculated into 200 mL LB medium containing 50 mg/L of kanamycin. The cells were induced at an A600 of ∼0.6 by addition of 0.5 mM isopropyl β-D-1- thiogalactopyranoside (IPTG) and incubated at 25 °C overnight. The cells were harvested by centrifugation and the pellets were stored at -80°C. 100 mL cell culture pellets were thawed and solubilized before removing the cellular debris by centrifugation. Proteins were purified from the supernatant via Ni-NTA resin (Qiagen) used standard procedures and eluted with approximately 100 μL elution buffer (600 mM imidazole, 1 mM dithiothreitol (DTT), 150 mM KCl, 50 mM HEPES, pH 7.5). Purified proteins were then flash frozen in small aliquots and stored at –80 °C. Protein concentrations were determined by Bradford assay with bovine serum albumin as a standard. Table 16. Amino acid sequences of target proteins used. Target Net Charge at pH 7.5 Component Sequence SEQ (Target protein + ID purification tag) NO. GFP-0 -5.1 Full Sequence MGHHHHHHSSASKGEELFTGVVPILVELDGDVNG 88 HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGY VQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVL LPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG IAANDENYALAA -359- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Affinity MGHHHHHHSS 89 purification tag GFP protein ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGE 90 GDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGV QCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGI Recognition AANDENYALAA 91 motif GFP-1 -5.1 Full Sequence MGHHHHHHSSASKGEELFTGVVPILVELDGDVNG 92 HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGY VQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVL LPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG IGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDE NYALAA Affinity MGHHHHHHSS 89 purification tag GFP protein ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGE 90 GDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGV QCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGI Stall motif GGGGGGGGGGGGS 93 Capture motif RRRRRRRRRRRRRRR 94 Recognition AANDENYALAA 91 motif GFP-2 -5.1 Full Sequence MGHHHHHHSSASKGEELFTGVVPILVELDGDVNG 95 HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGY VQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVL LPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG IRRRRRAANDENYALAA Affinity MGHHHHHHSS 89 purification tag GFP protein ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGE 90 GDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGV QCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGI Capture motif RRRRR 96 -360- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Recognition AANDENYALAA 91 motif GFP-3 -5.1 Full Sequence MGHHHHHHSSASKGEELFTGVVPILVELDGDVNG 97 HKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGY VQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELK GIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVL LPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG IGEGDGEGDGEGDAANDENYALAA Affinity MGHHHHHHSS 89 purification tag GFP protein ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGE 90 GDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGV QCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI LGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHN IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQ SALSKDPNEKRDHMVLLEFVTAAGI Stall/Capture GEGDGEGDGEGD 98 motif Recognition AANDENYALAA 91 motif MBP-1 -7.5 Full Sequence MHHHHHHSSPWKIEEGKLVIWINGDKGYNGLAEV 99 GKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGP DIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYP FTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNP PKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLT FLVDLIKNKHMNADTDYSIAEAAFNKGETAMTIN GPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGV LSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEI MPNIPQMSAFWYAVRTAVINAASGRQTVDEALKD AQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRR RRRAANDENYALAA Affinity MHHHHHHSS 89 purification tag MBP protein PWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTG 100 IKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDR FGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYN GKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPA LDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNK HMNADTDYSIAEAAFNKGETAMTINGPWAWSNID TSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAAS PNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA FWYAVRTAVINAASGRQTVDEALKDAQTRITKHM Stall/Capture GGGGGGGGGGGGSRRRRRRRRRRRRRRR 101 motif Recognition AANDENYALAA 91 motif -361- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 MBP-2 -7.5 Full Sequence MHHHHHHSSPWKIEEGKLVIWINGDKGYNGLAEV 102 GKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGP DIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYP FTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNP PKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLT FLVDLIKNKHMNADTDYSIAEAAFNKGETAMTIN GPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGV LSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEI MPNIPQMSAFWYAVRTAVINAASGRQTVDEALKD AQTRITKHMGGGGGGSRRRRRRRRRRRRRRRAAN DENYALAA Affinity MHHHHHHSS 89 purification tag MBP protein PWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTG 100 IKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDR FGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYN GKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPA LDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNK HMNADTDYSIAEAAFNKGETAMTINGPWAWSNID TSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAAS PNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA FWYAVRTAVINAASGRQTVDEALKDAQTRITKHM Stall/Capture GGGGGGSRRRRRRRRRRRRRRR 103 motif Recognition AANDENYALAA 91 motif MBP-4 -7.5 Full Sequence MHHHHHHSSPWKIEEGKLVIWINGDKGYNGLAEV 104 GKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGP DIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYP FTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNP PKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLT FLVDLIKNKHMNADTDYSIAEAAFNKGETAMTIN GPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGV LSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEI MPNIPQMSAFWYAVRTAVINAASGRQTVDEALKD AQTRITKHMGGGGGGGGGGGGGSDDDDDDDDDDA ANDENYALA Affinity MHHHHHHSS 89 purification tag MBP protein PWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTG 100 IKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDR FGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYN GKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPA LDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNK HMNADTDYSIAEAAFNKGETAMTINGPWAWSNID TSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAAS -362- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 PNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA FWYAVRTAVINAASGRQTVDEALKDAQTRITKHM Stall/Capture GGGGGGGGGGGGGSDDDDDDDDDD 105 motif Recognition AANDENYALA 91 motif [1251] Preloading of the translocase onto Target Protein. To improve the percentage of ClpX:target- protein complexes formed, the complexes were formed prior to addition to the nanopore system as illustrated schematically in FIG. 78. Pre-loading the ClpX translocase onto target protein was performed by mixing and incubating translocase with the given target protein in the presence of ATP and MgCl2 in a suitable binding buffer (e.g.50 mM Tris-HCl, 200 mM KCl, 25 mM MgCl2, 1 mM DTT, 1 mM EDTA, PH 7.5). A range of pre-loading conditions and incubation times was found to result in a high percentage of ClpX loaded complex. Preloading was typically performed at ratios of 1:1 up to 5:1 ClpX:target-protein. For the examples used herein the ClpX:Target-protein complexes were preloaded by mixing the components at a final concentration of about 10 µM ClpX, 5 µM target protein in a 2:1 ratio, in a solution containing 10 mM ATP and 25 mM MgCl2 in a final volume of 10 ul (50 mM Tris-HCl, 200 mM KCl, 10 mM ATP, 25 mM MgCl2, 1 mM DTT, 1 mM EDTA, PH 7.5). The mixture was then incubated for at least 10 minutes at room temperature to allow sufficient time for the ClpX to bind to the target proteins and translocate along the Leader construct sequence up to the stall motif. [1252] Preparation of CytK nanopores. CytK can pass current when inserted into a membrane. To identify CytK’s beta-barrel region, and the putative analyte recognition region, a homology model was built by mapping the CytK sequence to the sequence and structure of the alpha-hemolysin nanopore from Staphylococcus aureus (FIG.95A). The model shows the low net charge inside the nanopore from the water facing residues. The beta-barrel region was identified as comprising the stretch running from amino acid E112 to amino acid S134, and from amino acid S137 to amino acid K155. The even residues in the range E112-S130 and odd residues in the range S137-K155 were identified as the inward lumen water-facing residues most appropriate for engineering to alter ion-selectivity (FIG. 95A). Cytk was prepared as follows. A plasmid containing a gene encoding for the CytK gene elongated by six histidine residues at the C-terminus was transformed into BL21(DE3) cells by electroporation. Transformed cells were grown overnight at 37ºC on LB agar plates (1% glucose, 100 µg/ml ampicillin). Colonies were resuspended and grown in 200 mL 2YT medium at 37 ºC until OD6000.6-0.8, then expression was induced by addition of 0.5 mM IPTG and the culture was incubated overnight at 25 ºC. Cells were pelleted by centrifugation and stored at -80 ºC for at least 30 minutes. Cell pellets were lysed by resuspension in lysis buffer (150 mM NaCl, 20mM imidazole, 15 mM Tris pH 7.5, 1 mM MgCl2, 0.2 units/ml DNase1, ~1 mg of lysozyme), incubated for 30 minutes at RT, then sonicated -363- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 (Branson Sonifier 450, 2 minutes). Cellular debris was pelleted by centrifugation and the supernatant containing CytK was recovered. CytK was extracted from the supernatant and purified using Ni-NTA beads, with final elution in 200 µl aliquots (150 mM NaCl, 300 mM imidazole, 15mM Tris buffered at pH 7.5) before storage at 4 ºC. Table 17. Polypeptide sequences of His-tagged nanopore monomers. Description Sequence SEQ ID NO. Wildtype (WT) α- MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRT 106 hemolysin KGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMST LTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNN MVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATV ITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKE EMTNRGSGSSGGSSHHHHHH WT CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTG 107 SFMKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETT VTSSVSYQLGGSIKASVTPSGPSGESGATGQVTWSDSVSYKQTSYKTNLIDQTNKHVK WNVFFNGYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNS GFSPGMIAVVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTF NKSFVLDWKNKKLVEKKGSAHHHHHH CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTG 108 K128D/K155D/ SFMKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETT S120D/Q122D VTSSVDYDLGGSIDASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVK (CytK 4D2E) WNVFFNGYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNS GFSPGMIAVVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTF NKSFVLDWKNKKLVEKKGSAHHHHHH CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTG 109 K128D/K155D/S SFMKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETT 120D/T147D VTSSVDYQLGGSIDASVTPSGPSGESGATGQVDWSDSVSYDQTSYKTNLIDQTNKHVK WNVFFNGYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNS GFSPGMIAVVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTF NK SFVLDWKNKKLVEKKGSAHHHHHH CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTG 110 K128D/K155D/ SFMKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETT S120D/S151D VTSSVDYQLGGSIDASVTPSGPSGESGATGQVTWSDDVSYDQTSYKTNLIDQTNKHVK (CytK 4D2E_Alt) WNVFFNGYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNS GFSPGMIAVVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTF NKSFVLDWKNKKLVEKKGSAHHHHHH CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTG 111 K128F/S120D/ SFMKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETT Q122D VTSSVDYDLGGSIFASVTPSGPSGESGATGQVTWSDSVSYKQTSYKTNLIDQTNKHVK (CytK_2D1F2E) WNVFFNGYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNS GFSPGMIAVVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTF NKSFVLDWKNKKLVEKKGSAHHHHHH CytK MAQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTG 112 K128F/S120D/Q1 SFMKANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETT 22D/K155D VTSSVDYDLGGSIFASVTPSGPSGESGATGQVTWSDSVSYDQTSYKTNLIDQTNKHVK (CytK_3D1F2E) WNVFFNGYNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPTLTNS GFSPGMIAVVISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTF NKSFVLDWKNKKLVEKKGSAHHHHHH -364- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1253] Planar lipid bilayer electrophysiological recordings system. For each experiment a single nanopore was inserted in a planar lipid bilayer system. Briefly, the electrophysiology chamber consisted of two compartments separated by a 25 µm thick Teflon (Goodfellow Cambridge Ltd) membrane. The Teflon membrane contained an aperture with a diameter of approximately 100-200 µm. Lipid membranes were formed by first applying 5 µl of 5% hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane, near the aperture. The pentane was left to dry and 400 µl of the appropriate buffered solution was added to each compartment. 20 µl of a 6.25 mg/ml solution of DPhPC dissolved in pentane was added on top of the buffer on each side of the chamber. The chamber was left to dry for ~2 minutes to allow evaporation of pentane. Silver/silver chloride electrodes were attached to each compartment. The cis compartment was connected to the ground electrode and the trans was connected to the working electrode. Planar lipid bilayers were created using the Langmuir-Blodgett method. Purified nanopore solutions were added to the cis compartment to obtain insertion of single nanopores. Once a single nanopore had inserted the orientation and properties of the nanopore was confirmed by the asymmetry of the current-voltage relationship and compared to previous characterization metrics from multiple insertion tests to ensure that the nanopore was in the correct state. Analytes were then added to the cis or trans compartment of the chamber as required for the methods described herein. [1254] Recordings of ionic currents were obtained using Axopatch 200B patch clamp amplifiers (Axon Instruments) combined with Digidata 1550B A/D converters (Axon instruments). Recordings were typically acquired at 10 kHz with a 2 kHz Bessel filter, and recorded using Clampex 10 (Molecular Devices). Unless stated otherwise, all recordings were carried out at 22°C. [1255] All applied voltages stated herein follow the convention of stating the polarity at the active trans electrode: positive applied voltages have a positive voltage applied at the trans electrode relative to a ground electrode in the cis compartment. Example 6.1: CytK pores with ClpX translocase [1256] Example 6.1 examined whether it was possible to create a sufficiently strong electro-osmotic flow (FIG. 1) to capture and translocate complex polypeptides against opposing electrophoretic forces (EPF), and to control the movement of the translocated polypeptide through the nanopore (FIG. 2). To create a large electro-osmotic force (EOF) a wide range of engineered (mutated) CytK nanopores were generated with varying extents of ion- selectivity to create strong ion-selective current flow under high salt conditions. These were examined versus wild-type or mutated nanopores with low ion-selectively. These nanopores were all tested against their ability to translocate net negative substrate proteins, based on Maltose Binding Protein (MBP) and Green Fluorescent Protein (GFP). The Maltose Binding protein has a net charge of about -7.5 at -365- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 pH 7.5, and was tested in a system with an applied voltage where the net EPF would be acting to repel/eject the net negative protein from the nanopore (i.e. the net EPF is acting trans-to-cis on the negatively charged protein when a negative voltage is applied to the trans electrode as described herein). GFP was also tested, which likewise has a net negative charge of about -5.1 at pH 7.5, for the ability to translocate a structurally different and stable protein against the net EPF. [1257] Determination of nanopore system EOF from ion-selectivity and electro- osmotic flow. The ion- selectivity and electro-osmotic flow parameters of various nanopore systems were determined according the methods and GHK derived equations described earlier. [1258] The ion-permeability parameters (e.g. P(K+), P(Cl-), P(Glu-)) for the various ions (e.g. K+, Cl-, Glu- ) were determined as described earlier by carrying out ion- selectivity measurements using asymmetric salt concentrations of the appropriate ions to reveal the relative cation:anion selectivity of each nanopore system. Briefly, reverse voltage from ion-selectivity measurements were performed in the planar lipid bilayer electrophysiological recording system described. During reversal potential measurements, the electrodes were not in direct contact with the buffer solution but were connected via agarose bridges containing 2.5% agarose in a 3 M KCl solution. For reverse voltage measurements, both compartments were first filled with 500 μL of “solution-A”. The electrodes were balanced to zero offset under these symmetrical salt conditions, and the IV current-voltage curve was measured between −140 and +140 mV in steps of 20 mV. Afterwards, the concentration of the trans compartment was decreased by perfusion to “solution-B” to create the final asymmetrical salt condition. The permeability parameters P(K+) and P(Cl-) for K+ and Cl- were determined using a solution-A of 2 M KCl and a solution-B of 0.5 M KCl. The permeability parameters P(K+) and P(Glu- ) for K+ and Glu- were determined using a solution-A of 2 M KGlu and a solution-B of 0.5 M KGlu. [1259] The IV curve was measured between −140 and +140 mV in steps of 20 mV in the asymmetric solution- A:solution-B system, and the reversal potential (Vr), which is the voltage offset that achieves zero ionic current flow, was estimated by linear regression of the curve between −20 and +20 mV. The pores were measured in triplicates. The reversal voltage for a given system were then used in the following equation to determine the relative permeability ratio:
Figure imgf000368_0001
wherein P(X+) and P(Y-) denote the permeability of the nanopore system for cation species X and anion species Y respectively. [^^−] and [^^+ ] are the activity of ion Y and X respectively in the indicated compartment, calculated by multiplying the concentration with the mean ion activity coefficient. The activity -366- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 coefficients for KCl in 0.5 M and 2.0 M solutions were 0.649 and 0.573 respectively. The mean activity coefficients of KGlu in 0.5 M and 2.0 M were 0.68 and 0.719 respectively. The empirical ion-selectivity ratios (e.g. P(K+)/P(Cl-) and P(K+)/P(Glu-)) were then used in the GHK flux equations and applied to further experimental measurements of ionic current versus applied voltage (I-V curves) as described previously to determine the absolute values of P(K+), P(Cl-), and P(Glu-). [1260] To a first approximation (ignoring the minor component from other ionic species in the systems as very low concentration), the separate ionic current (Is) contributions from the flow of K+, Cl- and Glu- ions in systems containing various symmetric and asymmetric combinations of KCl and KGlu were calculated using the specific GHK flux equation for each ion of:
Figure imgf000369_0001
[1261] The predicted current ^ = ^(^+) + ^(^^−) + ^(^^^−) closely matched that measured current in electrical recordings. The net ionic current flowing cis-to-trans (^∆^→^), the total current flowing the nanopore (Itotal), and the relative proportion of net ionic current flowing cis-to-trans over the total current (Irel) was determined as described previously. The values determined for a number of nanopore systems are shown in Table 18. Table 18. Values for number of nanopore systems. Pore Voltage Salt in cis Salt in Irel P(+)/P(-) ClpX:MBP-1 (mV) trans events aHL_WT -80 to -180 1M KCl 1M KCl -0.12 0.78 No -80 to -180 1M KGlu 1M KGlu 0.30 1.86 No CytK_WT -80 to -180 1M KGlu 1M KGlu 0.20 1.51 No CytK_2D1F2E -120 1M KGlu 1M KGlu 0.39 2.28 Yes, C>T CytK_3D1F2E -80 1M KGlu 1M KGlu 0.49 2.89 Yes, C>T CytK_4D2E -80 1M KGlu 1M KGlu 0.47 2.78 Yes, C>T +80 1M KGlu 1M KGlu -0.47 2.78 Yes, T>C -80 1M KGlu 1M KCl 0.58 3.91 Yes, C>T CytK_4D2E_alt -80 1M KGlu 1M KGlu 0.57 3.68 Yes, C>T [1262] Recordings of protein translocation. Measurements of translocase controlled protein translocation were carried out according to the system schematically described in FIG. 76. Both compartments of the nanopore system were filled with an electrolyte solution (1 M potassium glutamate, 20 mM MgCl2 and 50 -367- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 mM Tris, buffered to pH 7.5). Unless stated otherwise, the appropriate purified CytK nanopore was added to the cis compartment to achieve a single inserted nanopore. After detecting the insertion of a single nanopore the open pore current was recorded at a range of voltages to assess the nanopore. Separately, translocase (ClpX) and target protein substrate (e.g. MBP-1) were preincubated, typically at a 2:1 translocase:target-protein molar ratio, for >10 minutes at room temperature in 10 mM ATP and 25 mM MgCl2. After pre-incubation, the translocase:target-protein complex was added to the cis-compartment (unless stated otherwise). [1263] FIG.74 demonstrates a nanopore system for translocating an analyte. A protein analyte comprising a leader, which is loaded with a protein translocase motor (with NTP powered translocase function in the direction away from the leader as indicated by subset arrow) to form a protein:translocase complex, is added to the cis side of a system containing nanopore(s) in a membrane (7401). The protein:translocase complex is captured by the nanopore, for example, via the leader construct. The entire complex is pulled into the nanopore by the combination of cis- to-trans EPF and/or EOF until the translocase motor encounters the top of the nanopore (7402), whereupon it prevents further uncontrolled translocation. The cis-to-trans EPF and/or EOF forces acting on the leading motifs of the polymer region within the nanopore pull the polymer through the translocase so that the translocase can overcome the stall and/or block regions in the leader region (7403), whereupon the translocase can continue to progress along the polymer analyte under chemical energy powered NTP hydrolysis, unfolding protein structures ahead as required, thus feeding the extruded polypeptide chain into the nanopore cis-to-trans in a controlled manner. The protein analyte is fully processed by the translocase (7404), which unbinds upon encountering the end of the molecule, releasing the polypeptide, which is then translocated to the trans compartment of the system, whereupon the nanopore is available to capture and process another protein:translocase complex as per A). [1264] No or weak EOF nanopore systems. Nanopore systems with weak or no ion-selectivity were found to be unable to generate a significant net electro-osmotic flow (Table 12), and thus a weak EOF, under symmetric salt conditions. Under low to moderate applied voltages the nanopores were unable to effectively capture either free MBP-1 substrate or the pre-loaded ClpX:MBP-1 complex. Effective capture was achieved with the application of much higher voltages (thus significantly increasing the EPF acting on the leader and protein), despite the protein analyte having a leader construct with a highly charged region of 15 consecutive arginine residues to aid electrophoretic capture. FIGs. 79A-79D and FIGs. 80A-80D show representative MBP-1 capture data for wild-type alpha-hemolysin (FIGs. 79A-79D) and wild-type CytK (FIGs. 80A-80D) nanopores acquired at voltages from -80 mV to -180 mV or any combination thereof. Measurements acquired with cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5. The cis compartment contained a final concentration of 0.1 µM MBP-1. Very little MBP-1 capture is observed at lower voltages. As the voltage is increased, blockades to almost 0 pA were observed due to capture of MBP-1. Some -368- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 captures spontaneously clear the pore, but many have to be removed by briefly reversing the applied voltage (marked with arrows). [1265] FIGs. 81A-81C shows representative sections of recordings from wild-type alpha-hemolysin (FIGs. 81A-81B), and wild-type CytK (CytK-WT) (FIG. 81C) respectively in systems containing pre-loaded ClpX:MBP-1 (added to a final concentration of 0.2 µM ClpX, 0.1 µM MBP-1, 2.5 mM ATP in the cis chamber). In all the above nanopore systems a range of blockade types were observed that, without being bound to a particular theory, could be attributed either to capture of ClpX:MBP-1 complex, capture or free MBP-1 or some pore gating (where the nanopores shutdown spontaneously under the applied voltage). The blockade events all display a mostly stable blockade current level that could be indicative of a static state where the polypeptide is not moving through the nanopore (e.g. under the control of the ClpX). Events corresponding to capture of ClpX:MBP-1 complex typically blocked the currently indefinitely, and could only be cleared from the nanopore by briefly reversing the voltage (marked by arrows in FIGs. 79-81), suggesting that there was no progression of the polypeptide through the nanopore. Pre-loaded MBP-1:ClpX complexes (preloaded according to FIG. 76) were added to the cis side (to a final concentration 0.2 µM ClpX, 0.1 µM MBP-1 and 2.5 mM ATP) of the nanopore systems (with cis and trans solutions of 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5). Voltage dependent capture of MBP-1:ClpX complexes were observed in these low EOF nanopores, resulting in ionic current blockades to almost 0 pA. However, no events progress to a translocating peptide stage, and events remain blocked indefinitely unless ejected by a brief reversal of the applied voltage (marked by arrows). [1266] Strong EOF nanopore systems. Nanopore systems designed to have a strong EOF by employing mutated CytK nanopores with a strong ion-selectivity of >3.0 P(+)/P(-) were tested. Clear and consistent capture and subsequent ClpX-controlled translocation of MBP-1 was observed. For example, FIG. 82A and FIG. 82B show representative sections of electrical recordings acquired at -80 mV from CytK-4D2E nanopores (CytK K128D K155D S120D Q122D) in a system set up according to FIG. 74, where pre-loaded ClpX:MBP-1 complex was added to the cis compartment (preloaded according to FIG. 76 and as described herein, then added to a final concentration of 0.2 µM ClpX, 0.1 µM MBP-1, 2.5 mM ATP in the chamber). FIG. 82A shows a representative example with 3 consecutive ClpX controlled MBP-1 translocation events labelled 1-3. FIG.82B shows a further example from a separate experiment with 4 identifiable events labelled 1-4 that were interspersed between some of the other blockades that were observed (e.g. blockades from unproductive enzyme complexes or free MBP- 1 without ClpX bound). The capture and translocation events all had the same characteristic and consistent patterns. The translocations of the target proteins proceed via similar current patterns and share similar characteristic features: All events start with an immediate almost full block of the ionic current from the open pore level (IO) at about -65 pA to a blockade level (IB) of almost 0 -369- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 pA as a result of the capture of the MBP-1:ClpX complex. After a short period of time, the translocase overcomes the stall and begins to unfold the protein and pass the polypeptide into the nanopore, giving rise to changes in current levels as the polypeptide progresses through the pore as a result of the varying amino acid composition within the nanopore at any one time. Upon reaching the end of the protein, the translocase releases the substrate through the nanopore, resulting in a return to unoccupied open pore current IO. [1267] Events started with an almost instant blockage of the current from the open pore level (IO) of about - 65 pA at -80 mV (state i in FIG.83) to a blocked level of about 0 to 5 pA (S1 in FIG.83) that corresponds to the initial capture of the ClpX:MBP-1 complex. This was followed by a brief static level that , without being bound by a particular theory, may have corresponded to the ClpX:MBP-1 complex being held on top of the pore where the motor remained stalled by the leader motifs so that the polypeptide within the nanopore was held in place (state ii in FIG. 83). After a brief period the event proceeded (S2 in FIG. 83) to a highly characteristic pattern of changing current levels (state iii in FIG. 83) corresponding to the motor overcoming the stall (possibly due to a combination of the EPF force acting on the charged section of the leader and/or the EOF acting on the leader) and continuing to unfold and translocate along the protein, in the process passing the polypeptide through the nanopore from the cis to the trans side. Without being bound by a particular theory, it was postulated that the strong EOF pull on the extruded polypeptide retained the translocase against the top of the pore during the proposed events. The proposed events would end when the translocase reaches the end of the protein (S3 in FIG.83) and the polypeptide was fully translocated from the cis to the trans compartment (state iv in FIG.83), whereupon the ClpX was no longer retained on top of the pore, which returned to the open pore level where it was available to capture a fresh ClpX:MBP-1 complex. [1268] FIGs.84A-84B illustrate the highly consistent nature of the MBP-1 protein capture and translocation events. FIGs.84A-84B show selected representative events, and connects easily recognizable features in the current levels with dotted lines to aid the eye. The ionic current flowing through a nanopore can be dependent on the composition and structure of the molecules that are within the recognition region of the nanopore. It was expected to observe changes in current level due to the progression of differing amino acid composition as the polypeptide translocated through the nanopore. A highly similar pattern of current levels was observed when comparing between separate MBP-1 events. This suggested that the translocation of separate proteins sharing the identical underlying amino acid sequence altered the ionic current in a consistent and characteristic manner. The majority of events started with an almost full blockade to almost 0 pA, which was attributed to the polyarginine motif of the leader being in the nanopore channel, and then progressed through the same start and end current levels. This suggested that the events were full length protein translocations, demonstrating the effectiveness of the stall elements of the Leader construct at retaining the ClpX translocase to the start of the protein. Without being bound to a particular theory, if the stall were not effective at preventing the -370- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 translocation of ClpX in solution, it would be expected that a large number of events would have been observed where the current levels started part way through due to capture of a complex with the translocase at a random point along the protein. The effectiveness of the stall was confirmed by gel assays comparing degradation of GFP-0 (no stall) to GFP-1 when mixed with ClpX and ClpP, which showed that GFP-0 could be digested while GFP-1 was stable. [1269] FIGs. 84A-84B and FIG. 88 also illustrate that the duration and step-size of the polypeptide translocation is highly consistent and reproducible. FIG. 88 shows a histogram of the duration of ClpX controlled MBP-1 events from a single experiment. The event durations were normally distributed with a mean duration of about 30 seconds. Without being bound to a particular theory, it was proposed that this distribution could only be achieved when the movement of the polypeptide through the nanopore was tightly controlled by the ClpX translocase translocating along the polypeptide via NTP hydrolysis at an approximately constant rate (about 12 - 15 aa/second in these conditions). This was supported by control experiments which replaced ATP with 2.5 mM Gamma-S-ATP (FIG.87), which is a minimally hydrolysable analog of ATP. These experiments showed capture of ClpX:MBP-1 complexes that resulted in long blockades that never progressed to the changing current levels phase associated with ClpX translocase controlled movement of polypeptide that is observed under regular ATP conditions, and events could only be cleared from the nanopore by briefly reversing the applied voltage. Pre-loaded MBP-1:ClpX complexes (preloaded with standard 10 mM ATP as described herein) were added to the cis side of the nanopore system at -80 mV (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5 containing 0.2 µM ClpX:0.1 µM MBP-1, 2.5 mM gamma-S-ATP and 0.25 mM ATP; trans: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5). A) Blockade events from the capture of MBP-1:ClpX complexes were observed; a typical zoom example is shown in B). The events display the usual initial block to almost 0 pA (i), which is then followed by an increase to -20 pA (ii). However the events never progress to the translocase controlled movement of polypeptide phase that is observed under regular ATP conditions, resulting in indefinite blockades that have to be cleared by a brief reversal of applied voltage (iii). [1270] FIGs. 85A-85D show some example ClpX translocase controlled MBP-1 events from different nanopores with varying degrees of medium to high ion- selectivity, and thus varying levels of EOF in symmetrical salt conditions. Table 12 summarizes data for further systems, along with determinations of ion- selectivity and the relative net ionic flow that creates the EOF. While the patterns of current level changes were different for each nanopore, the events all possessed the same characteristic changes in current levels, indicating that sufficiently strong EOF produced (and determined) by a wide range of nanopore mutations can create systems capable of ClpX controlled polypeptide transport. Panels include subset schematic showing the -371- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 location of the net negative charges in the respective nanopores (where a negative residue is balanced by an adjacent positive residue neither are shown). [1271] Alternative substrates. To further prove that the pattern of current level changes were characteristic of the protein’s amino-acid composition, the ClpX controlled translocation of the protein GFP was tested under the same conditions described above for MBP-1. FIGs. 86A-86B show representative events from ClpX controlled translocation of MBP-1 (FIG. 86A) and GFP-1 (FIG. 86B), illustrating the different pattern of current levels that were observed for the proteins. MBP-1 and GFP-1 were translocated through CytK K128D K155D S120D Q122D (CytK 4D2E) nanopores at -80 mV. These data illustrated that the current levels were unique to the underlying amino acid sequence, such that the protein targets could be unambiguously identified by comparing the data to previously acquired signals. In principle it would be possible to train software to learn the underlying sequence-to-signal relationship to characterize target proteins, and, in principle, to determine the amino-acid sequence of an analyte. [1272] Pre-loading ClpX:target protein complexes. It can be optimal to bind proteins under conditions that may be different from those employed in nanopore systems (which may be optimized for ionic current signal). For example, the high salt concentrations often employed in nanopore systems can inhibit protein-substrate binding. It may be preferable to improve binding efficiency by mixing the components at high relative concentration, however, the amount of a target protein may be limited (e.g. in a real-world sample) and/or a nanopore system may not require high concentrations of substrate for efficient capture. It may be preferable to pre-incubate the components at high relative concentration in a small volume before addition into the nanopore system in a diluted form. In this example, preloading of translocases onto target proteins was explored by incubating the components under optimal binding conditions (i.e. optimal temperature, pH, salt content, salt concentration, cofactor concentration, or any combination thereof) at high relative translocase and target protein concentrations (> 1 µM) prior to addition to the nanopore system. In all nanopore systems, preloading significantly improved the proportion of translocase:target-protein complexes captured versus free target- protein captured when compared to systems where the components were added to the system separately (where they bind in the bulk solution). FIGs. 89A-89B illustrate the difference between addition of preloaded ClpX:MBP-1 (FIG.89B) versus separate addition of ClpX and MBP-1 (FIG.89A). Without pre- loading, the system was dominated by the capture of free MBP-1 substrate (no translocase bound), while preloading yielded a higher percentage of MBP- 1:ClpX complex capture that led to ClpX controlled MBP-1 translocations. FIG. 89A showed ClpX (0.2 µM final concentration) and MBP-1 (0.1 µM final concentration) were added to the cis compartment separately. FIG.89B showed ClpX and MBP-1 pre-incubated (10 µM ClpX, 5 µM ClpX, 10 mM ATP, 25 mM MgCl2) in a 10 µL volume before addition to cis compartment (0.2 µM ClpX, 0.1 µM MBP- 1 final concentration). Arrows mark voltage reversals, and stars mark ClpX controlled translocations. -372- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1273] Trans-to-cis translocations. As described herein, EOF from net electro-osmotic flow can be created in either the cis-to-trans direction or the trans- to-cis direction across the nanopore relative to the polarity of the applied voltage and the normal orientation of the nanopore. Compared to examples above (net cis-to-trans flow is created with a negative voltage at the trans electrode), if the voltage polarity is reversed, and the pore is also inserted from the trans compartment, then an equal and directly equivalent net trans-to-cis flow is created (in the case that the cis and trans salts are symmetrical), such that polypeptides can be translocated trans-to-cis when added to the trans compartment. To test the ability to translocate a polypeptide through the a nanopore in the reverse direction with respect the nanopore orientation (FIG. 90), CytK_4D2E nanopores (inserted from cis) were tested at positive applied voltage in trans to create a net trans- to-cis electro-osmotic flow, and preloaded ClpX:MBP-1 complexes were added to the trans compartment. Characteristic ClpX controlled MBP-1 translocation events trans-to-cis were observed under the strong trans-to-cis electroosmotic flow (FIG.90). A different pattern of current levels from the polypeptide translocation were observed, which was expected since the orientation of the polypeptide relative to the pore was reversed. Different orientations of analyte in a nanopore can change discrimination. This example illustrated that the direction of the EOF could be set up to direct polypeptide translocation across a nanopore in either direction relative to the nanopore. [1274] Since nanopores can often be asymmetrical, it can be advantageous to exploit other properties of their shape that change aspects such as capture of analyte, unwanted capture, location and orientation of nanopore constriction, and what surface the translocase interacts with. [1275] Alternative salts. Tests Assays of ClpX in KCl demonstrated that ClpX unfoldase function was inhibited in greater than 0.3 M KCl, while function was largely retained in 1M KGlu (FIGs. 91A-91B). Therefore, for the examples herein, experiments employed KGlu at high 1M concentration in the cis compartment to maximize the net cis-to-trans electro-osmotic flow that is dominated by the flux of K+ ions cis to trans. [1276] Asymmetric nanopore systems containing a different salt on the cis and trans sides can also be employed. For example, ClpX controlled MBP-1 translocations were obtained from CytK 4D2E nanopores systems with 1M KGlu in the cis and 1 M KCl in trans due to the strong cis-to-trans EOF (FIG. 92). The nanopore system contained 1M KGlu in the cis compartment and 1M KCl in the trans compartment (cis: 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, pH 7.5 containing 0.2 µM ClpX:0.1 µM MBP-1, 2.5 mM ATP; trans: 1 M potassium chloride, 50 mM Tris, 25 mM MgCl2, pH 7.5). Example 6.2: Cis-to-Cis protein measurements. [1277] Example 6.2 describes a system and method for characterization of a target protein by capturing it from the cis side into the nanopore with high net cis-to-trans EOF, in conjunction with a protein translocase motor that then pulls the polypeptide back through the same nanopore to the cis side. This process (also referred -373- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 to as “Out mode”) is illustrated schematically in FIGs. 93A-93E. In the schematic, a protein translocase (8) binds to a target protein (7), which is modified with terminal tags that can promote binding to the translocase (1) and capture in the nanopore (2), to create a translocase:target protein complex (FIG. 93A). The translocase:target protein complex is captured in a nanopore via the terminal capture tag via a combination of EPF and/or EOF forces (FIG. 93B) so that the target protein portion of the complex translocates partially through the nanopore from the cis side to trans side, until the bound translocase encounters the top of the nanopore (FIG. 93C). The force of the net cis-to-trans EOF acting on the polypeptide passing through the nanopore pulls on the translocase:target protein complex and retains the translocase against the top of the pore. The translocase continues to move along the target protein under NTP powered hydrolysis in the direction of the sub-arrow (in the cis-to-trans direction), and in doing so pulls the polypeptide back out of the nanopore in the trans-to-cis direction (FIG. 93D). Upon reaching the end of the target protein molecule the translocase unbinds from the target protein (FIG. 93E) and both the translocase and the target protein may be released back into the cis side. [1278] A target protein of interest was provided on the C-terminus with the ssrA capture motif AANDENYALAA to facilitate binding with a ClpX translocase. The target protein was also provided on the N-terminus with a >30 amino acid-length tag that was designed to have no secondary structure, a high cation content to improve efficient capture in a nanopore, and no ability to bind or stall ClpX translocase. A suitable tag was composed of repeating (SR)n residues. [1279] The adapted target protein was premixed with ClpX translocase under preloading conditions (0.4 uM ClpX, 0.2 uM adapted target protein, 10 mM ATP and 25 mM MgCl2 in a final volume of 10 ul) and incubated for at least 10 minutes for loading of the ClpX onto the target protein. The preloaded ClpX:target protein complex was then added to this cis compartment (final concentration of 0.2 µM ClpX, 0.1 µM target protein) of a system containing a single CytK-4D2E nanopore in a membrane under a negative applied voltage (-60 mV to -120 mV) at the trans electrode (1M KGlu cis, 1M KGlu trans, 50 mM TRIS, 2.5 mM ATP, 25mM MgCl2, pH 7.5). [1280] Without being bound by a particular theory, ClpX:target protein complex captures were suggested by a sharp blockade of the ionic current passing through the open nanopore, indicating that the capture motif on the N- termini of the target protein and then a portion of the target protein itself were partially translocated through the pore nanopore from cis to trans as a result of the strong cis-to-trans EOF acting on the polypeptide (and initially EPF acting on the polycation capture tag) (FIG. 94). The initial fast translocation of the polypeptide was theorized to stop when the bound translocase encountered the top of the nanopore, and then the strong cis-to- trans EOF would have continued to pull on the polypeptide within the nanopore and thus kept the translocase atop the nanopore. A sequence of amino-acid dependent changes in current levels were -374- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 observed as the translocase atop the nanopore continued to move along the polypeptide (i.e. C>N according to this protein design, and in the cis-to-trans direction relative to the nanopore system) under the power of ATP hydrolysis, in the process pulling the translocated portion of the polypeptide in the nanopore and trans compartment back out of the nanopore from trans-to-cis against the EOF. A return to the open pore current level (IO) was observed when the translocase motor would have reached the end of the adapted target protein, pulling it fully back through the pore trans-to-cis, where it would have unbound and both components would have been released into the cis solution. Example 7. Protein identification and long proteins [1281] This example describes a system and method to that enabled the identification of proteins based on their unique ionic current signature during translocation. The target proteins were captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolded and transported the polypeptide through the pore. [1282] Target protein analytes of interest were provided with a long >30 amino acid tag that had an AANDENYALAA capture recognition motif that facilitated binding with a ClpX translocase, a domain with high cation content that enabled efficient capture into the nanopore (RRRRRRRRRRRRRRR; SEQ ID NO: 94) and a domain to that stalled the ClpX translocase (GGGGGGGGGGGG; SEQ ID NO.: 127). [1283] The adapted target protein was added to the cis compartment in a concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system with a single CytK-4D2E nanopore in a membrane under – 80 mV applied potential. The ionic current measurements were then performed using the standard methods described herein. [1284] Four different substrates ({GFP}-{MBP-1}, {LIVBP}-{MBP-1}, {SpuE}-{MBP-1} and {GBP}- {MBP-1}) were shown to each have similar ionic current signals in the initial section corresponding to MBP- 1, and unique ionic current signals in the respective attached proteins during translocation (FIGs.96A-96E). Measurements obtained with CytK_4D2E nanopore in 1 M potassium glutamate, 50 mM Tris, 25 MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5 at -80 mV. The cis compartment had a concentration of 0.2 µM ClpX, 0.1 µM of the respective indicated substrate, and 2.5 mM ATP. Different (but reproducible) ionic current levels were observed for the regions of difference between the substrates, which indicated that the ionic current that was measured reflected the amino acid composition of the proteins. This example demonstrated the ability of such a system to identify proteins based on their ionic current characteristics. [1285] Additionally, MBP-MBP-1 was a genetic fusion protein of two MBP proteins with a total length of 805 amino acids and a molecular weight of 88 kDa. Despite its large size, full length reads of the protein were reliably obtained (FIG. 97), which indicated that this system was able to read long proteins from C-terminus to N-terminus during translocation events. Measurements obtained with a CytK_4D2E nanopore in 1 M -375- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 potassium glutamate, 50 mM Tris, 25 MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5 at -80 mV. The cis compartment had a concentration of 0.2 µM ClpX, 0.1 µM MBP-MBP and 2.5 mM ATP. Table 19. Amino acid sequences of proteins used in Example 7 Protein Amino acid sequence SEQ ID NO. {GFP}- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF 113 {MBP-1} PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPI AVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAF KYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPW AWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLE AVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAAS GRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA {LIVBP}- MHHHHHHSSGEDIKVAVVGAMSGPVAQYGDQEFTGAEQAVADINAKGGIKGNKLQIVKYDD 114 {MBP-1} ACDPKQAVAVANKVVNDGIKYVIGHLCSSSTQPASDIYEDEGILMITPAATAPELTARGYQ LILRTTGLDSDQGPTAAKYILEKVKPQRIAIVHDKQQYGEGLARAVQDGLKKGNANVVFFD GITAGEKDFSTLVARLKKENIDFVYYGGYHPEMGQILRQARAAGLKTQFMGPEGVANVSLS NIAGESAEGLLVTKPKNYDQVPANKPIVDAIKAKKQDPSGAFVWTTYAALQSLQAGLNQSD DPAEIAKYLKANSVDTVMGPLTWDEKGDLKGFEFGVFDWHANGTATDAKVKIEEGKLVIWI NGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQ SGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIP ALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTF LVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPS KPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPR IAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGG GGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA {SpuE}- MHHHHHHSSGEKKSLHIYNWTDYIAPTTLKDFTKESGIDVSYDVFDSNETLEGKLVSGHSG 115 {MBP-1} YDIVVPSNNFLGKQIQAGAFQKLDKSKLPNWKNLDPALLKQLEVSDPGNQYAVPYLWGTNG IGYNVAKVKEVLGDQPIDSWAILFEPENMKKLAKCGVAFMDSGDEMLPAALNYLGLDPNTH DPKDYKKAEEVLTKVRPYVSYFHSSKYISDLANGNICVAFGYSGDVFQAAARAEEAGKGID IQYVIPKEGANLWFDLMAIPADAKAADNAYAFIDYLLRPEVIAKVSDYVGYANAIPGARPL MDKSVSDSEEVYPPQAVLDKLYVSAVLPAKVLRLQTRTWTRIKTGKLEKIEEGKLVIWING DKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPAL DKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLV DLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKP FVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIA ATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGG GGGGGSRRRRRRRRRRRRRRRAANDENYALAA {GBP}- MHHHHHHSSGADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQ 116 {MBP-1} IDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDS KESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKT EQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFG VDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPY VGVDKDNLAEFSKKGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYP IAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYA FKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGP WAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGL -376- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 EAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAA SGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA {MBP}- MGSSHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEEDTGIKVTVEHP 117 {MBP-1} DKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNG KLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETA MTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYL LTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRT AVINAASGRQTVDEALKDAQTRITKGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGI KVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTW DAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAA FNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAK EFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA FWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALA Example 8: Stalling of unfoldase at higher temperatures using blocking domains [1286] This example demonstrates improved stalling of unfoldases, such as at higher temperatures, using a block motif. [1287] Fluorescence assay for the stalling efficiency of block motif [1288] 141 µL of a solution containing 1M KGlu, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1mM EDTA was added in two different wells of a 96-well black plate for fluorescent assays. Then, 1 µL of a solution with 10 µM of a Green Fluorescent Protein carrying the ssrA tag at the C-terminus was added to each well. Subsequently, 8 µL of a solution with 5 µM of ClpX was added to one well, to achieve the desired volume of 150 µL and the desired molar ratio of 1:4, substrate : ClpX. The well where ClpX was not added was used as positive control. The plate was then inserted in a plate reader set at an incubation temperature of 37ºC. [1289] The measurement of fluorescence over time was carried out one well at a time. To measure fluorescence, the well was excited with a light pulse of optimal excitation wavelength for the analyzed fluorescent protein, and the emitted light was collected at the optimal emission wavelength using an appropriate bandwidth filter. After an initial shaking of 30 seconds to ensure proper mixing of the solution, fluorescence was tracked for 1 minute to ensure no spontaneous loss of fluorescence. Subsequently, 16 µL of a solution -377- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 with 100 mM ATP and 200 mM KOH was automatically injected into the well, to achieve a concentration of 10 mM ATP. The fluorescence was then measured for 8 minutes at intervals of 30 seconds. Table 20. Amino acid sequence of the proteins used. Protein Amino acid sequence SEQ ID NO. GFP-1 MGHHHHHHSSASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFIC 118 TTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNY KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGIGGGGGGGGGGGGSRRRRRRRRRRAANDENYALAA mNG- MKHHHHHHGVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLK 119 alpha_helix STKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASL TVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFK WSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA FTDVMGMDELYKGGSGSGDYMERWYRYYNEFSGGVAANDENYALAA mNG-HTH MKHHHHHHGVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLK 120 STKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASL TVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFK WSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA FTDVMGMDELYKGGSGSGDELAQLERELMKLKAQGVDSDELEALARKLAMLARSGGVA ANDENYALAA mNG- MKHHHHHHGVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLK 121 beta_hairpin STKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASL TVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFK WSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKA FTDVMGMDELYKGGSGSGRGKITVNGKTYEGRSGGVAANDENYALAA [1290] Fluorescence experiments adding ClpX to the fluorescent protein GFP (also comprising a ssrA recognition sequence) at 37ºC resulted in a loss of fluorescence, indicative of the unfoldase’s ability to proceed through the substrate and unfold the protein (FIG.98A). [1291] However, the insertion of block motifs comprising high stability protein structures, such as alpha- helices, helix-turn-helix structures and/or beta-hairpins, were able to stall ClpX to prevent it proceeding through the attached fluorescent protein, thus resulting in no decrease in fluorescence (FIGs. 98B-D). This example demonstrated that such stable structures can effectively stall ClpX when placed between the recognition tag and the protein of interest Such stable blocking motifs can help to ensure that the unfoldase was placed at the start of the target polypeptide before the unfoldase-substrate complex was captured by the pore, which increased the probability of obtaining full-length reads of the protein of interest. Measurements were performed with a 1:4 substrate:unfoldase molar ratio (0.065 µM substrate and 0.26 µM ClpX) in the -378- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 presence of 10 mM ATP in a solution containing 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. Example 9: Protein unfolding and translocation using VAT-ΔN protein translocase. [1292] This example describes a system in which the translocase was based on the VAT unfoldase from Thermoplasma acidophilum. VAT unfoldase used herein (VAT-ΔN) had a N-terminal truncation to remove a regulatory domain, which increased the unfoldase activity. This VAT-ΔN, where 183 amino acids of the N- terminus were truncated, was used to test its ability to unfold and translocate proteins across the nanopore. [1293] Preparation of VAT-ΔN unfoldase [1294] A plasmid with a gene encoding the VAT-ΔN gene elongated by six histidine residues was transformed into E. coli BL21(DE3) cells by electroporation. Expressed VAT-ΔN protein was extracted from the supernatant and purified using Ni-NTA beads, with elution in 200 µl aliquots (150 mM NaCl, 300 mM imidazole, 15mM Tris buffered at pH 7.5) before storage at 4oC. The sequence of VAT-ΔN protein is shown in Table 21. Table 21. Amino acid sequence of the translocase used in this example. Protein Amino acid sequence SEQ ID NO. VAT-ΔN MGSSHHHHHHGSGLVPRGSAGEVSRISYEDIGGLSEQLGKIREMIELPLKHPELFERLGIT 122 PPKGVILYGPPGTGKTLIARAVANESGANFLSINGPEIMSKYYGQSEQKLREIFSKAEETA PSIIFIDEIDSIAPKREEVQGEVERRVVAQLLTLMDGMKERGHVIVIGATNRIDAIDPALR RPGRFDREIEIGVPDRNGRKEILMIHTRNMPLGMSEEEKNKFLEEMADYTYGFVGADLAAL VRESAMNALRRYLPEIDLDKPIPTEILEKMVVTEDDFKNALKSIEPSSLREVMVEVPNVHW DDIGGLEDVKREIKETVELPLLKPDVFKRLGIRPSKGFLLYGPPGVGKTLLAKAVATESNA NFISIKGPEVLSKWVGESEKAIREIFKKAKQVAPAIVFLDEIDSIAPRRGTTSDSGVTERI VNQLLTSLDGIEVMNGVVVIGATNRPDIMDPALLRAGRFDKLIYIPPPDKEARLSILKVHT KNMPLAPDVDLNDIAQRTEGYVGADLENLCREAGMNAYRENPDATSVSQKNFLDALKTIRP SVDEEVIKFYRTLSETMSKSVSERRKQLQDQGLYL [1295] MBP-1 target proteins of interest (as described previously) were provided with a long >30 amino acid tag that had an AANDENYALAA (SEQ ID NO.: 91) capture motif that facilitated binding with a VAT translocase, a domain with high cation content that enabled efficient capture into the nanopore. [1296] Electrophysiology measurements of protein translocation were performed as described herein. Briefly, the adapted MBP-1 target protein was added to the cis compartment in a concentration of 0.1 µM together with 0.2 µM VAT-ΔN and 2.5 mM ATP in a system containing a nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein. FIG. 130 shows a representative electrophysiology read of VAT controlled translocation of MBP-1 protein, showing characteristic pattern of changing current level similar to that obtained for ClpX translocase. -379- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1297] VAT-ΔN, originating from a thermophilic organism, was active at increasing temperatures. Therefore, the temperature of the system was increased to 37°C. This further showed that various types of protein translocases, in particular AAA+ protein unfoldases, can be used in a method or system provided herein. Example 10: N-terminus to N-terminus linked proteins [1298] To test the ability of the system to process proteins with different linkages, such as a N-terminus to N- terminus linkage, a C-MBP construct was measured. The C-MBP construct was a MBP-variant with a cysteine on the third residue from the N-terminus of the protein. The C-MBP construct protein naturally formed homodimers in non-reducing conditions (confirmed by appearance of a dimer band with SDS-PAGE). These dimers were covalently linked and had proteins that were coupled N-terminus to N-terminus (FIGs.99A-99B). [1299] The C-MBP target protein dimer solution was added to the cis compartment of the nanopore system in a concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a nanopore system with a single CytK-4D2E nanopore in a membrane under –80 mV applied potential. The ionic current measurements were then performed using the standard methods described herein. [1300] FIG.132 shows a representative electrophysiology read from the translocation of the linked substrate, showing a clear pattern of level for both the first C-N section and the second N-C section after passing through the linker. After reading the first monomer of C-MBP, the unfoldase was able to pass through the disulfide bond, after which it reads the second C-MBP monomer in the N- to C-terminus direction. This demonstrates the translocase reading in both the C-to-N and N-to-C direction through the protein analytes. This also demonstrates that the EOF of the system enables the translocase to process through unnatural chemical linkages. The sequence of C-MBP is shown in Table 22. Table 22. Amino acid sequence of the proteins used in this study Protein Amino acid sequence SEQ ID NO. C-MBP MGCHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEEDTGIKVTVEHPD 123 KLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAM TINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLL TDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA VINAASGRQTVDEALKDAQTRITKSS Example 11: Tagging of the C-terminus of a maleimide-peptide tag [1301] This example demonstrated the ability to tag a target protein substrate to improve translocase loading and subsequent capture in a nanopore. -380- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1302] A MBP target protein comprising a C-terminal cysteine was chemically tagged on the C-terminus by chemical coupling to a maleimide modified leader as described below to create a cysteine-maleimide linkage between the tag and target protein. The leader tag had an AANDENYALAA recognition motif to facilitate binding with a ClpX translocase, a domain with high cation content to enable efficient capture into the nanopore (RRRRRRRRRRRRRRR; SEQ ID NO.: 94), and a domain to stall the ClpX translocase (GGGGGGGGGGGG; SEQ ID NO.: 127). [1303] The tagged target protein was added to the cis compartment of the nanopore system in a concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a nanopore system with a single CytK-4D2E nanopore in a membrane under –80 mV applied potential. The ionic current measurements were then performed using the standard methods described in this patent. See FIG. 100 for an exemplary electrophysiology reads. This example showed that tagged target proteins were captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolded and transported the polypeptide through the pore (translocase controlled translocations are marked with arrows). Measurements obtained with a CytK_4D2E nanopore in 1 M potassium glutamate, 50 mM Tris, 25 MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5 at -80 mV. The cis compartment contained has a concentration of 0.2 µM ClpX, 0.1 µM tagged-MBP and 2.5 mM ATP. [1304] Tagging of MBP-C: [1305] MBP-C was an N-terminally His-tagged MBP protein with a C-terminal extension of SSC. The Cysteine was engineered there to serve as a handle to modify MBP with a maleimide-peptide. MBP-C was expressed in E. coli BL21(DE3)(pET28a_MBP-C). Cells were grown in LB with 50 ug/ml kanamycin. Expression was induced by adding 1 mM IPTG concentration when the culture reached OD600 of 0.8. The cells were harvested after overnight growth at 25 °C. Pellet was resuspended in MBP binding buffer (200 mM Tris-HCl pH 7.4, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) with protease inhibitors and the cells were sonicated. The cell extract was centrifuged for 30 min at 8000 x g and the supernatant was filtered over a 0.45 um filter. The filtered extract was loaded on a 1 ml MBPtrap column, that was preequilibrated with MBP binding buffer. The column was washed with 5 ml MBP binding buffer.0.5 ml of 1 mg/ml solution of Peptide- 2 (Maleimide-G12SR15-ssrA) in MBP binding buffer was applied to the column. The column was left overnight at room temperature to let the reaction proceed. Next day, the column was washed with MBP binding buffer to remove excess Peptide-2 tag. MBP protein was eluted from the column with MBP elution buffer (MBP binding buffer + 10 mM maltose). The tagged protein was further purified using the 1 ml HiTrap SP column. The Peptide-2 tag which had a polyarginine sequence has high affinity for cation exchange column. 0.5 ml of MBP column elution was mixed with 1 ml salt buffer (1.5 M NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 8.0). The HiTrap SP column (preequilibrated with 1 M NaCl buffer) was loaded with the protein sample -381- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 and washed with 3 ml 1 M NaCl buffer (1 M NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) to wash away untagged protein. The tagged protein was eluted with 1.5 M NaCl (1.5 M NaCl, 1 mM EDTA, 10 mM Tris- HCl pH 8.0). Subsequent analysis of the fractions with SDS-PAGE showed that a small percentage of protein was tagged. The sequence of MBP-C, comprising a C-terminal cysteine residue, is shown in Table 23. Table 23: Amino acid sequence of the proteins used in this study Protein/peptideAmino acid sequence SEQ ID NO. MBP-C MGSSHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEEDTGIKVTVEH 124 PDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRY NGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNK GETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQTRITKSSC Peptide-2 Tag GGGGGGGGGGGGRRRRRRRRRRRRRRRSGGVAANDENYALAA 125 (Maleimide) Example 12: Nanopore with a Molecular Adaptor [1306] The methods and systems disclosed herein can be used to determine (e.g., sequence) a non-nucleic acid based polymer analyte (e.g., polypeptide). [1307] Preparation of nanopores with adaptor [1308] The alpha-haemolysin (aHL) nanopore of the present example are prepared to have a chemical adaptor coupled to the channel of the nanopore. Wildtype aHL nanopores are designed with glutamate mutations to the inwards facing residues (positions 119/121/123/125) of the beta-strands of the beta barrel to increase the net negative charge inside the barrel of the nanopore. To introduce an attachment site within aHL nanopores one of the subunits also comprises a L135C mutation. [1309] The amino acid sequence of aHL is set forth as: ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRV YSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYDFDGDVDGDDTGKIG GCIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFM KTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTST NWKGTNTKDKWTDRSSERYKIDWEKEEMTN (SEQ ID NO.: 126) [1310] Alpha-haemolysin (aHL) nanopores are expressed, assembled, and purified for use in the nanopore system. To prepare the heteroheptamers, a sequence encoding a C-terminal tail of eight aspartate residues is added to the genes of the cysteine mutant nanopores. The aspartate tail allows the resolution of heptamers with different combinations of subunits by protein electrophoresis (e.g., SDS-PAGE), enabling heteroheptamers containing a single cysteine modified subunit to be excised from the final protein (e.g., SDS-PAGE) gel. -382- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1311] A modified beta-cyclodextrin (heptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropanoyl-b- cyclodextrin, bCD) with a reactive side-arm linker is additionally prepared. The modified beta-cyclodextrin then attaches to the channel of the generated aHL nanopore. [1312] Recordings of protein translocation [1313] Measurements of translocase controlled protein translocation were carried out according to the system schematically described in FIG. 74. Experiments are run on a nanopore system (e.g., axopatch planar bilayer system) as described herein at room temperature. Both compartments of the nanopore system are filled with 0.4 mL of an electrolyte solution (1 M potassium glutamate, 20 mM MgCl2 and 50 mM Tris, buffered to pH 7.5).1uL of a preparation of purified aHL nanopore solution is added to the cis compartment with mixing to achieve a single inserted nanopore. The insertion of a single nanopores is detected by the characteristic step- wise change in open pore current. The nanopore is characterised at a range of voltages to assess the quality of the nanopore to ensure suitability for the experiment. bCD is added to the trans compartment. bCD binding to the nanopore is evident by ionic current blockades under an applied voltage (variable from -180 mV to +180 mV), and permanent covalent reaction of the bCD to the nanopore is evident by the permanence of the reduced ionic current bCD blockade level. Reacted bCD-aHL nanopores with an open-pore current of >20pA at 100mV are selected for protein translocation experiments. Separately, ClpX translocase (prepared as described herein) and target MBP-1 protein analyte (prepared as described herein) are preincubated as described at a 2:1 translocase:target-protein molar ratio, for >10 minutes at room temperature in a preloading solution (e.g., 10 mM ATP and 25 mM MgCl2). After pre-incubation, the translocase:target-protein complex is added to the cis- compartment. [1314] Electrical recordings are acquired over a range of voltages from -60 mV to -180 mV. ClpX:MBP-1 complex translocation events through bCD-aHL nanopores are evident by their characteristic blockade reduction in ionic current flowing through the nanopore, followed by a characteristic pattern of amino-acid dependent changes in current levels lasting for about 10-30 seconds before the events end and the ionic current returns to the open-pore level. Example 13. Nanopore with a protein adapter [1315] Preparation of ClpX Translocase [1316] E. coli ClpX was employed as exemplary translocase to control the movement of the polypeptide through the nanopore (FIGs.2 and 74). ClpX was selected as a AAA+ translocase systems, and can unfold and translocate along a wide variety of proteins, generating a high force through NTP hydrolysis. The monomer and covalently linked trimer of N-terminal truncated ClpX variants (residues 61–423) were purified as with minor modifications and used for ClpX nanopore experiments. Specifically, the gene encoding for monomer -383- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 and trimer of ClpX-ΔN were separately transformed into electrocompetent cells (e.g., E. coli BL21 (DE3) electrocompetent cells). Transformants were selected after overnight growth at 37 °C on agar plates(e.g., lysogeny broth (LB) agar plates) supplemented with ampicillin (100 mg/L). The resulting colonies were inoculated into 200 mL culture media (e.g., LB medium) containing 100 mg/L of ampicillin. The ClpX protein expression was induced at an A600 of ∼0.6 by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 25 °C overnight. The cells were harvested by centrifugation for 20 min (4000 x g) at 4°C and the pellets were stored at -80°C. About 100 mL of cell culture pellet was thawed and solubilized with ~20 mL lysis buffer (e.g., 50 mM HEPES, pH 7.5, 300 mM KCl, 20 mM imidazole, 1 mM dithiothreitol (DTT), 0.1 units/mL DNase I, 10 µg/mL lysozyme) and stirred with a vortex shaker for 1 hour at 4°C. The bacteria were then lysed by sonication (duty cycle 10%, output control 3, Branson Sonifier 450). The lysate was subsequently centrifuged at 6000 x g at 4 °C for 20 min and the cellular debris discarded. The supernatant was mixed with 100 μL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was pre-equilibrated with wash buffer (e.g., 50 mM HEPES, pH 7.5, 300 mM KCl, 20 mM imidazole, 1 mM dithiothreitol (DTT)). Proteins were purified from the supernatant via Ni-NTA resin (Qiagen) using standard procedures and eluted with approximately 600 μL elution buffer (e.g., 600 mM imidazole, 1 mM dithiothreitol (DTT), 100 uM EDTA, 200 mM KCl, 25 mM MgCl2, 50 mM Tris, pH 7.5). The proteins were further purified using a Superose 6 column Increase 10/600 GL and eluted in 200 ul fractions in elution buffer 2 (e.g., 1 mM dithiothreitol (DTT), 100 uM EDTA, 200 mM KCl, 25 mM MgCl2, 50 mM Tris, pH 7.5). The fractions with pure protein were concentrated using Amicon Ultra Centrifugal Filters. Purified proteins were then flash frozen in small aliquots supplemented with 30 % glycerol and stored at –80 °C. Protein concentrations were determined by Bradford assay with bovine serum albumin as a standard. [1317] Preparation of Protein Analytes [1318] The analyte MBP-1 based on the well-known model protein Maltose Binding Protein (MBP) was used to test protein translocation through the nanopores with adapters. MBP-1 comprised a long >30 amino acid leader with a AANDENYALAA capture recognition motif that facilitated binding with ClpX translocase, a domain with high cation content that enabled efficient capture into the nanopore (RRRRRRRRRRRRRRR; SEQ ID NO: 94) and a domain to that stalled the ClpX translocase (GGGGGGGGGGGG; SEQ ID NO: 127). [1319] Briefly, MBP-1 was prepared transforming the gene encoding MBP-1 into electrocompetent cells (e.g., E. coli BL21 (DE3) electrocompetent cells). Transformants were selected after overnight growth at 37 °C on agar plates (e.g., lysogeny broth (LB) agar plates) supplemented with kanamycin (50 mg/L). The resulting colonies were inoculated into 200 mL culture media (e.g., LB medium) with 50 mg/L of kanamycin. The cells were induced at an A600 of ∼0.6 by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and incubated at 25 °C overnight. The cells were harvested by centrifugation and the pellets were stored at -80°C. -384- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 100 mL cell culture pellets were thawed and solubilized before removing the cellular debris by centrifugation. Proteins were purified from the supernatant via Ni-NTA resin (Qiagen) used standard procedures and eluted with approximately 100 μL elution buffer (e.g., 600 mM imidazole, 1 mM dithiothreitol (DTT), 150 mM KCl, 50 mM HEPES, pH 7.5). Purified protein as then flash frozen in small aliquots and stored at –80 °C. Protein concentrations were determined by Bradford assay with bovine serum albumin as a standard. Table 24. Sequences of MBP-1 analyte Analyte Component Sequence SEQ ID NO. MBP-1 Affinity MHHHHHHSS 89 purification tag MBP protein PWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF 100 PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDA VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKS ALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTF LVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYG VTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEA VNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWY AVRTAVINAASGRQTVDEALKDAQTRITKHM Stall/capture GGGGGGGGGGGGSRRRRRRRRRRRRRRR 101 motif Recognition AANDENYALAA 91 motif [1320] Preloading of the translocase onto Target Protein [1321] To improve the percentage of ClpX:target-protein complexes formed, the complexes are formed prior to addition to the nanopore system. ClpX:Target-protein complexes are preloaded by mixing the components at a concentration of about 10 µM ClpX, 5 µM target protein in a 2:1 ratio, in a solution with 10 mM ATP and 25 mM MgCl2 in a volume of 10 ul (50 mM Tris-HCl, 200 mM KCl, 10 mM ATP, 25 mM MgCl2, 1 mM DTT, 1 mM EDTA, PH 7.5). The mixture is incubated for at least 10 minutes at room temperature to allow sufficient time for the ClpX to bind to the target proteins. [1322] Preparation of CsgG/F nanopores [1323] CsgG-F56D/CsgF nanopores are prepared. Briefly, E. coli cells is transformed with genes coding for CsgG-F56D and CsgF subunits are resuspended in 50 mM Tris–HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 0.4 mM AEBSF, 1 µg ml−1 leupeptin, 0.5 mg ml−1 DNase I and 0.1 mg ml−1 lysozyme. The cells are lysed and then incubated for 30 min with 1% n-dodecyl-β-D-maltopyranoside (DDM) to extract the outer membrane components. Cell debris is removed by ultracentrifugation at 100,000g for 40 min and supernatant is loaded onto a 5-ml HisTrap column (GE Healthcare) equilibrated in buffer A (e.g., 25 mM Tris pH 8, 200 mM NaCl, 10 mM imidazole, 10% sucrose and 0.06% DDM). The column is washed with >10 -385- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 column volumes of 5% buffer B (e.g., 25 mM Tris pH 8, 200 mM NaCl, 500 mM imidazole, 10% sucrose and 0.06% DDM) in buffer A, and elutes with a gradient of 5–100% buffer B over 60 ml. The eluate is diluted twofold before loading overnight on a 5-ml Strep-Tactin column (IBA GmbH) equilibrated with buffer C (e.g., 25 mM Tris pH 8, 200 mM NaCl, 10% sucrose and 0.06% DDM). The column is washed with >10 column volumes of buffer C and the bound protein is eluted in buffer C complemented with 2.5 mM desthiobiotin. The co-expressed complex is injected on a Superose 610/30 column (GE Healthcare) equilibrated with buffer F (e.g., 25 mM Tris pH 8, 200 mM NaCl and 0.03% DDM) and run at 0.5 ml min−1.The CsgG–CsgF complexes are digested at room temperature overnight with TEV protease in buffer F. The mixture is then run back through a 5-ml HisTrap (GE Healthcare) column and the flow-through is collected, heated at 60  oC for 15 min and centrifuged at 21,000g for 10 min before use in electrophysiology. Protein concentrations are determined on the basis of calculated absorbance at 280 nm and assuming 1/1 stoichiometry. [1324] The CsgG amino acid sequence is set forth as: CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNDSTAVPQSATAM LVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSIIGYESNVKS GGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGE VGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES (SEQ ID NO.: 128). [1325] The CsgF amino acid sequence is set forth as: GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNSYKDPSYNDDFGIETPSALDNFTQAIQSQILGGLLSNI NTGKPGRMVTNDYIVDIANRDGQLQLNVTDRKTGQTSTIQVSGLQNNSTDF (SEQ ID NO.: 129). [1326] Planar lipid bilayer electrophysiological recordings system [1327] For each experiment a single nanopore is inserted in a planar lipid bilayer system. Briefly, an electrophysiology chamber with two compartments separated by a 25 µm thick membrane (e.g., Teflon (Goodfellow Cambridge Ltd) membrane) is used. The Teflon membrane has an aperture with a diameter of approximately 100 µm. Lipid membranes are formed by first applying 5 µl of 5% hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane, near the aperture. The pentane is left to dry and 400 µl of buffered solution (1 M potassium glutamate, 20 mM MgCl2 and 50 mM Tris, buffered to pH 7.5)is added to each compartment.20 µl of a 6.25 mg/ml solution of DPhPC dissolved in pentane is added on top of the buffer on each side of the chamber. The chamber is left to dry for ~2 minutes to allow evaporation of pentane. Silver/silver chloride electrodes are attached to each compartment. The cis compartment is connected to the ground electrode and the trans is connected to the working electrode. Planar lipid bilayers are created using the Langmuir-Blodgett method. Purified nanopore solutions are added to the cis compartment to obtain -386- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 insertion of single nanopores. Once a single nanopore is inserted the orientation and properties of the nanopore are confirmed by the asymmetry of the current-voltage relationship. [1328] Recordings of ionic currents are obtained using an amplifier (Axopatch 200B patch clamp amplifiers (Axon Instruments)) combined with a A/D converter (Digidata 1550B A/D converters (Axon instruments)). Recordings are acquired at 10 kHz with a 2 kHz Bessel filter, and recorded using Clampex 10 (Molecular Devices) at 22oC. [1329] Recordings of protein translocation [1330] Measurements of translocase controlled protein translocation are carried out according to the system schematically described in FIG.74. Both compartments of the nanopore system are filled with 0.4 mL of an electrolyte solution (e.g., 1 M potassium glutamate, 20 mM MgCl2 and 50 mM Tris, buffered to pH 7.5).1uL of a preparation of purified CgsG/F nanopore solution is added to the cis compartment with mixing to achieve a single inserted nanopore. After detecting the insertion of a single nanopore by the characteristic step-wise change in open pore current, the nanopore is characterised at a range of voltages to assess the quality of the nanopore to ensure suitability for the experiment. Nanopores with an open-pore current of >40pA at 180mV are selected for protein translocation experiments. Separately, ClpX translocase (prepared as described above) and MBP-1 target protein substrate (prepared as described) are preincubated as described at a 2:1 translocase:target-protein molar ratio, for >10 minutes at room temperature in 10 mM ATP and 25 mM MgCl2. After pre-incubation, the ClpX:MBP-1 complex is added to the cis-compartment. [1331] Electrical recordings are acquired over a range of voltages from -60 mV to -200 mV. ClpX:MBP-1 complex translocation events are evident by their characteristic blockade reduction in ionic current flowing through the nanopore, followed by a characteristic pattern of amino-acid dependent changes in current levels which lasts for about 10-30 seconds before the events end and the ionic current returns to the open-pore level. [1332] Expression and purification of pAzF-modified nanobodies [1333] The DNA encoding for Ty152, nb2255, 2Rs15d and 2Rb17c54 nanobodies were cloned into PET22b (+) plasmid (Addgene) respectively, with a pelB leader sequence at the N-terminal and a hexahistidine tag (6xHis) at the C-terminal. An amber stop codon (TAG) was added before the 6xHis to incorporate UAA into the nanobody. The production of pAzF-modified nanobody was conducted by following an established protocol. Firstly, the constructed plasmid was transformed into BL21 E.coli cells. Cells were cultured in 1 L TB medium supplemented with 100 mL salt buffer (0.17 M KH2PO4, 0.72 M K2HPO4), 1 mL 2 M MgCl2, 1 mL 100 mg/mL ampicillin, 1 mL 50 mg/ml spectinomycin, 10 mL 10% glucose and 250 mg 4-azido-L-phenylalanine at 37 °C at 200 rpm. When the OD600 reached at 0.6-0.9, IPTG with a final concentration of 1 mM was added. The protein induction was completed at 25 °C by overnight shaking. The cells were harvested by centrifuging at 4°C and 4500 rpm for 15 min, which were then resuspended in 24 mL cold TES buffer (0.2 M Tris, pH 8, 0.5 -387- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 mM EDTA, 0.5 M sucrose). The suspension was incubated at 4 °C and 200 rpm (horizontal rotator) for 6 h, followed by the addition of 48 mL 1/4 TES buffer and incubation at 4°C and 200 rpm for overnight. Subsequently, the cell suspension was centrifuged at 4°C and 12000 g for 30 min. The supernatant was collected and supplemented with 5 mM MgCl2, followed by a further purification by FPLC (GE Healthcare) using a 5 mL HisTrap column (GE Healthcare). Binding buffer and elution buffer used here were 20 mM and 500 mM imidazole, respectively, both were supplemented with 20 mM sodium phosphate pH 7.4, 500 mM NaCl. The purity of the protein was analysed on 4%-12% SDS-PAGE gel. [1334] Conjugation of nanobody with f’-oligo [1335] Oligo f’ ordered with amine group at 5’ end (NH2-C6-5’-ATCCGCGGGTGTCGGG-3’) was firstly reacted with 20 fold excess of NHS-DBCO in 60% DMSO at pH 8.0 and 25°C for overnight. After being purified by ethanol precipitation and subsequent reverse-phase HPLC, the DBCO-oligo was incubated with azide-modified nanobody in PBS at 25°C for overnight. The reaction was optimized by adding different ratio of nanobody and f’-DBCO oligo. When the molar ratio was 5:1, the conjugation yield was above 70%. Therefore, this ratio was applied for the conjugation of all four nanobodies. Subsequently, the nanobody-f’ conjugates were purified by ion-exchange chromatography and verified by either 16% denaturing urea polyacrylamide gel electrophoresis or SDS-PAGE. [1336] Expression and purification of ClyA-S110C nanopore [1337] The ClyA-S110C construct was prepared by mutating the serine on position 110 to a cysteine in the cysteine-free variant ClyA-CS as previously reported. The constructed plasmid was transformed into E.coli BL21 (DE3) electrocompetent cell by electroporation. Cells were cultured in 2x YT medium containing 100 µg/mL ampicillin at 37°C and 200 rpm until OD600 reaches 0.8-1. Protein expression was induced by adding 0.5 mM IPTG and incubating at 20°C and 200 rpm for overnight. Cells were harvested by centrifugation at 6500 rpm and 4°C for 15 min. The pellets were stored in -80°C freezer for at least 1h and then thawed at 37°C, followed by resuspension in 20 mL lysis buffer (10 mM imidazole pH 8.0, 150 mM NaCl, 50 mM Tris.HCl, pH 7.5, 1 mM MgCl2, 5 mM TCEP) supplemented with 0.2 mg/mL of lysozyme. After incubating at 4°C for 25 min on a rotator, the cells were further lysed by sonication. The lysate was then centrifuged at 6500 rpm and 4°C for 30 min, and the supernatant was collected and incubated with Ni-NTA beads (Qiagen) at room temperature for 1 h on a rotator. Non-specific binding protein was removed by at least 20 column volumes of wash buffer (10 mM imidazole pH 8.0, 150 mM NaCl, 50 mM Tris.HCl, pH 7.5) and the protein was eluted from the beads in elution buffer (200 mM EDTA pH 7.5, 150 mM NaCl, 50 mM Tris.HCl, pH 7.5). The purity of the protein was analyzed on 4%-12% SDS-PAGE gel. [1338] Preparation of ClyA-f-nb nanopore -388- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1339] The freshly purified ClyA-S110C was firstly incubated with 20x molar excess of DBCO-PEG4- maleimide at pH of 7.5 and 4°C overnight, gently shaking. Unreacted DBCO-PEG4-maleimide was removed in standard buffer (150 mM NaCl, 50 mM Tris.HCl, pH 7.5) using a 3 kDa cut-off Amicon filter (Millipore). The purified ClyA-PEG4-DBCO was then incubated with 1.5-fold excess of f-azide oligo, at 4°C overnight, gently shaking, to create the ssDNA modified “ClyA-f” monomers. The f-azide oligo Linker was prepared by reaction of an oligo with an amino modification at the 5’ end (NH2-C6-5’-CCCGACACCCGCGGAT-3’) with an azidobutyric acid NHS ester. SDS-PAGE gel was utilized to check the click reaction efficiency. The ClyA- f monomer was oligomerized in the presence of 0.2% n-dodecyl-β-D-maltoside (DDM) by incubating at 37°C for 30 minutes. Subsequently, the oligomerized ClyA-S110C and ClyA-f were analyzed and purified by blue native polyacrylamide gel electrophoresis (BN-PAGE, Bio-Rad). Due to the negative charges of DNA oligos, ClyA-f oligomers migrated slightly faster than unmodified ClyA-S110C oligomers. According to the previous study, the lowest oligomeric bands of ClyA-S110C and ClyA-f were type-I nanopores (12-mer). Therefore, ClyA-S110C and ClyA-f dodecamers were obtained by slicing these bands from the gel. After eluting from the gel pieces using 30 µL of the standard buffer with the presence of 0.02% DDM, the ClyA-f oligomers solution was aliquoted into 5 µL/tube. The concentration of ClyA-f dodecamers eluted from gel was too low to be measured by neither Nanodrop nor Bradford assay. Therefore, prior to the single-channel recording experiments, excess of nanobody-f’ (~40 pmol) was incubated with 5 µL of the ClyA-f oligomers at room temperature for at least 30 mins to ensure each ClyA nanopore was maximally modified with duplexed nanobodies. [1340] Single-channel recording experiment [1341] Electrical recordings were performed using a vertical planar lipid membrane set-up as described previously. Briefly, a 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, purchased from Avanti Polar Lipids) lipid bilayer was formed on the aperture of the Teflon membrane separating the first side (e.g., cis side) and the second side (e.g., trans side) of the fluid chamber of a recording chamber. After being connected to a patch-clamp amplifier (Axopatch 200B, Axon Instruments) using Ag/AgCl electrodes, both the trans- and the first side of the chamber were filled with electrolyte buffer 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. ClyA nanopores were added into the first side(e.g., cis side) of the chamber, which was connected to the ground electrode. After pore insertion, excess ClyA was removed by buffer exchange. DNaseI (Sigma-Aldrich), BSA, muPA (kindly provided by Emil Oldenburg), Her2 (acquired from SinoBiological) and various concentration of Spike proteins (SARS-CoV-2 S protein, purchased from ACROBiosystems) were all added to the first side (e.g., cis side) if not specifically stated. All recordings were conducted using a Bessel low-pass filter of 2 kHz and a sampling rate of 10 kHz. All electrical recording current signals were filtered by Gaussian lowpass filter with a cutoff of 1 kHz prior to analysis. The data analysis software used in this study was Clampfit. -389- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Example 14: Functionalization of ClyA nanopore with nanobody [1342] To specifically detect proteins with various sizes, a ClyA nanopore was designed, functionalized with multiple nanobodies via a 16 base pair DNA duplex linker to the wide end of the pore. It was hypothesized that the binding of proteins to the nanobodies would alter the ionic flux through the nanopore, thus inducing a distinguishable current signal indicating protein detection. To enable site-specific attachment of the DNA linker to ClyA, a ClyA-CS variant was mutated by substituting a serine with a cysteine at position 110 (ClyA- S110C, FIG.101A). Then, a 16nt DNA oligonucleotide with an azide group at 3’ end (f-azide) was attached to ClyA-S110C by using a maleimide-PEG4-DBCO linker (FIG. 101B). FIG. 101B shows that a 16mer oligonucleotide, named f, was conjugated to ClyA monomer via a maleimide-PEG4-DBCO linker, where the maleimide reacted with –SH group on the protein and DBCO was clicked to the azide group on the oligo. ClyA-f monomer then oligomerized to ClyA-f oligomer in the presence of 0.2% DDM at 37℃. With the addition of 20-fold excess of the linkers to the ClyA-S110C, the band of the product was entirely upshifted compared to ClyA-S110C in the SDS-PAGE gel, indicating a high yield of ClyA-DBCO products (FIG. 101C). Subsequently, the purified ClyA-DBCO reacted with 1.5-fold excess of f-azide, leading to a full yield of ClyA-f constructs (FIG.101C). Furthermore, after the self-assembly in the presence of detergent to form oligomerized pores, ClyA-S110C and ClyA-f dodecamers (FIG.101D, band I) were extracted from the blue native polyacrylamide gel. On the basis of the high conjugation efficiency of ClyA-f monomers and homogeneity of the oligomerization, it can be assumed that on each ClyA-f dodecamer, there are approximately 12 oligos available for nanobody attachment. [1343] To allow nanobodies to anchor on ClyA nanopores, nanobodies were produced with an azide group at N-terminal through unnatural amino acid incorporation by amber codon suppression38 and conjugated to a complementary strand of oligo f containing a DBCO group (f’-DBCO) at 5’ end, via click chemistry. As a proof-of-concept, Ty1 nanobody, that can reversibly bind the receptor binding domain (RBD) of SARS-CoV- 2 Spike proteins, was conjugated with f’. The binding activity of the oligo-attached nanobody was examined using the bio-layer interferometry assay, which showed that the attachment of oligos did not affect the binding affinity of Ty1 nanobody to the RBD (data not shown). Furthermore, to test the feasibility of the attachment of nanobodies to ClyA, the ClyA-f monomers were incubated with 5-fold excess of the Ty1-f’ conjugates and analyzed by SDS-polyacrylamide gel. It displayed that ClyA-f had a clear mobility shift due to nanobody attachment and suggested that the attachment efficiency was up to 100% (data not shown). Eventually, the nanobody-functionalized ClyA nanopores (ClyA-f-nb) were prepared by incubating the ClyA-f dodecamers with the respective nanobody-f’ modules. Example 15 : Characterization of nanobody-functionalized ClyA nanopore. -390- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1344] Firstly, the electrical characterization of ClyA-S110C, ClyA-f, and Ty1-modified ClyA (ClyA-f-Ty1) was conducted at different applied potentials using single-channel recording system, to investigate the effect of the attachment of ssDNA and nanobodies. A schematic of the functionalization of the ClyA nanopore with Ty1 nanobody is shown in FIG. 102A. The Ty1-f’ (R) was immobilized on ClyA-f nanopore (N) by DNA strand (L) hybridization. At applied potentials of ± 35 mV, the current signals of ClyA-f were similar to that of ClyA-S110C and no specific signal caused by the attached oligos entering the nanopore was observed (data not shown). However, the I-V curves showed that the open pore current of ClyA-f was slightly smaller than that of ClyA-S110C at positive bias ranging from 10 mV to 90 mV (FIG.102B), which indicated that driven by the applied positive potential, the attached ssDNA entering ClyA, partially blocked the pore. Nevertheless, the conductive behavior of ClyA-f was not affected by the attachment of ssDNA at negative bias (FIG.102B). On contrary, the attachment of Ty1 nanobodies had no effect on the ClyA-f-Ty1 pore at a positive potential (+35 mV), whereas the pore was partially blocked compared to ClyA-f when a negative potential (-35 mV) was applied. When lowering the applied potential to -20 mV, transient and reversible blockade signals were observed (FIG. 102D). These signals are composed of two current levels (in and out), where one (out) is similar to the one expected for the open pore current, while the other is consistent with the entry of one nanobody inside the nanopore. At the same applied potential, the current blockade percentage ((Io-Ib)/Io x 100 (or ΔI/Io x 100, Io is the open pore current and Ib is the blocked pore current) of these signals was 14.2 ± 0.3% (n=3) and the dwell time of the blockade signals (tin) was 21.09 ± 1.06 ms (n=3). By fitting the all-point histogram of the current signals using the Gaussian function and calculating the proportion of the area under the curve, the open probability of ClyA-f-Ty1 at -20 mV was 51% (FIG.102E). Moreover, by measuring the blocked pore current at different applied potentials, the I-V curve of ClyA-f-Ty1 was obtained, which demonstrated that the current of ClyA-f-Ty1 was smaller than that of the non-nanobody attached ClyA-f at negative bias ranging from -10 to -90 mV (FIG. 102B). As a result, the conductance of ClyA-f-Ty1 (1.71 ± 0.01 nS, n=22) at -35 mV was smaller than that of ClyA-f (1.92 ± 0.01 nS, n=22) (FIG.102C). These results showed that the attachment of Ty1 resulted in voltage-dependent gating of ClyA nanopores. All of the experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. [1345] To further confirm that these blockade signals were caused by the movement of the attached nanobodies, the dependency of these blockade signals on the applied potentials was investigated. With the increase of the applied potential from -10 mV to -40 mV, the blockade probability of the pore and the dwell time of the blockade signals (tin) remarkably increased, whereas the interval time of the pore remaining open (tout) greatly decreased (FIGs.103A-103F). These experiments were performed in 150 mM NaCl, 50 mM Tris- HCl, pH 7.5. -391- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1346] For example, at a potential of -50 mV and above, for this specific nanobody the ClyA pore was almost permanently blocked. However, by reversing applied potential, ClyA-f-Ty1 nanopore can return to an unblocked state. These results indicated the blockade signals were not caused by molecule translocation as higher voltage usually drives molecules to translocate through nanopore faster (translocation would be evidenced by shorter blockade dwell times at higher voltages). It is known that ClyA-AS generates a strong electroosmotic flow43, which under negative applied potentials induces the capture of a variety of proteins. Given the small size of a nanobody (a diameter of 2.5 nm and a height of 4 nm44) and the flexible linkage of the 16 bp DNA duplex (a length of about 5.5 nm), the current blockades are the result of coupled Ty1 nanobodies entering inside the nanopore in close proximity to the constriction region of the pore (FIG.102F). [1347] In addition, irreversible opening of the pore was observed after adding 5 U of DNase I to the first side (e.g., a cis side) of the chamber in the presence of Mg2+ for about 30 mins at -20 mV (FIGs.104A-104B), as a result of cleavage of the dsDNA linker. This result confirmed that the nanobodies were successfully attached on the ClyA nanopore by DNA duplex formation and it offered additional evidence for the interpretation of the blockade signals. Herein, “in” and “out” are used to define the position of the nanobodies either inside the nanopore vestibule or outside the nanopore vestibule respectively, and tin and tout to represent the time that the nanobodies stay inside and outside of the nanopore, respectively. These results demonstrate attachment of nanobodies to the ClyA nanopore via a flexible oligonucleotide linker that enables the coupled nanobody to dynamically move in and out of the nanopore, partially blocking the ionic current when in the nanopore. Further, the results demonstrate the ability to control the dynamics between the in and the out states through applied voltage. The experiments were performed in 150 mM NaCl, 2.5 mM MgCl2, 50 mM Tris-HCl, pH 7.5. Example 16 : Real-time detection of SARS-CoV-2 Spike protein [1348] Bovine serum albumin (BSA) has been widely used as a blocking agent in sensing techniques like ELISA to eliminate the nonspecific interaction such as protein-protein or protein-surface. In our case, with the addition of BSA to the first side (e.g., a cis side) of ClyA-f-Ty1 nanopore, no additional blockade signals caused by BSA translocation were observed. Surprisingly, it was found that both tout and open probability of ClyA-f-Ty1 (the probability of being in the “out” state) decreased with the increasing concentration of BSA (FIG.105, FIGs.106A-106G). For example, in the presence of 6 µM BSA and at a bias of -20 mV, the tout of ClyA-f-Ty1 decreased from 42.9 ± 38.9 ms to 4.64 ± 0.38 ms and the probability of ClyA-f-Ty1 being in the open-state decreased from 14.2 ± 7.5% to 2.1 ± 0.8%. These results suggested that the presence of BSA drastically reduced the time in which the ClyA nanopore is unoccupied by a coupled Ty1 nanobody. Given that BSA possesses a dimension of about 14 x 4 x 4 nm and a pI of 4.7 in aqueous solution, it was very likely -392- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 BSA created a crowded environment outside of the nanopore, hence increasing the chance of nanobodies entering the pore. Similar crowding effects have been used for enhancing the capture of macromolecules in previous studies9 48 49. Moreover, the addition of BSA greatly minimized the pore-to-pore variance of ClyA-f- Ty1 (Fig. 6E, 6G). Therefore, for further sensing applications, 6 µM BSA was added to the first side of the fluid chamber to minimize the background signal. The experiments of FIG. 105 were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. [1349] Multivalent interaction has been widely exploited to improve binding affinity and enhance sensing sensitivity. It was reported that the binding affinity between the SARS-CoV-2 Spike protein and Ty1 was dramatically increased by multimerization of the nanobody. Given the dodecamer structure and well-defined distance, the ClyA nanopore is predicted to be an optimal scaffold for multimerizing nanobodies in close proximity to each other so that multiple nanobodies can bind to a single protein simultaneously, to increase the sensitivity of spike protein recognition. To test the feasibility of this sensing system, SARS-CoV-2 Spike protein was added to the first side (e.g., cis side) of the fluid chamber of the ClyA-f-Ty1 nanopore system at a final concentration of 2.3 nM in the presence of 6 µM BSA. Remarkably, after about 1 min, the frequency of the blockade signals started to decrease and the tout increased (FIG. 105, FIG. 107A). Shortly thereafter the current signals became almost entirely locked into the “out” state, which was in accordance with the ClyA-f- Ty1 nanopore recovering to open pore current state (~-38 pA) (FIG. 105). In a recording time of about 25 minutes post addition of the Spike protein, the probability of Ty1 locating outside ClyA lumen (open probability) increased from 3.9 ± 0.4% to 98.9 ± 0.6% (n=3, FIGs. 107A-107E), which suggested that upon being captured by Spike, Ty1 nanobodies were retained outside of ClyA lumen. Moreover, with the presence of 2.3 nM Spike, the histogram of the logarithm of tout showed two peaks (FIG. 107E) with an average interevent time of 5.03 ± 1.34 ms and 20230.19 ± 1.95 ms (n=3), respectively. The interevent duration of the first peak was very close to that of before adding Spike protein (4.48 ± 1.32 ms, n=3), indicating those events were attributed to the unbound Ty1 nanobodies non-specifically locating inside and outside the nanopore, whereas the events of the second peak were likely caused by the binding of Spike to the nanobodies. Compared to the first peak, the time of the second peak increased by 3 orders of magnitude, which suggested that the binding interaction between the Spike trimer and the multimerized Ty1 nanobodies was very strong. These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 with the presence of 6 µM BSA. [1350] To make a calibration curve for Spike detection and to further investigate the binding kinetics of the trimeric Spike with the multimerized Ty1 nanobodies, the response of ClyA-f-Ty1 nanopore to different concentrations of Spike protein was tested. At lower concentrations (0-460 pM), the open probability of ClyA- f-Ty1 increased with increasing Spike concentration over the entire range (FIGs. 108A-108B). The time of Ty1 locating outside the pore (tout) increased approximately linearly with increasing concentration of Spike -393- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 protein, while the time of Ty1 lodging inside the nanopore (tin) was independent of the concentration (from 100 to 500 pM, FIGs. 109A-109H). These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 with the presence of 6 µM BSA. This confirmed that the increased open probability was indeed caused by Spike protein associating with Ty1 nanobodies. As the Spike is trimeric protein that can interact with three Ty1 nanobodies, any of the 12 Ty1 on a ClyA nanopore being occupied by one Spike would modulate the ionic flow. Thus, this makes our platform capable to sensitively detect Spike at picomolar concentration. When further increasing the Spike concentration, the open probability had a positive correlation with the concentration and it reached a plateau at around 2 nM (FIG.108C). The data can be fitted by Hill-Langmuir equation with a hill coefficient greater than 1 (n=1.31), indicating that the binding between the trimeric Spike and the multimerized Ty1 was with positive cooperativity. Moreover, the previously observed long interevent duration caused by Spike binding and the small dissociation constant (Kd = 760.6 pM) was consistent with the fact that cooperative binding between multiple ligands and the same receptor can create a much stronger binding affinity. FIG.108D shows a schematic model depicting the dynamics of the interaction between ClyA- f-Ty1 and Spike protein. Ty1 nanobodies dynamically move in and out of ClyA nanopore under applied potential. Spike protein reversibly interact with the Ty1 nanobodies attached on the nanopore presumably in a multivalent fashion at high concentration of Spike trimers. The experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 in the presence of 6 µM BSA. Therefore, it was concluded that nanopores with multiple binding ligands to the same protein have great potential for highly sensitive detection. Example 17: Detection of SARS-CoV-2 Spike protein in blood [1351] For sensing application in the clinic, it is crucial that the sensing efficiency and specificity of the sensor is not affected by blood components such as proteins, red and white blood cells and platelets. To test the influence of blood components using our ClyA-f-Ty1 pore sensor, 1 µL (final concentration: 0.2% v/v) of defibrinated sheep blood was added to the first- side (e.g., cis side) of the fluid chamber in the presence of BSA (FIG.110A). Rewardingly, the conductive behavior of ClyA-f-Ty1 nanopore was only slightly affected by the blood and the membrane remained stable (FIGs.110B-110C). No obvious blood-induced blockade was observed, except for very few transient blockade signals with current blockage of around 31.5% ± 0.1% (FIG. 110E, level 2). However, the dwell time of those events were very short (~ 0.6 ms), suggesting it might be due to transient collision by proteins or platelets in the blood. Moreover, the changes of the open probability, the dwell time and the interevent time of the ClyA-f-Ty1 nanopore before and after the addition of blood were negligible (FIGs. 110D-110H). These experiments were performed in electrolyte buffer 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 in the presence of 6 µM BSA. -394- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1352] After the addition of 2.3 nM Spike, the nanopore largely transited to open state due to binding of the Ty1 nanobodies. Some large and seconds-long blockade events were also observed in this state (FIGs.111A- 111B). Likely, in the absence of Spike proteins, the steric hindrance of nanobodies on ClyA prevented blood components from entering the pore, whereas upon Spike protein binding to the nanobodies, the pore remained open so that some large proteins in blood occasionally entered the nanopore. The experiment was performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 in the presence of 6 µM BSA. [1353] It is worth noting that unlike other approaches, in this system the protein does not need to enter the nanopore to be detected. This is important, because the applied potential required for protein detection in this assay was just -20 mV, which was much lower than that required for capturing proteins into nanopores. The lower voltage reduces the chances of capturing unwanted background contaminants in the nanopore. Furthermore, the coupled nanobodies at the entrance to the nanopore further prevent capture and interference of unwanted proteins and contaminants (e.g. background proteins in blood) in the nanopore, which drastically increases the selectivity of the nanopore for the proteins. Example 18: General applicability of nanobody-functionalized nanopores as protein sensors. [1354] This example demonstrates that the concept exemplified for Spike protein detection is widely applicable to various other proteins when using the appropriate nanobody. Nanobodies have similar characteristics in sizes and shapes34. It is expected, therefore, that a variety of nanobodies can provoke similar transient blockage signals when immobilized on ClyA nanopore, thus allowing the detection of variable-sized proteins. Taking advantage of the modularity of our approach, ClyA nanopores were functionalized with nanobodies 2Rs15d (ClyA-f-15d), 2Rb17c (ClyA-f-17c) and nb22 (ClyA-f-nb22), respectively. Among these nanobodies, 2Rs15d and 2Rb17c54 recognize N-terminal half and C-terminal half of human epidermal growth factor receptor 2 (HER2) proteins that are highly expressed in breast cancer. Nanobody nb2255 recognizes murine urokinase-type plasminogen activator (muPA) which is a biomarker associated with cancer progression. [1355] All of the three nanobodies were successfully conjugated with the oligo f’ and these nanobodies could be functionalized on ClyA with high attachment efficiency. Due to their similarity in size, shape and surface charge, these nanobodies may have similar effects on the electrical behavior of ClyA as Ty1. Indeed, all of the nanobody-conjugated ClyA nanopores induced similar blockage signals at an applied potential of -20 mV (FIGs.112A-112B). [1356] In the presence of 6 µM BSA, the blockade percentage caused by 2Rs15d, 2Rb17c, and nb22 were 11.7% ± 0.1%, 14.2% ± 0.4%, and 13.7% ± 0.1%, respectively. To verify the protein sensing capability, recombinant soluble protein Her2-hFc (96 kDa) was added to the ClyA-f-15d and ClyA-f-17c pores, -395- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 respectively. Similar to the phenomenon observed for the interaction of Spike with ClyA-f-Ty1, both nanobody-functionalized nanopores showed significantly increased open probability after the addition of Her2- hFc as a result of the protein binding to the coupled nanobodies (FIGs.112A-112B). Reported binding affinity of 2Rs15d to Her2: kon = 2.14 x 105 M-1 s-1, koff = 5.71 x 10-4 s-1, KD = 2.7 nM. Reported binding affinity of 2Rb17c to Her2: kon = 7.6 x 106 M-1 s-1, koff = 4.58 x 10-2 s-1, KD = 6 nM. These experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 in the presence of 6 µM BSA. [1357] Furthermore, the feasibility of ClyA-f-nb22 for protein sensing was tested. Interestingly, after protein muPA (48 kDa, pI 8.53, FIG. 113A) was added to the first side (e.g., cis side) of ClyA-f-nb22 pores (FIG. 113B), at a potential of -15, a new class of blockade events (levels 2 and 3) was observed in addition to the open-pore level (level 0) and events provoked by the nanobody (level 1). At -15 mV the new Level 3 blockade showed a current block of 63.6 ± 0.1% and was relatively long in duration of 45.45 ± 1.50 ms, while Level 2 blocked to 34.1 ± 0.5% and was very short in duration of 1.60 ± 0.43 ms. Level 3 blockades were not observed before adding muPA (FIG. 113B, left panel) or when muPA was added to ClyA-f or ClyA-f-Ty1 nanopores (data not shown), suggesting that the level 3 events were not caused by free muPA protein itself nor by non- specific interaction between nanobodies and the proteins. More likely, level 3 blockades reflect the entry of nb22:muPA complex inside the nanopore. In addition, as the applied potential increased from -5 mV to -15 mV, the dwell time of the level 3 events increased by around 1.5 orders of magnitude (data not shown), which was consistent with the fact that positively charged muPA:nb22 complex tends to reside in the nanopore for a longer time at higher negative potential. FIG.113C shows enlarged representative current signals after adding 3 nM muPA at -15 mV. The signals consisted of three blockade levels in addition to the open pore level (Level 0) with current blocking percentages of 13.7% ± 0.1% (Level 1), 34.1% ± 0.5% (Level 2), and 63.6% ± 0.1% (Level 3), respectively. These results further confirm that muPA complexing with nb22 enters the ClyA pore, provoking the level 3 blockade events. [1358] In comparison, Level 2 blockades did not significantly change with applied voltage. Given the short dwell time and non-voltage dependency, Level 2 blockade may reflect the transient collision of nb22:muPA complex with ClyA nanopore rather than full entry into the nanopore. [1359] Based on the above analysis, a model was constructed (FIG. 113E) displaying the conformational transitions of ClyA-f-nb22 in response to muPA, which corresponded to the observed multiple current levels. Taken together these results demonstrate the ability to detect smaller analytes inside the nanopore through binding to the coupled nanobody. The experiments were performed in 150 mM NaCl, 50 mM Tris-HCl, pH 7.5 with the presence of 6 µM BSA. [1360] In another embodiment the linker L on the nanopore is first protected, so that it is only activated when desired. For example, the nanopore is initially duplexed to a blank protecting strand, which is removed on a -396- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 channel-by-channel basis on a chip array containing multiple nanopores. For example, the blank protecting oligonucleotide strand can be removed from a desired nanopore on a channel-by-channel basis by applying voltage to the selected channel containing a nanopore so as to capture and electrophoretically strip the protecting strand from the nanopore (FIGs.115A-115D). [1361] In some embodiments, different orientations are possible to locate R favorably relative to the L attachment point on the nanopore and to the nanopore entrance (FIGs. 114A-114C). For example, N and R might be coupled to the same end of an oligonucleotide duplex linker L . Alternatively, N and R might be coupled to the mid points or far ends of an oligonucleotide duplex linker L. [1362] FIG.114A-114C illustrates 3 possible options for coupling the components. FIG.114A shows the N and R components are located at opposite ends of the duplex linker L. For example, this can easily be achieved by coupling the components to both 5’ ends of each strand, or to both 3’ ends. The distance “d” between the N and R coupling locations is principally controlled by the length of the duplexed oligonucleotide strands, and the flexibility of the system (which determines the ability of R to enter the nanopore) is in part dependent on the flexibility of duplex oligonucleotide (e.g. dsDNA), which is less flexible than single-strand oligonucleotide. FIG.114B shows the N and R components are located at the same end of the hybridized duplex linker L. E.g. one component is coupled to the 5’ end of strand 1 and the other to the 3’ end of strand 2, or vice versa. This method of coupling is favorable for positioning the coupling points of N and R closer together to reduce the distance “d”, while still allowing for much longer oligonucleotide strands if required. FIG. 114C shows one or both the N and R components are coupled to the oligonucleotide strands of the hybridized duplex linker L at an internal position along the strand (FIG.114C shows R coupled at a mid-point for simplicity), for example via coupling to the backbone or a base of the polynucleotide. [1363] In all cases above, the oligonucleotides can have sections of non-duplexed single strand (e.g. ssDNA overhangs) to further control distances and optimize the flexibility of the coupled components. Example 19: Use of high electro-osmotic force (EOF) system for characterization of proteins. [1364] The following example disclosed herein demonstrates the ability to further characterize proteins using strong EOF nanopore systems to characterize a range of proteins. Preparation of nanopores [1365] A plasmid containing a gene encoding mutant MspA_D90N elongated by a strep-tag was transformed into E. coli BL21(DE3) cells by electroporation. Afterwards, the cells were incubated in 500 µl LB medium supplemented with 1% glucose for 1 hour at 37°C. The transformed cells were then spread on LB-agar plates (containing LB-medium supplemented with 1% glucose and 100 µg/mL ampicillin) and incubated overnight -397- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 at 37°C. On the next day, the grown colonies were used to inoculate 200 mL of 2YT medium containing 100 µg/mL ampicillin. The culture was grown for 2-4 hours at 37°C while shaking until an OD600 of 0.8-1 is reached, at which point 0.1 mM IPTG is added to induce protein expression. The cultures were then incubated overnight at 20°C while shaking. On the next day, the cells were harvested by centrifugation for 15 minutes at 6500 rpm. Cell pellets were first put at -80°C for at least 30 minutes and afterwards thawed on ice. Cell pellets from 50 mL culture were resuspended in 5 mL lysis buffer containing 150 mM NaCl, 15 mM Tris-HCl, 1 mM MgCl2, buffered to pH 7.5, supplemented with 1µg/µL DNasI and 0.2 mg/mL lysozyme. The lysate was incubated for 10 minutes at 37°C on a tube shaker. Afterwards 0.8% DDM (n-Dodecyl β-D-maltoside) was added, and the solution is incubated for an additional 20 minutes at 37°C on a tube shaker. The cells were sonicated on ice using a Brandon sonicator for 3 x 40 seconds at 25% output. After sonication, cell debris was pelleted by centrifugation for 20 minutes at 6500 rpm. The supernatant was incubated with 200 µl of pre- washed Strep-Tactin beads suspension for 1 hour at 4°C on a tube rotator. The bead solution was loaded on a gravity flow column and afterwards extensively washed with wash buffer containing 150 mM NaCl and 15 mM Tris, buffered to pH 7.5. MspA oligomers were eluted by 100 µL of elution buffer (150 mM NaCl, 15 mM Tris, pH 7.5 supplemented with 2.5 mM desthiobiotin and 0.02% DDM). The MspA oligomers were stored at 4°C. [1366] The mutant MspA-based nanopore, MspA_D90N, utilized in the Example described herein comprises the following amino acid sequence: MGLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADEFEGTLEL GYQIGFPWSLGVGINFSYTTPNILINDGDITAPPFGLNSVITPNLFPGVSISADLGNGPGIQEVATFSVD VSGAEGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDSVTTYGEPWNMNGSAGSAWSHPQFEK* (SEQ ID NO: 132). [1367] The sequence of the wild-type CytK monomer is set forth in SEQ ID NO.: 107. [1368] Preparation of the ClpX translocase, preparation of protein analytes, and the setup of the planar lipid bilayer electrophysiological recordings system (single chamber) followed those methods described in Example 6. [1369] Recordings of ClpX controlled protein translocation [1370] Measurements of translocase controlled protein translocation were carried out according to the system schematically described in FIG. 74. Both compartments of the nanopore system described above were filled with an electrolyte solution (e.g.1 M potassium glutamate, 20 mM MgCl2 and 50 mM Tris, buffered to pH 7.5 unless stated otherwise). Unless stated otherwise, the appropriate purified MspA or CytK nanopore prepared as described herein was added to the cis compartment to achieve a single inserted nanopore. After detecting the insertion of a single nanopore the open pore current was recorded at a range of voltages to assess the -398- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 nanopore. Separately, translocase (ClpX) and target protein substrate (e.g. MBP-1 unless stated otherwise) were preincubated for about 10 minutes before adding to the system (or otherwise added to the system separately if stated), at room temperature in 10 mM ATP and 25 mM MgCl2. ClpX translocase and target proteins (or proteins) were added to the system at a 2:1 translocase:protein molar ratio unless stated otherwise. After pre-incubation, the translocase: protein complex was added to the cis-compartment (unless stated otherwise). [1371] The amino acid sequences of analytes (substrates) used in Example 19 are shown in Table 26. Table 26. Amino acid sequences of peptide and protein analytes. Peptide / Amino acid sequence SEQ ID protein NO. A0A0H3C8 MHHHHHHSSGGLVPRGSHMEVVVSFNDLSQPFFVAMRRELEDEAAKLGVKVQVLDAQNNS 133 34_CAUVN SKQISDLQAAAVQGAKVVIVAPTDSKALAGAADDLVEQGVAVISVDRNIAGGKTAVPHVG ADNVAGGRAMADWVVKTYPAGARVVVITNDPGSSSSIERVKGVHDGLAAGGPAFKIVTEQ TANSKRDQALTVTQNILTSMRDTPPDVILCLNDDMAMGALEAVRAAGLDSAKVKVIGFDA IPEALARIKAGEMVATVEQNPGLQIRTALRQAVDKIKSGAALKSVSLKPVLITSGNLTEA SRIGEMGSSGSLRSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAY PIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGG YAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLT DEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA VINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDEN YALAA A0A1S4NY MHHHHHHSSGVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKS 134 F2_BRALA TKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVN YRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTT GNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGM DELYKGSLRSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQV AATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAV EALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFK YENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPW AWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGL EAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINA ASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALA A DNAK_EC MAWSHPQFEKGSSGKIIGIDLGTTNSCVAIMDGTTPRVLENAEGDRTTPSIIAYTQDGET 135 OLI LVGQPAKRQAVTNPQNTLFAIKRLIGRRFQDEEVQRDVSIMPFKIIAADNGDAWVEVKGQ KMAPPQISAEVLKKMKKTAEDYLGEPVTEAVITVPAYFNDAQRQATKDAGRIAGLEVKRI INEPTAAALAYGLDKGTGNRTIAVYDLGGGTFDISIIEIDEVDGEKTFEVLATNGDTHLG GEDFDSRLINYLVEEFKKDQGIDLRNDPLAMQRLKEAAEKAKIELSSAQQTDVNLPYITA DATGPKHMNIKVTRAKLESLVEDLVNRSIEPLKVALQDAGLSVSDIDDVILVGGQTRMPM VQKKVAEFFGKEPRKDVNPDEAVAIGAAVQGGVLTGDVKDVLLLDVTPLSLGIETMGGVM TTLIAKNTTIPTKHSQVFSTAEDNQSAVTIHVLQGERKRAADNKSLGQFNLDGINPAPRG MPQIEVTFDIDADGILHVSAKDKNSGKEQKITIKASSGLNEDEIQKMVRDAEANAEADRK FEELVQTRNQGDHLLHSTRKQVEEAGDKLPADDKTAIESALTALETALKGEDKAAIEAKM QELAQVSQKLMEIAQQQHAQQQTAGADASANNAKDDDVVDAEFEEVKDKKGSLRPSKIEE GKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAH DRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPN PPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVD NAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGV -399- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 TVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDA QTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA DYR_ECO MHHHHHHSSGISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIG 136 LI RPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLY LTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERRGSLRSKIEEGKL VIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRF GGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPK TWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAG AKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVL PTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSY EEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTR ITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA EFTS_ECO MAWSHPQFEKGSSAEITASLVKELRERTGAGMMDCKKALTEANGDIELAIENMRKSGAIK 137 LI AAKKAGNVAADGVIKTKIDGNYGIILEVNCQTDFVAKDAGFQAFADKVLDAAVAGKITDV EVLKAQFEEERVALVAKIGENINIRRVAALEGDVLGSYQHGARIGVLVAAKGADEELVKH IAMHVAASKPEFIKPEDVSAEVVEKEYQVQLDIAMQSGKPKEIAEKMVEGRMKKFTGEVS LTGQPFVMEPSKTVGQLLKEHNAEVTGFIRFEVGEGIEKVETDFAAEVAAMSKQSGSLRP SKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDI IFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNK DLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIK DVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSK VNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPL GAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDE ALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA G3P1_ECO MAWSHPQFEKGSSTIKVGINGFGRIGRIVFRAAQKRSDIEIVAINDLLDADYMAYMLKYD 138 LI STHGRFDGTVEVKDGHLIVNGKKIRVTAERDPANLKWDEVGVDVVAEATGLFLTDETARK HITAGAKKVVMTGPSKDNTPMFVKGANFDKYAGQDIVSNASCTTNCLAPLAKVINDNFGI IEGLMTTVHATTATQKTVDGPSHKDWRGGRGASQNIIPSSTGAAKAVGKVLPELNGKLTG MAFRVPTPNVSVVDLTVRLEKAATYEQIKAAVKAAAEGEMKGVLGYTEDDVVSTDFNGEV CTSVFDAKAGIALNDNFVKLVSWYDNETGYSNKVLDLIAHISKGSLRPSKIEEGKLVIWI NGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYA QSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEE IPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAG LTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFK GQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEEL AKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKH MGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA G6PD_ECO MAWSHPQFEKGSSAVTQTAQACDLVIFGAKGDLARRKLLPSLYQLEKAGQLNPDTRIIGV 139 LI GRADWDKAAYTKVVREALETFMKETIDEGLWDTLSARLDFCNLDVNDTAAFSRLGAMLDQ KNRITINYFAMPPSTFGAICKGLGEAKLNAKPARVVMEKPLGTSLATSQEINDQVGEYFE ECQVYRIDHYLGKETVLNLLALRFANSLFVNNWDNRTIDHVEITVAEEVGIEGRWGYFDK AGQMRDMIQNHLLQILCMIAMSPPSDLSADSIRDEKVKVLKSLRRIDRSNVREKTVRGQY TAGFAQGKKVPGYLEEEGANKSSNTETFVAIRVDIDNWRWAGVPFYLRTGKRLPTKCSEV VVYFKTPELNLFKESWQDLPQNKLTIRLQPDEGVDIQVLNKVPGLDHKHNLQITKLDLSY SETFNQTHLADAYERLLLETMRGIQALFVRRDEVEEAWKWVDSITEAWAMDNDAPKPYQA GTWGPVASVAMITRDGRSWNEFEGSLRPSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDT GIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYP FTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNL QEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYS IAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASP NKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMP NIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRR RRRRRRRRRAANDENYALAA -400- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 GLYA_EC MAWSHPQFEKGSSLKREMNIADYDAELWQAMEQEKVRQEEHIELIASENYTSPRVMQAQG 140 OLI SQLTNKYAEGYPGKRYYGGCEYVDIVEQLAIDRAKELFGADYANVQPHSGSQANFAVYTA LLEPGDTVLGMNLAHGGHLTHGSPVNFSGKLYNIVPYGIDATGHIDYADLEKQAKEHKPK MIIGGFSAYSGVVDWAKMREIADSIGAYLFVDMAHVAGLVAAGVYPNPVPHAHVVTTTTH KTLAGPRGGLILAKGGSEELYKKLNSAVFPGGQGGPLMHVIAGKAVALKEAMEPEFKTYQ QQVAKNAKAMVEVFLERGYKVVSGGTDNHLFLVDLVDKNLTGKEADAALGRANITVNKNS VPNDPKSPFVTSGIRVGTPAITRRGFKEAEAKELAGWMCDVLDSINDEAVIERIKGKVLD ICARYPVYAGSLRPSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIA YPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMT INGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLL TDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRT AVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDE NYALAA GPMA_EC MAWSHPQFEKGSSAVTKLVLVRHGESQWNKENRFTGWYDVDLSEKGVSEAKAAGKLLKEE 141 OLI GYSFDFAYTSVLKRAIHTLWNVLDELDQAWLPVEKSWKLNERHYGALQGLNKAETAEKYG DEQVKQWRRGFAVTPPELTKDDERYPGHDPRYAKLSEKELPLTESLALTIDRVIPYWNET ILPRMKSGERVIIAAHGNSLRALVKYLDNMSEEEILELNIPTGVPLVYEFDENFKPLKRY YLGNADEIAAKAAAVANQGKAKGSLRPSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTG IKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPF TWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQ EPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSI AEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPN KELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPN IPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRR RRRRRRRRAANDENYALAA LIVJ_ECOL MHHHHHHSSGEDIKVAVVGAMSGPVAQYGDQEFTGAEQAVADINAKGGIKGNKLQIVKYD 142 I DACDPKQAVAVANKVVNDGIKYVIGHLCSSSTQPASDIYEDEGILMITPAATAPELTARG YQLILRTTGLDSDQGPTAAKYILEKVKPQRIAIVHDKQQYGEGLARAVQDGLKKGNANVV FFDGITAGEKDFSTLVARLKKENIDFVYYGGYHPEMGQILRQARAAGLKTQFMGPEGVAN VSLSNIAGESAEGLLVTKPKNYDQVPANKPIVDAIKAKKQDPSGAFVWTTYAALQSLQAG LNQSDDPAEIAKYLKANSVDTVMGPLTWDEKGDLKGFEFGVFDWHANGTATDAKVKIEEG KLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHD RFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNP PKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDN AGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVT VLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALK SYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQ TRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA LIVK_ECO MHHHHHHSSGDDIKVAVVGAMSGPIAQWGDMEFNGARQAIKDINAKGGIKGDKLVGVEYD 143 LI DACDPKQAVAVANKIVNDGIKYVIGHLCSSSTQPASDIYEDEGILMISPGATNPELTQRG YQHIMRTAGLDSSQGPTAAKYILETVKPQRIAIIHDKQQYGEGLARSVQDGLKAANANVV FFDGITAGEKDFSALIARLKKENIDFVYYGGYYPEMGQMLRQARSVGLKTQFMGPEGVGN ASLSNIAGDAAEGMLVTMPKRYDQDPANQGIVDALKADKKDPSGPYVWITYAAVQSLATA LERTGSDEPLALVKDLKANGANTVIGPLNWDEKGDLKGFDFGVFQWHADGSSTAAKGSSG SLRSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDG PDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLI YNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKY DIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNID TSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKD KPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA MGLB_EC MHHHHHHSSGADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQND 144 OLI QIDVLLAKGVKALAINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGT -401- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 DSKESGIIQGDLIAKHWAANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKG IKTEQLQLDTAMWDTAQAKDKMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSI PVFGVDALPEALALVKSGALAGTVLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKV VRVPYVGVDKDNLAEFSKKGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHP DKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYN GKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPL IAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKG ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRR AANDENYALAA PGK_ECOL MAWSHPQFEKGSSSVIKMTDLDLAGKRVFIRADLNVPVKDGKVTSDARIRASLPTIELAL 145 I KQGAKVMVTSHLGRPTEGEYNEEFSLLPVVNYLKDKLSNPVRLVKDYLDGVDVAEGELVV LENVRFNKGEKKDDETLSKKYAALCDVFVMDAFGTAHRAQASTHGIGKFADVACAGPLLA AELDALGKALKEPARPMVAIVGGSKVSTKLTVLDSLSKIADQLIVGGGIANTFIAAQGHD VGKSLYEADLVDEAKRLLTTCNIPVPSDVRVATEFSETAPATLKSVNDVKADEQILDIGD ASAQELAEILKNAKTILWNGPVGVFEFPNFRKGTEIVANAIADSEAFSIAGGGDTLAAID LFGIADKISYISTGGGAFLEFVEGKVLPAVAMLEERAKKGSSGLRSKIEEGKLVIWINGD KGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSG LLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPA LDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTF LVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQP SKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKD PRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGG GGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA PNC1_YEA MHHHHHHSSGKTLIVVDMQNDFISPLGSLTVPKGEELINPISDLMQDADRDWHRIVVTRD 146 ST WHPSRHISFAKNHKDKEPYSTYTYHSPRPGDDSTQEGILWPVHCVKNTWGSQLVDQIMDQ VVTKHIKIVDKGFLTDREYYSAFHDIWNFHKTDMNKYLEKHHTDEVYIVGVALEYCVKAT AISAAELGYKTTVLLDYTRPISDDPEVINKVKEELKAHNINVVDKLEGSLRSKIEEGKLV IWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFG GYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKT WEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGA KAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLP TFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYE EELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRI TKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA Q9CES5_L MHHHHHHSSGGENLYFQGMATPKKDVYTIASDNSFAPFEFQNDDKQFTGIDVDLLNAIAK 147 ACLA NQGFKLKWNFIGFQAAVDSVQSGHADGMMSGMSITDARKQVFDYGSPYYSSNLTIATSST DDSIKSWKDLKGKTLGAKNGTASFDYLNAHAKEYGYTVKTFTDATTMYSSLNNGSINALM DDEPVIKYAIKQGQKFATPIKPIPDGQYGFAVKKGSNPELIEMFNNGLANLRANGEYDKI IDKYLESDAGSLRSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAY PIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGG YAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLT DEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA VINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDEN YALAA Q9CKB5_P MHHHHHHSSGADYDLKFGMVAGPSSNEYKAVEFFAKEVKEKSNGKIDVAIFPSSQLGDDR 148 ASMU VMIKQLKDGALDFTLGESARFQIYFPEAEVFALPYMIPNFETSKKALLDTKFGQGLLKKI DKELNVQVLSVAYNGTRQTTSNRAINSIEDMKGLKLRVPNAATNLAYAKYVGAAPTPMAF SEVYLALQTNSVDGQENPLPTIQAQKFYEVQKYLALTNHILNDQLYLISNDTLADLPEDL QKVVKDAAAKAAEYHTKLFVDGENSLVEFFKSQGVTVTQPDLKPFKAALTPYYDEYLKKN GEVGKMAIEEISNLAKLGSSGSLRSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKV TVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD -402- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEA AFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQ MSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRR RRRRRAANDENYALAA THIB_ECO MHHHHHHSSGSSGLVPRGSHMKPVLTVYTYDSFAADWGPGPVVKKAFEADCNCELKLVAL 149 LI EDGVSLLNRLRMEGKNSKADVVLGLDNNLLDAASKTGLFAKSGVAADAVNVPGGWNNDTF VPFDYGYFAFVYDKNKLKNPPQSLKELVESDQNWRVIYQDPRTSTPGLGLLLWMQKVYGD DAPQAWQKLAKKTVTVTKGWSEAYGLFLKGESDLVLSYTTSPAYHILEEKKDNYAAANFS EGHYLQVEVAARTAASKQPELAQKFLQFMVSPAFQNAIPTGNWMYPVANVTLPAGFEKLT KPATTLEFTPAEVAAQRQAWISEWQRAVSRGSLRSKIEEGKLVIWINGDKGYNGLAEVGK KFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAF QDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKS ALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMN ADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAG INAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQ KGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGS RRRRRRRRRRRRRRRAANDENYALAA IDH_ECOL MAWSHPQFEKGSSESKVVVPAQGKKITLQNGKLNVPENPIIPYIEGDGIGVDVTPAMLKV 172 I VDAAVEKAYKGERKISWMEIYTGEKSTQVYGQDVWLPAETLDLIREYRVAIKGPLTTPVG GGIRSLNVALRQELDLYICLRPVRYYQGTPSPVKHPELTDMVIFRENSEDIYAGIEWKAD SADAEKVIKFLREEMGVKKIRFPEHCGIGIKPCSEEGTKRLVRAAIEYAIANDRDSVTLV HKGNIMKFTEGAFKDWGYQLAREEFGGELIDGGPWLKVKNPNTGKEIVIKDVIADAFLQQ ILLRPAEYDVIACMNLNGDYISDALAAQVGGIGIAPGANIGDECALFEATHGTAPKYAGQ DKVNPGSIILSAEMMLRHMGWTEAADLIVKGMEGAINAKTVTYDFERLMDGAKLLKCSEF GDAIIENMGSLRPSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAY PIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGG YAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLT DEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA VINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDEN YALAA Analysis [1372] Analysis: Preprocessing reads (Extraction, segmentation, and denoising) [1373] A signal from the electrophysiological measurements was produced by the device. Depending on the configuration of the device the signal may be a measurement of the current, the voltage, resistance, or other physical characteristics of the system affected by translocation of the analyte; herein, the signal is amplitude of the current but the techniques presented may be applied to other measurements either individually or in combination. Much of the preprocessing can be performed in either hardware or software, with different trade- offs between speed and flexibility. [1374] A putative read is a contiguous portion of the signal relating to a putative event of interest, which may be further divided into segments and denoised. The signal was preprocessed to determine the location and length of putative reads related to putative events and then the corresponding region of measurements extracted. Putative reads were then filtered based on a set of metrics to filter out reads that do not meet the -403- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 criteria for a good nanopore translocation events. The remaining reads were then split into regions with similar characteristics and optionally smoothed – the processes of segmentation and denoising respectively. For the example used herein the process of preprocessing the electrophysiological signal into reads is shown as a flowchart in FIG.117A and illustratively in FIG.117B. [1375] To determine the location of and to extract putative reads, the current signal from the electrophysiological measurements was split into contiguous chunks of constant applied voltage (FIG 117A.1). Chunks corresponding to voltage flicks (i.e., where zero or positive applied voltage is detected) were discarded. An additional 0.1s of data were removed from the beginning of each chunk to account for capacitive effects as the system recovers from the voltage change. Each chunk was then smoothed using Total Variational Denoising (described in L. Condat, "A Direct Algorithm for 1-D Total Variation Denoising," in IEEE Signal Processing Letters, vol.20, no.11, pp.1054-1057, Nov.2013, doi: 10.1109/LSP.2013.2278339, the contents of which are hereby incorporated by reference in its entirety) with a parameter of 250. Putative reads are those regions where a greater than 10 pA change from and then back to the open-pore current longer than 0.5s was observed (FIG 117A.2). The location and extraction of reads described may be performed “off-line” after acquisition of current trace, or “on-line” concurrent to acquisition. [1376] Information about regions of the current that are not putative reads may also be stored for further reference: examples of useful information include, but are not limited to, estimates of open-pore current from the smoothing of a contiguous chunk of current can be helpful in determining how a read should be scaled, the time between reads provides a measure of sample concentration, location length and noise of current blocks or regions of high frequency noise are informative about the performance of system, or the time of voltage flicks or membrane ruptures than may affect measurements in other wells. [1377] To determine whether a putative read will be processed further, the following metrics are calculated: median current, median absolute deviation of the current, the median absolute deviation of the difference between adjacent current samples, the number of runs of current values above or below the median current, the length of read. At this point reads with unusual values of metrics were filtered out and discarded as outliers (FIG 117A.3). Many methods of outlier detection are known in the art, including but not limited to: comparison of the metrics to predetermined thresholds, pretrained machine-learning classifiers, unsupervised learning to locate clusters of similar reads, auto-encoder and variational autoencoder techniques to detect outlier, nearest-neighbor techniques. If further analysis confirms or contradicts the classification of a read, this information can be used to update the classification criteria for other reads. [1378] Herein, reads were selected off-line by consideration of the metrics for all putative reads from a signal using a matrix of scatter plots, plotting each metric against each other metric, and looking for a clusters. -404- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Comparison was also made with metrics for reads from other signals acquired under similar conditions. Quality Control was completed to confirm the reads. [1379] Segmentation of each read into regions of current with similar properties proceeds by performing a Haar discrete wavelet decomposition to the fifth level and discarding the detail (high frequency) components (FIG 117A.4). The inverse discrete wavelet transform is then calculated, leaving an ordered series of piecewise constant values and these form the segmentation: an ordered series of regions covering the read with an associated length and parameters summarizing the characteristics of the segment, non-limitative examples of which include: the mean or median of the current, measures of noise or range like the standard deviation or median absolute deviation, the autocorrelation between samples or other frequency derived components. [1380] Optionally, the levels for the ordered series of segments were further denoised by applying the Total Variation Denoising procedure with a parameter of 50 to the series of piecewise constant values, to produce a new series of piecewise constant values which form a new segmentation (FIG 117A.5a). Smoothing may be performed for a series of segments. To correct for possible reduction in the range of the new segmentation compared to the original, the level for each new segment was set to the median of those segments in the original which it covers. Due to transient blocks or spikes in the measurement, some segments may have values outside of the normal range of the read; these are discarded based in thresholds chosen from prior analysis of reads from other experiments made under similar conditions. [1381] Optionally, neighboring segments can be merged by similarity (FIG 117A.5b). All pairs of neighboring segments were scored for similarity and then the most similar were merged; the resulting segment having a equal to the sum of the lengths of the constituent segment lengths and a level being the length- weighted average of the levels of the constituent segments. Merging of neighboring segments continues until the measure of similarity for the most similar neighboring segments reaches a pre-defined threshold signifying that the segments are considered distinct. [1382] Herein, neighboring segments were scored for similarity by dividing the square of the difference in their levels by the geometric mean of their lengths. To correct for small sample bias, a correction factor of (n- 4) / (n-2) was applied where n is the sum of the lengths of the two segments. The threshold for the similarity score below which two segments are merged was 0.5. Neighboring segments whose combined length is less than four are always merged. Other examples of scoring metrics may be, but are not limited to, the difference in mean and/or median of the consistent samples of the two segments, statistics which adjust for sample-size and variation in the background noise such as the t-statistic, order-based statistics such as Wilcoxon Rank-Sum or Mann-Whitney statistics, distribution based statistics such as likelihood-ratio test statistic or score statistic or Lagrange multiplier statistic, tests of sequential independence such as Wald-Wolfowitz test, classification techniques such as that from a machine learning model (examples include but are not limited to, convolutional -405- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 neural network, hidden Markov model, recurrent neural network, transformer model or any combination thereof), or any combination thereof. [1383] After segmentation (FIG. 117A.4) and, optionally denoising and merging (FIG. 117A.5a and FIG. 117A.5b), the reads were further filtered to discard those that did not appear to be from complete translocations or contained long stalls (FIG.117A.6). [1384] Analysis: Creating references and databases – Data Selection [1385] Reads between the same nanopores within an experiment over time, between different nanopores (different nanopores on the same array chip or different nanopores in different experiments) are generally highly similar and can be combined into datasets to increase the number of reads to improve analyses. [1386] The preprocessed reads were aggregated in data datasets from either: 1) single experiments with single nanopore systems (e.g., with a system comprising a single nanopore as described herein), or 2) single experiments with multiple nanopore systems (with a system comprising an array of nanopores), or 3) from multiple experiments with single nanopores systems (e.g., with a system comprising a single nanopore as described herein), or 4) from multiple experiments with multiple nanopore systems (with a system comprising an array of nanopores). [1387] Different read datasets were used for training models as described in detail herein. Other read datasets were informatically analyzed (by comparison to references for example) to test the ability to characterize features of the molecules or the samples they were derived from. [1388] Analysis: Creating references and databases – Model Structure [1389] This section describes building a model from a set of training sequences (e.g., the amino-acid sequences of the measured training substrate) that is capable of generating predicted reference squiggles for sets of input sequences (e.g., the amino-acid sequences of the measured test substrate). [1390] Many neural network architectures are appropriate for predicting a squiggle from an input sequence: for example, but not limited to: convolution networks, recurrent neural networks, transformer or attention based networks, networks that include these networks as components. The specific architecture used herein is shown in FIG. 118, and has the following specific features: (N.1) the amino acid residues of the reference sequence are embedded in to an n-dimensional space, so each identity of residue is assigned a point in this space representing its properties; (N.2) the series of points are convolved over length by multiple filters to produce a representation of the local properties of the residues in the sequence; (N.3) an elementwise non- linearity is applied; (N.4) multiple sequential processing blocks (“Block 1”); (N.5) a pooling layer to down- sample the length of reference sequence to position in the reference squiggle; (N.6, N.7) a feed-forward layer to process each position; (N.8) a final position-wise linear transformation to predict the level for each position in the reference squiggle. -406- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1391] The processing block is labelled in FIG. 118 as “Block 1” and consists of several sequential layers: (B1.1) a convolution of input features over length using a number of filters equal to the number of input features; (B1.2) a per-feature normalization over length; (B1.3) an elementwise non-linearity is applied; and a linear transform layer. These sequential layers are wrapping in a residual connection (B1.5, point-wise addition of the input to the block) and then (B1.6) an elementwise non-linearity is applied. [1392] The model architecture is parameterized as: (N.1) 5 embedding features; (N.2) convolution using 64 filters of 5 features with a window length of 7 plus a bias; (N.3) Sigmoidal Linear Unit (SiLU); (N.4) 6 repetitions of Block1, each independently parameterized; (N.5) max pooling with a stride of 2; (N.6) linear transform of 64 to 64 with a bias; (N.7) SiLU non-linearity; (N.8) linear transform of 64 to 1 with bias. Block 1 is parameterized as: (B1.1) convolution using 64 filters of 64 features with a window length of 7 plus a bias; (B1.3) SiLU non-linearity; (B1.4) linear transform of 64 to 64 with a bias; (B1.6) exponential linear unit (ELU). For training, a dropout layer, randomly zeroing the entire length for 5% of the features was added to the beginning of Block 1. [1393] Analysis: Creating references and databases – Training [1394] The model was trained using standard stochastic techniques from the field of machine learning, starting from randomly initialized model parameter values and optimized over multiple pass through the entire training set (“epochs”). For each epoch, the training data is split randomly into batches of 16 read-reference sequence pairs and training proceeds by calculating the value of an objective function and then updating the parameters of model based on the objective function’s value and gradients. Training can comprise the model generating a squiggle from the sequence. A read can be scored against the sequence. The model may be improved and/or trained based on the scoring. The read-pairs can provide input for the training. [1395] The contribution of each read in a batch to the objective is based on the score that read against its reference squiggle predicted by the current model from the corresponding reference squiggle; the method to score a read against a reference is described in Analysis: Scoring read against a reference. The contribution of each read to the objective was the score for that read adjusted by adding a penalty term and then dividing by the length of the read; the penalty term was 0.05 multiplied by the sum of the absolute differences of neighboring positions in the predicted squiggle. The value of the objective function for each batch was the mean of contributions of the individual reads. [1396] The squiggle model was trained for 4000 epochs and model parameters were updated using the AdamW optimiser with beta parameters of 0.9 and 0.999, and a weight decay of 0.005; gradients were calculated using automatic differentiation techniques, as implemented in PyTorch software, and clipped when their absolute values reached 5. The OneCycleLR learning rate scheduler was used to adjust the learning rate, increasing for 400 epochs to a maximum learning rate of 0.002 and then decreasing to zero. -407- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1397] Shift and scale parameters of the reference squiggle for each reads are determined at epochs 500, 1000, 1500, 2000, 2500, 3000 and 3500 using the method described in “Analysis: Scoring and/or Aligning a Reference”. [1398] Analysis: Creating references and databases – Validation and Generalization [1399] The training procedure described in Analysis: Creating references and databases – Training results in a model that is likely to perform well for the sequences of reads it was trained on, particularly validation reads from substrates in the training set are likely to closely agree with the predicted squiggle. As well as performing well on a validation set, it is desirable that the performance of the trained squiggle model generalizes to previously unseen sequence. [1400] The models were tested against validation sets.. The validation sets, consisted of events from analytes that were withheld from training, so the model has neither seen the constituent events nor their underlying sequence, were used to determine for example to determine that the models were not overtrained and to test how well the trained models generalize to unknown proteins. [1401] The ability of the trained models to generalized to unknown protein sequence was assessed by comparing their performance on this second validation set. Methods of comparison include, but are not limited to, the score the trained model assigns to reads against the reference sequence or the rank of the ability to discriminate the reference sequence from a set of alternative sequences. The ability of the trained model to generalize was measured using the sum of the scores of the reads against their corresponding reference. [1402] Analysis: Creating references and databases – Usage [1403] To create a database of reference squiggles from a set of reference sequences (e.g., the amino-acid sequences of the measured substrate/s), the trained squiggle model is applied to each reference sequence to predict its reference squiggle. Each reference sequence must be fully determined and cannot contain symbols representing ambiguous residues or other non-canonical amino acids except those explicitly accounted for by the model. [1404] The database of reference squiggles may be updated during an analysis, removing squiggles or adding new squiggles by applying the trained squiggle model to new reference sequence. The database of reference squiggles may be constructed online, or in a just-in-time fashion, by applying the trained squiggle model to reference sequences as and when they are required. [1405] Analysis: Scoring and/or Aligning a Reference [1406] Because of the stochastic nature of how an analyte translocates through a pore during measurement, the alignment between a read and a reference squiggle is not known and so segment values and squiggle levels cannot be directly compared. To score a read against a reference, multiple different alignments should be considered but not all alignments are equally likely. Here a non-limiting example of a method is described to -408- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 calculate a score based on over all possible alignments between the read and reference squiggle, and it is shown how the method can be adapted to find the highest scoring alignment. [1407] For the purpose of exposition, segments are numbered 1 through T, first to last; positions in the reference squiggle are numbered 1 through P, first to last. [1408] The cost c(t,p) for state (t, p), alignment of segment t to position p of the reference squiggle, is calculated using an emission density based on the difference between the segment value v(t) and the squiggle level l(p). [1409] Emission densities are defined by location-scale families
Figure imgf000411_0001
log ^ [1411] where s is the noise scale of reference squiggle, a trained parameter of the squiggle model used create the reference squiggle, and the kernel function k(x) may be one of: [1412] Equation, Normal
Figure imgf000411_0002
[1414] Equation, Laplace [1415] log ^(^) = −|^| − ^ [1416] Equation, Fair [1417] log ^(^) = log(1 + |^|) − |^| − ^ [1418] where K is the normalising constant for the distribution (0.9189385332046727, 0.6931471805599453 and 1.3862943611198906 for the Normal, Laplace and Fair kernels respectively). For the purposes of this example, the Fair kernel was used. [1419] To allow for different segment lengths and the possibility that the value of the segment is an outlier values, the costs were further modified by a factor equal to the segment length n(t) and a minimum cost M that limits how badly a segment can fit a level of the squiggle; this can be interpreted as mixing the emission density with an improper uniform prior. [1420] ^(^, ^) = ^(^) log^^^^(^) + ^^^^^(^, ^)^^ [1421] The minimum cost M was set to -20. [1422] There are three transition weights T(stay) T(step) and T(skip) are defined, representing, respectively, the cost of that the segment stayed in the same position as the previous, stepped in the next position from the previous, or skipped a position (next position but one from the previous segment). The transition weights were the logarithms of (0.9, 0.095 and 0.005) for stay step and skip respectively when the reads were segmented but not merged (see “Analysis: Preprocessing reads (extraction, segmentation, denoising)”; where read segments were merged, the stay step and skip transition weights were the logarithms of (0.095, 0.9, 0.005) respectively. -409- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1423] Two sets of prior weights were defined over positions: f(p) representing the distribution of the position in the reference squiggle that the first segment of the read matches; b(p) representing the distribution of the position in the reference squiggle that the last segment of the read matches. The weights f(p) are set to equal to the logarithm of the probabilities from a geometric distribution with mean 0.1; values for negative positions being set to negative infinity. The weights b(1 + P - p) are set to equal to the logarithm of the probabilities from a geometric distribution with mean 0.1 (the distribution is translated and reflected, so the final position P has the greatest weight and the weights decrease towards the first position). [1424] The overall score S(T) is calculated recursively by dynamic programming using the following equations:
Figure imgf000412_0001
[1426] One non-limiting example, where the operation (+) is a function called the log-sum-exp (the logarithm of the sum of the exponentials of the arguments) and the operation (x) is addition of its arguments then the score calculated represents the sum over all possible alignments; these are the preferred operators when a read is scored against a reference but an alignment is not required. Referred to as the forward algorithm; also referred to herein as soft alignment. [1427] Another non-limiting example, where the operation (+) is the maximum of its arguments and the operation (x) is addition of its arguments then the score calculated is that of the highest scoring alignment. Referred to as the Viterbi Algorithm. [1428] To determine an alignment between the read and the reference squiggle, the score of the highest scoring alignment was calculated as described. At each step in the dynamic programming recursion used to calculate this score, the element that is maximal defines the transition into each state – for example state (t+1, p) may be reached by a step from state (t, p-1), or a skip from state (t, p-2) and these transitions are recorded. By construction, each state has exactly one transition recorded for it and this transition must come from the previous segment. [1429] Letting A be the position that had maximal score at the Termination step of the recursion, above, determination of alignment proceeds by starting at state (T, A) and back-tracking through the recorded transitions to form a path. For example, annotating each state with the transition recorded, a path might look like (T, A, step) -> (T-1, A-1, stay) -> (T-2, A-1, skip) -> (T-3, A-3, step) … and so on until the first segment is reached; note that the segment ordinal decreases by exactly one at each step. After reversing the path. so the order of segments is first to last, the series defines the alignment. -410- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1430] Analysis: Scaling a Reference to a Read [1431] Due to intra- and inter- experimental variation, the absolute level and range of reads for the same substrate may fluctuate relative to each other. It is desirable to correct for range changes to avoid false positive differences and to reveal differences that would be disguised. [1432] For the examples used herein scaling of reads and reference squiggles was performed as indicated by the workflow in FIG. 119. The read (FIG. 119, 119 R1) was denoised and segmented as described in “Analysis: Preprocessing reads (extraction, segmentation, denoising)” (FIG.119, 119 R2) it was then shifted and scaled so its median is zero and its median absolute deviation is one (FIG.119, 119 R3)If the reference is a reference sequence, then the trained model was applied to produce a reference squiggle (FIG.119, 119 S1). The initial estimates of the scaling factors are set so the shift is zero and a scale was one unless better values are available (FIG. 119, 119 S2); herein, the better values are available during model training because the scaling factors from previous rounds of scaling may be available. [1433] Estimating the scaling of a reference squiggle to a read proceeds in a iterative fashion. The current estimates of the scaling factors were used to scale the reference squiggle (FIG.119, 119 S3) and this was then scored against the read using the process described in “Analysis: Scoring and/or aligning a reference” (FIG. 119, 119 R4). After calculating the score, the iteration may be stopped (FIG. 119, 119 R5) and a scaled reference with associated scale are available (FIG. 119, 119 R6); alternative the scaling factors may be updated. [1434] While the reference squiggle was scaled prior to calculating the score, the cost c(t, p; x, y) was rewritten to explicitly incorporate x to improve the exposition herein. [1435] ^(^, ^; ^, ^) = log
Figure imgf000413_0001
log ^ [1436] The weights contributing to the score have a probabilistic interpretation and the derivative of the score with respect to the (exponential of the) cost was the posterior probability Pr(t,p) that segment t was aligned to position p of the reference. Using these probabilities, the expectation of the cost was calculated (FIG. 119, 119 S4) as [1437] ^(^, ^) = ∑^,^ ^^(^, ^)^(^, ^; ^, ^) [1438] and the scaling factors for reference squiggle are the shift and scale that maximize L(x, y) and these can be found to a given tolerance using numerical optimization (FIG.119, 119 S5) ; other techniques such as Majorise-Minorisation or Iteratively Reweighted Least Squares could also be used.. Where the cost was based on the Normal kernel, a closed form solution for x and y can easily be found and so x and y can be explicit calculated. -411- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1439] The posterior probabilities depend on the previous values of the scaling factors, and hence so does the expectation of the cost and hence the new values of the scaling factors. The process of scaling the reference squiggle, scoring the reads against it, forming the expectation of the cost, and estimating new scaling factors by maximization can be repeated to improve the estimates. Iterating the expectation and maximization uses the algorithm known as Expectation-Maximisation in the literature. [1440] Iterative estimation of scale factors was stopped after a fixed number of iterations (FIG.119, 119 R5); other stopping criteria, such as change in the score of the read to the reference squiggle being small, or the change in the value of estimated scaling factor from the previous estimate being small. For model training, the scaling factors are estimated multiple times during the training process and five iterations are used to fit the scaling factors each time; in other examples where a reference squiggle is scaled to a reads, ten iterations are used. [1441] Analysis: Consensus Analysis (Mutations, Post-translational modifications, and variants) [1442] To directly compare reads to each other, it is desirable to transform them into coordinates that have been time-warped to a common reference squiggle; such a transformation enables reads to be piled-up for comparison and enables the formation of a consensus read. After preprocessing, as described in “Analysis: Preprocessing reads (extraction, segmentation, denoising)”, the common reference squiggle (FIG. 122, 122 S1) was scaled to each read using the process described in “Analysis: Scaling a reference to a read” (FIG.122, 122 R2) and an alignment of the read to this scaled reference squiggle was determined using the process described in “Analysis: Alignments to reference” (FIG. 122, 122 R3). The alignment assigns each segment of each read to a position, and because the reference squiggle is common then these positions form a common coordinate system for the reads. As the reference squiggle has been shifted and scaled to the read, the range is not comparable; the shift and scale is inverted and applied to the levels of each segment of the read so the values of all segments are put on a scale that is common to all reads (FIG.122, 122 R4). [1443] For a particular read, the levels of all segments aligned to a given position can be averaged to create a new ordered series of levels; the average was the mean level weighted by the segment lengths. The new ordered series of segment levels can be compared to the original reference squiggle to determine positions of similarity or difference, differences being indicative of the presence of a mutation, PTM or other variant. The technique known as Student’s t-test was applied at each position to determine statistically significant positions (FIG. 122, 122 R5). [1444] In a similar fashion to that of a single read, the aligned segments from multiple reads can be averaged at each position to create a consensus of multiple reads (FIG.122, 122 S2). The average was the mean level weighted by the segment lengths. Analogously to the procedure described for a single read, standard statistical tests can applied at each position to determine whether an estimated level differs from that of the reference -412- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 squiggle and so whether and where there are significant differences between the read and the reference squiggle. [1445] The consensus of multiple reads has an interpretation as reference squiggle and the procedure outline above could be reapplied for further refinement (FIG.122, 122 S3). [1446] For visualizing how many reads, plotting the level of each segment against its assigned position in the reference squiggle obscures many details as all segments for a given position are overlayed; specifically their temporal order is lost. Instead, for each read, the segments aligned to each squiggle position can assigned time-warped coordinates that are uniformly spaced between that squiggle position and the next (FIG.122, 122 R6). Plotting the time-warped coordinates of multiple reads as a pile-up reveals the behavior at each position in the context of the behavior at the surrounding positions and helps elucidate the individual reads. A non- limiting example of such a plot is shown in FIG. 128B highlighting two populations of reads that have been time-warped to a common reference squiggle. [1447] Analysis: Identification of Single-Molecule [1448] This section describes identification (ID) of an individual read, for example by comparison to a predicted reference-squiggle from a trained model as described above, and the process is shown as a flowchart in FIG 120. [1449] An electrophysiological read (FIG. 120, 120 R1) was denoised and segmented using the processes described in “Analysis: Preprocessing reads (extraction, segmentation, denoising)” and the segments further merged and denoised as described in the optional steps (FIG.120, 120 R2). A database of reference squiggles was synthesized from reference sequences (FIG.120, 120 S1) by a trained squiggle model using the process described in “Analysis: Creating references and databases – usage” (FIG.120, 120 S2). [1450] Every reference was scaled to the read using the process described in “Analysis: Scaling a reference to a read” (FIG. 120, 120 R4) and finally the read was scored against the scaled reference squiggle (see “Analysis: Scoring and/or aligning a reference” but note the required score was calculated as part of the scaling process) . The scores can additionally incorporate a weight to adjust them to reflect prior information about the identity of read. Where the scores represent the log-probability of the read given the reference squiggle, an appropriate way of incorporating the weight would be to add the log of the prior probability of the identity, which was applied in this example (FIG.120, 120 R5). [1451] Finally, the scores for the scaled reference squiggles are ranked and the read is assigned the identity of the highest ranked squiggle (FIG.120, 120 R6). [1452] The accuracy of single-molecule identity in the context of repeated experiments (e.g. asking out of 100 reads, what percentage would be correct?) was estimated by applying the procedure above to a plurality of reads with a known identity. For example, to obtain the average accuracy, the number of reads correctly -413- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 identified is counted and then divided by the total number of reads to obtain the proportion of reads correctly identified. [1453] Analysis: Identification of a Plurality of Molecules [1454] In addition or instead of estimating the identity of individual reads, it is desirable to aggregate information from all reads known to or believed to be from the same substrate into a single estimate of identity, for example: analysis of sample containing a single protein, or analysis of a sample containing a mixture of proteins where the reads have been clustered by similarity. The scores for all reads are aggregated together for each putative reference squiggle before estimating the identity from these aggregated scores. [1455] Methods of aggregation include, but are not limited to: the sum of the scores, the weighted sum of scores, the median of the scores, the maximum or minimum of the scores, a L-statistic (linear combination of order statistics), a M-estimator. The scores may be normalized by read length or substrate length before aggregation. [1456] For example, the aggregated score for each putative reference squiggle was calculated by first normalizing each score by the length of the read and then taking the median over all reads being aggregated. The identity was estimated as that of the reference squiggle with the highest aggregated score. [1457] Confidence in the estimate of identity can be assessed by bootstrap method (Efron, B., Tibshirani, R., & Tibshirani, R. J. (1994). An introduction to the bootstrap. Chapman & Hall/CRC. https://doi.org/10.1007/978-1-4899-4541-9 the contents of which are hereby incorporated by reference in its entirety. Reads were resampled with replacement to produce 10,000 pseudo-replicate experiments each for which the identity of the substrate can be estimated on aggregate according to the method described. The proportion of these pseudo-replicates where the protein was correctly identified gives a measure of accuracy of identification over repeated experiments. [1458] Analysis: Quantification [1459] Given a sample containing a mixture of proteins, it is desirable to determine what proteins are present in the sample and to quantify their absolute or relative abundance and the process to achieve this is described herein and shown as a workflow in FIG.121. [1460] Exemplified herein, reads were obtained from the electrophysiological signal according to the method described in Analysis: Preprocessing reads (extraction, segmentation, denoising) (FIG. 121, 121 R1) and identity assigned to the individual reads by applying the procedure described in “Analysis: ID (single- molecule)” (FIG. 121, 121 R2) with a database containing the references for the proteins of interest in the mixture (FIG.121, 121 S1). The number of reads assigned to each unique identity were counted, quantifying the sample (FIG.121, 121 R3). -414- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1461] The counts of identity may be normalized to create an estimate of the composition of the sample and one method of normalization is to divide the count of each identity by the total over all identities. The normalization was constrained by prior knowledge of the sample composition or biology, for example: it might be known that two or more peptide chains must be present in a known ratio and so their counts may be combined during normalization; or a previous measurement of the composition may be available (FIG. 119, 119 R4). [1462] The estimate of quantification can be determined (FIG.121, 121 R5) or be iteratively improved (FIG. 121, 121 S3) by reapplying the procedure to identify each read using the estimated composition as a prior probability to weight the reference squiggles (FIG. 121, 121 S2) and hence their scores (see Analysis: ID (single-molecule)), discouraging or preventing assignment to identities that are not supported by other reads in the sample. A new estimate of quantification, and hence composition, can be derived from these identities and this could be used for further refinement of the estimate. [1463] Analysis: Empirical squiggle & Time Warped coordinates [1464] After adjusting a reference squiggle to a set of reads, it may be referred to as an empirical squiggle since its levels are now derived from observed values of a particular experiment or set of experiments. [1465] To directly compare reads to each other, it is desirable to convert them into coordinates that have been time-warped to a common squiggle. Each read is aligned to the squiggle and the segments, or raw current samples, corresponding to each squiggle position are assigned time-warped coordinates that are uniformly spaced between that squiggle position and the next, preserving the original time ordering. Plotting the reads using these time-warped coordinates results in all the features corresponding to each position being overlaid, revealing differences and similarities in their surrounding context. The time-warped coordinates are also suitable for many other analyses. Example 20. Peptide/Polypeptide/Protein Identification [1466] This example demonstrates the ability to accurately identify proteins versus a database of references, where the references are reference squiggles generated by a Machine Learning model. This example demonstrates the ability to determine the identity of protein translocation reads obtained from known proteins previously used to train models. The example further demonstrates that the trained models generalize and are able to accurately de novo identify unknown proteins that were not in the training. Data selection and Preprocess [1467] The 35 protein substrates listed in Table 36 were prepared and measured as described in the methods of this example. Briefly, the proteins of interest were engineered as genetic fusions of the E. coli sequence plus a >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation content to enable efficient capture into the nanopore and a domain to -415- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 stall the ClpX translocase. The proteins were prepared in E.coli as described herein. In separate experiments, the protein substrates were added to the cis compartment of a single chamber electrophysiology system as described herein (final concentration of 0.1 µM protein substrate, 0.2 µM ClpX, 2.5 mM ATP) containing a single inserted MspA-D90N nanopore. Protein translocation measurements were performed as described herein under –60 mV applied potential. [1468] Multiple experiments were run for each substrate sample (on different nanopores in a fresh electrophysiology chamber for each experiment) to acquire multiple extracted reads of protein translocation. As long as experimental conditions are kept controlled, nanopore experiments are highly reproducible, with measured reads from the same sample showing a high level of consistency between nanopores and/or experiments. For this example, for all protein substrates in both the training and the validation datasets, sets of reads for each substrate were aggregated from two or more separate experiments and showed a high level of reproducibility. [1469] After acquisition the reads were extracted, denoised and segmented according to Analysis: Preprocessing reads (segmentation, denoising). In total 1763 reads were acquired over 253 experiments and these were split into 1573 reads for training and 190 reads for validation (10 from each of 19 substrates, one set discarded due to naming issues). The substrates chosen for validation were those for which the most reads had been acquired, so moving some of these reads into the validation helped balance the training set; validation reads for each substrate were chosen randomly from those available. Model Structure [1470] The model structure used was as described in Analysis: Creating references and databases – Model Structure. Model Training & Validation [1471] Training was performed as described in Analysis: Creating references and databases – Training. The training dataset used for model training is detailed in Table 36. Table 36. Training dataset. Column A. Column B. Substrate No. training Reads A0A0H3C834_CAUVN_MBP1 59 A0A1S4NYF2_BRALA_MBP1 52 CH60_ECOLI_MBP1 38 DLDH_ECOLI_MBP1 39 DNAK_ECOLI_MBP1 35 DYR_ECOLI_MBP1 43 -416- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 EFTS_ECOLI_MBP1 47 EFTU1_ECOLI_MBP1 31 ENO_ECOLI_MBP1 35 G3P1_ECOLI_MBP1 64 G6PD_ECOLI_MBP1 36 G6PI_ECOLI_MBP1 25 GLNA_ECOLI_MBP1 31 GLYA_ECOLI_MBP1 49 GPMA_ECOLI_MBP1 102 IDH_ECOLI_MBP1 49 IMDH_ECOLI_MBP1 43 LIVJ_ECOLI_MBP1 55 LIVK_ECOLI_MBP1 94 MBP1 45 MBP1_KNKv2_PKA_ENGv2_CK2 42 MBP1_N175D 14 MBP1_Q255E 26 MBP_E174Q 32 MBP_N203D 28 MGLB_ECOLI_MBP1 59 ODP2_ECOLI_MBP1 43 PGK_ECOLI_MBP1 46 PNC1_YEAST_MBP1 53 Q9CES5_LACLA_MBP1 52 Q9CKB5_PASMU_MBP1 37 SKY1_ECOLI_MBP1 38 THIB_ECOLI_MBP1 52 TKT1_ECOLI_MBP1 39 TYPH_ECOLI_MBP1 40 [1472] To validate the trained models and determine the best performing model and the optimal values for the training meta-parameters (e.g. the learning rate and weight decay), the performance of the models was -417- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 tested against a set of validation reads which were withheld from training. In this example, multiple trained models (303 different models obtained from different parameterized training runs performed on the same set of training reads) were tested by using them to generate a database of predicted reference squiggles, which were then compared against the validation set of reads from the proteins listed in Table YY. Model performance was compared by aggregating the scores (e.g. the mean or median), over the entire validation set and determining the best model from the highest score. Alternatively, other methods could be used. The number of events in the validation set should be large enough to ensure statistically significant decisions to be made, with the additional requirement that they should also be from sequences diverse enough to ensure that the result reflects for all proteins. In this example the validation set consisted of 12% of all events, evenly covering 63% of the analytes trained from. [1473] Table 37 outlines the performance of the model that scored the highest against the reads from the validation set. The table outlines the ability of this model to accurately characterize the protein identity of reads obtained from nanopore translocations. Then ground truth identity of the validation reads is known as the reads were acquired from separate samples of known expressed and purified protein. This example demonstrates that the methods and system of the invention were able to accurately characterize the peptide/polypeptide/protein identify the reads acquired from the nanopore translocations of the proteins. [1474] Table 37 – Validation dataset and peptide/polypeptide/protein identification results: Column (a) tabulates the 19 proteins included in the first validation set, named according to their Uniprot ID. The validation set is formed of 10 randomly selected electrophysiological reads selected from much larger sets of reads (aggregated over 2 or more runs for each samples) for each of the 19 protein substrates. All reads in the validation set were withheld from the training set. [1475] Table 37 tabulates the summary identification results from comparing and scoring all the reads against all the reference squiggles (as described herein in the section Analysis: Scoring and/or aligning a reference) in the reference squiggle database, where the predicted reference squiggles in the database are from the model that scored the highest against the reads from the validation set. In this example, the reference squiggles database was generated from 4180 E. coli references (inclusive of the 19 protein sequences corresponding to the substrate of the validation set), therefore offering the possibility of the reads scoring against 4179 other spurious proteins (i.e. 4180 minus the correct protein). The 4180 protein sequences used to generate the database represents almost all proteins from the reference K12 E. coli proteome (lightly filtered to remove proteins longer than 800 residues) comprises >90% of the proteins in the E.coli proteome. [1476] The number of reads where the top scoring sequence corresponded to the correct protein is shown in column (b). The bracketed number in column (b) tabulates the accuracy of single-molecule identification, which determines the identity of each read without further aggregated information from other reads in the -418- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 dataset. The table illustrates the ability to accurately identify proteins at the single-molecule level at greater than 80% for most proteins. In this example, the reference squiggles database was generated from 4180 E. coli references, therefore offering the possibility of the reads scoring against 4179 other spurious proteins (4180 minus the correct protein), further demonstrating the ability of the trained model to generalize to complex input samples and large libraries (such as those that would be obtained from real world samples such as cell lysates, blood plasma, etc.) This demonstrates that protein translocation reads from the methods and system of the invention can be identified with high accuracy (average identification accuracy = 84%, Median accuracy = 90% across the 19 proteins) against an almost complete cellular proteome (e.g., proteomes containing 1000’s of possible proteins) as long as the proteins were part of the training. Furthermore, since the identity is measured with high accuracy at the single-molecule level on a read-by-read basis, and it was previously demonstrated the reproducibility of signal herein that enables reads from different runs to be combined into a dataset to effectively represent data from mixtures in a single run, the 19 different proteins that comprise this validation example can be considered as a complex mixture that demonstrates the ability to identify proteins in real-world complex mixtures (e.g samples obtained from cell lysates, bio-fluids, etc). [1477] Each read can retain the information about other scoring possibilities against some or all of the other proteins in the full squiggle database of 4180 proteins, and this information can be encoded in the read results file. Column (c) tabulates the number of reads where the correct protein was among the top 5 highest scoring proteins. This demonstrates that even incorrectly identified reads almost always rank the correct protein near the top of the possible candidates. Furthermore, column (d) tabulates the results of position of the correct protein (1 = best, 4180 = worst) when aggregating the scores of all the possible protein identity assignments (Sum or median of all scores against each reference protein/sequence both produced the same results). This demonstrates that an aggregation analysis can be performed on clusters of reads using the underlying scores against other “off-target” proteins to improve the accuracy of identification. The brackets in column (d) tabulates the accuracy of identification for the aggregate set (accuracy determined by bootstrap analysis as described in Analysis: Identification of a Plurality of Molecules). Aggregate analyses can be used to improve identification in samples with one or more unknown proteins. For example, in one embodiment reads from a sample containing single experiment on an unknown protein, or reads obtained from a mixture of unknown proteins where reads are clustered based in similarity) that all protein sets determine the correct reference protein/sequence. Table 37 – Validation dataset and peptide/polypeptide/protein identification results. Column A Reads Column B. Column C. Column D. Substrate No. reads protein = No. reads protein = Aggregate rank top hit Top 5 (% accuracy) -419- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 (single-molecule % accuracy) A0A0H3C834_CAUVN 10 6 (60%) 10 1 (98%) A0A1S4NYF2_BRALA 10 10 (100%) 10 1 (>99.9%) DNAK_ECOLI 10 10 (100%) 10 1 (>99.9%) DYR_ECOLI 10 8 (80%) 10 1 (>99.9%) EFTS_ECOLI 10 8 (80%) 10 1 (>99.9%) G3P1_ECOLI 10 10 (100%) 10 1 (>99.9%) G6PD_ECOLI 10 10 (100%) 10 1 (>99.9%) GLYA_ECOLI 10 8 (80%) 10 1 (>99.9%) GPMA_ECOLI 10 10 (100%) 10 1 (>99.9%) IDH_ECOLI 10 8 (80%) 10 1 (>99.9%) LIVJ_ECOLI 10 9 (90%) 10 1 (>99.9%) LIVK_ECOLI 10 9 (90%) 10 1 (>99.9%) MGLB_ECOLI 10 9 (90%) 10 1 (>99.9%) PGK_ECOLI 10 9 (90%) 10 1 (>99.9%) PNC1_YEAST 10 4 (40%) 10 1 (99.6%) Q9CES5_LACLA 10 5 (50%) 10 1 (>99.9%) Q9CKB5_PASMU 10 9 (90%) 10 1 (>99.9%) THIB_ECOLI 10 10 (100%) 10 1 (>99.9%) TYPH_ECOLI 10 8 (80%) 9 1 (>99.9%) Model Generalization and de novo identification [1478] It is desirable that a trained model make good predictions for sequences which were not part of its training set, i.e. the model may perform well on the validation set of protein sequences that formed part of the training but fail to generalize to protein sequences not in the training. To find a trained model that generalizes well, multiple models are trained and then assessed on their ability to generalize against reads from proteins -420- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 never used in the training. The best generalizing model can then be chosen as the squiggle model for generating reference squiggle databases. [1479] In this example, the models trained in the previous step were tested against a second validation, consisting of reads from protein substrates that were withheld from training, so the model has neither seen the constituent events nor their underlying sequence. This process was used to validate that how well the trained models generalize to unknown proteins. Like the first validation set, this second validation was chosen to contain sufficient events and sequence diversity to ensure a statistically significant choice between models can be made. [1480] The ability of the trained models to generalize to unknown protein sequence was assessed by comparing their performance on this second validation set; methods of comparison include but are not limited to the score the trained model assigns to events against the reference sequence or the rank of the ability to discriminate the reference sequence from a set of alternative sequences. S combination of these methods was used to select the highest scoring model, the results of which are shown in Tables 38 and 39 against the second set of de novo validation proteins tabulated in column (a) by their Uniprot ID. This example follows the same process as that described for the first validation set. Tables 38 and 39 outline the ability of this model to accurately characterize the protein identity of reads obtained from nanopore translocations. [1481] Table 38 shows de novo Validation dataset and peptide/polypeptide/protein identification results: Column (a) tabulates 4 proteins included in the second validation set, named according to their Uniprot ID. The validation set is formed of randomly selected electrophysiological selected from larger sets of reads (aggregated single or multiple experiments for each samples) for each of the 4 protein substrates. Both the underlying sequences and the reads in the second validation set were withheld from the training set. Each read was segmented and denoised. [1482] Electrophysiological reads of ClpX controlled nanopore translocation were obtained for each of 4 different substrates in column (a), labelled by their assigned Uniprot name, which were withheld from the model training set. Measurements were obtained from single or multiple experiments on the single chamber system described in the example herein, with MspA_D90N nanopores in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5 at -60 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM substrate and 2.5 mM ATP. [1483] Tables 38 and 39 tabulate the summary identification results from comparing and scoring all the reads against all the reference squiggles (as described herein in the section Analysis: Scoring and/or aligning a reference) in a medium sized reference squiggle database and a large sized reference squiggle database respectively, where the predicted reference squiggles in the database are from the model that scored the highest against the reads from the second validation set. The data in Table 38 is from comparison of reads to a reference -421- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 squiggles database generated from 120 E.coli references (inclusive of the training reference sequences and the validation reference sequences), therefore offering the possibility of the reads scoring against 119 other spurious proteins (i.e. 120 minus the correct protein). The data in Table 39 is from comparison of reads to a reference squiggles database generated from 4180 E. coli references (inclusive of the training reference sequences and the validation reference sequences), therefore offering the possibility of the reads scoring against 4179 other spurious proteins (i.e.4180 minus the correct protein). [1484] Each read from the second validation set was scored as described against all reference squiggles from the database and identity was assigned as that of the best scoring squiggle. Scores were retained for all squiggles. Columns (b) in Tables 38 and 39 show the number of reads for each substrate whose identity was correctly assigned on a single-molecule basis. Single-molecule accuracy “SM accuracy” is shown in brackets. Although the single-molecule accuracy is lower than for proteins included in the training sets, the reads are still assigned with high accuracy for 2 of the 4 proteins. This demonstrates that the model has generalized, and that a limited library of training proteins can be used to train models that are able to generate reference squiggles that can be used to accurately de novo identify nanopore protein translocation reads from unknown proteins against medium (100s) and large proteomes (1000s). This demonstrates that it is possible to build generalized models from limited libraries of training proteins (e.g., 10s, 100s, 1000s of unique proteins) that will enable downstream identification of unknown proteins obtained from samples with much larger proteomes, thus avoiding the requirement to pre-train the models with all proteins that were expected to be detected. [1485] When aggregating the reads, Column (c) in Table 38 shows the Median of rank (out of 120, 1 being best) that the correct protein was scored. Column (c) shows that the reads score the correct protein either 100% correct or in top 10% of protein references. Further, column (d) shows that when the scores for each substrate were combined together to make an aggregate prediction, every substrate was correctly identified (100% accuracy) by this metric (Rank 1). To assess the accuracy of the aggregated prediction, a bootstrap analysis may be used -- 10,000 pseudo-replicates were sampled and the aggregate score found for each. The accuracy can be estimated as the proportion of the bootstrap pseudo-replicates that correctly identify the substrate and Tables 38 and 39 show that the aggregate prediction often exceeds 99.9% accuracy for all but one substrate “Aggregate rank (accuracy)”. This demonstrates that all four proteins can be identified correctly at above 90% to above 99% when analyzing using multiple reads in a cluster, demonstrating the ability to de novo identify unknown proteins from mixtures of polypeptides containing a plurality of different peptides. [1486] Similarly, Table 39 shows that aggregation over multiple reads improves accuracy for a very large proteome of more than 4000 protein references. All of the substates were identified with high accuracy (100% accuracy for two of the proteins) when the criterion for identification was relaxed to include those where the -422- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 correct identification was ranked in the top 5 (column (C)). Further, column (d) shows that when the scores for each substrate were combined together to make an aggregate prediction, two of the four proteins are called 100% correctly, and the other two are called in the top 0.25% of all protein references in the proteome. It is clear to a person of skill in the art that accuracies will significantly improve both for single-molecule and aggregate analyses as the training is extended to include larger datasets (e.g. more reads per substrate) and more different proteins. Table 38. de novo validation dataset and peptide/polypeptide/protein identification results (120 reference library database). Column A. Reads Column B. Column C. Column D. Column E. Substrate No. reads protein Median rank Aggregate rank Bootstrap = top hit (accuracy) confidence (single-mol % accuracy) HTPG_ECOLI 16 12 (75%) 1/120 1 (100%) 96% PURA_ECOLI 3 1 (33%) 3/120 1 71% TALB_ECOLI 21 2 (10%) 10/120 1 9% TIG_ECOLI 20 15 (75%) 1/120 1 (100%) 99..6% Table 39. de novo validation dataset and peptide/polypeptide/protein identification results (4180 reference library database). Column A. Reads Column B. Column C. Column D. Column E. Substrate No. reads protein No. reads protein Aggregate rank Bootstrap = top hit = Top 5 (accuracy) confidence (single-mol % (% of reads) accuracy) HTPG_ECOLI 16 9 (56%) 16 (100%) 1 (100%) 99.70% PURA_ECOLI 3 0 (0%) 2 (66%) 3 (>99.9%) ~0% TALB_ECOLI 21 1 (5%) 13 (62%) 11 (99.8%) 5% TIG_ECOLI 20 10 (50%) 20 (100%) 1 (100%) 99.70% Example 21. Peptide/Polypeptide/Protein Identification Quantitation. [1487] This example demonstrates the ability to measure a plurality of polypeptides from a mixture of different proteins and to characterize properties of the sample, for example to quantitate the relative amount of -423- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 the different protein types in the mixture. The sequences of the analytes of this Example are shown in Table 26. [1488] A mixture of three substrates with sequences of the proteins with Uniprot identifiers: LIVJ_ECOLI, LIVK_ECOLI and A0A1S4NYF2_BRALA was prepared separately as described in this example; the proteins LIVJ_ECOLI and LIVK_ECOLI have similar length, 367 and 369 respectively, and 70% sequence identity, and so are relatively more difficult to discriminate from each other to further test the capabilities of the methods described herein are robust to real proteins. [1489] The proteins were prepared separately in E.coli as described herein, and extracted from the E.coli lysates via Hit-tag affinity extraction and no further purification. After preparation the concentration of the prepared proteins was determined by the well-known Bradford assay using conventional means. The concentrations of the samples were determined as 2.2 uM, 2.4 uM and 2.1uM for LIVJ_ECOLI, LIVK_ECOLI and A0A1S4NYF2_BRALA respectively. The proteins were mixed together into a single sample at a 1:1:1 ratio by volume. The final real relative concentration was determined based on the concentration from the Bradford assay. Table 27, Column A tabulates the expected proportions from the measured concentration from the Bradford assay, which were 33% of all protein, 36% of all protein and 31% of all protein for LIVJ_ECOLI, LIVK_ECOLI and A0A1S4NYF2_BRALA respectively. The protein mixture prepared from the complex lysates contained the proteins of interest plus a complex background of other lysate proteins, molecules and other impurities from the original biological sample (these were evident by other types of current signal events and blockades in the measured nanopore signal resulting from other proteins and impurities interacting with the nanopore). [1490] Electrophysiological measurements were performed from the sample containing the 3 protein mixture of substrates as described herein. Measurements were obtained from a single experiment on the single chamber system described in the example herein, with MspA_D90N nanopores in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5 at -60 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM substrates (combined concentration of all proteins in mixture) and 2.5 mM ATP. The extracted putative reads corresponding to putative target analytes were denoised and segmented, and then each read scored against a database of reference squiggles (generated from the best model built and validated in the previous section above) containing one squiggle for each of the three possible substrates as described herein. The identity for each read was assigned as that of the best scoring reference squiggle, and all unassigned reads (e.g. as a result of impurities) were discarded. Assignments were further verified by manual inspection. [1491] Table 27, Column B shows the number of reads identified as being from each of the substrates in the sample over the total number of reads measured, and normalized to a percentage in brackets. The relative -424- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 concentration estimates obtained from the electrophysiological measurements and identity analyses agree very closely with the ground truth proportions of each substrate based on the Bradford assays. The absolute error in % difference between these two measurements is given in Table 27, Column C, showing that the difference in expected % in the sample versus measured differs by less than 3%, and for one protein was 100% accurate. [1492] The absolute error can more meaningfully be estimated by a sum of the absolute mis-identifications divided by the total number of reads. [1493] |16 – 0.3333 * 48| + |16 – 0.36 * 48| + |16 – 0.31* 48| = 2.39 [1494] Error: 2.39 / 48 = 5% [1495] Thus, the accuracy of measurement can be estimated as 95% (100% - Error). [1496] This example demonstrates the ability to correctly identify proteins from a real complex mixture of proteins derived from a natural biological sample, and, by analyzing a plurality of translocation reads and counting the number assigned into each identity, accurately determine the relative concentration of the substrates versus a well-known gold standard measurement. Table 27. Output of reads. Protein Column A. Column B. Column C. Percent in sample (relative Number Absolute difference in % concentration in sample) reads/total reads (percentage of all reads) LIVJ_ECOLI (2.2uM/6.7uM) 33% 16/48 (33%) 0% LIVK_ECOLI (2.4uM/6.7uM) 36% 16/48 (33%) 3% A0A1S4NYF2_BRALA (2.1uM/6.7uM) 31% 16/48 (33%) 2% Example 22. Detection of variants and single amino acid differences in proteins. (Point mutations). [1497] This example demonstrates the ability of the nanopore system of the invention to discriminate between small differences in the target proteins, including as little as one amino acid difference. Target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the pore. [1498] Target protein variants of interest were provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation content to enable efficient capture into the nanopore and a domain to stall the ClpX translocase. The proteins were prepared as described herein. [1499] The adapted target protein was added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a -425- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 membrane under –80 mV applied potential. The ionic current measurements are then performed using the standard methods described herein. [1500] To test whether changes in ionic current signal during translocation events could be observed for small changes in protein sequence, several variants of MBP-1 (Uniprot: MALE_ECOLI) comprising mutations at residues 214-216 (KNK motif) were prepared (see Table 28) and tested in the nanopore system in separate experiments. Table 28 outlines the four proteins and highlights the mutations in the sequence in the region of interest at positions 214-216 (underlined amino acid residues). MBP-1 is considered the original sequence with a KNK motif in the region of interest, MBP_ENK comprises a KNK -> ENK mutation, MBP_KWK comprises a KNK-> KWK mutation, MBP_WWW comprises a KNK -> WWW mutation. Table 28. Amino acid sequences of target proteins used. Protein Amino acid sequence SEQ ID NO. MBP- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE 150 1{KNK} KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALAA MBP- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE 151 1{KWK} KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKWKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALAA MBP- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE 152 1{WWW} KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIWWWHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALAA MBP- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE 153 1{ENK} KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIENKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALAA -426- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 MBP- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE 154 1{ENE} KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIENEHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALAA [1501] The nanopore translocation reads for the MBP-1 variants each showed different and reproducible ionic current features in the translocation events (FIGs. 124A-124E). For instance, MBP-1{KWK} or MBP- 1{ENK} differ from MBP-1{KNK} by only one amino acid, yet their ionic current signals are remarkably different. This example thus shows that differences as subtle as a single amino acid substitution can be detected by changes in the ionic current at the single-molecule level. [1502] FIGs.125A-125B further illustrate the difference in ionic current signal from the point mutations in a bioinformatic analysis that overlays multiple reads upon each other to form a consensus. To analyze a plurality of reads the reads were clustered and then each read was aligned to a reference squiggle of MBP-1 as described herein in the Consensus analyses section, which yields a consensus “pile-up” of the plurality of reads all scaled and aligned by index so that they are overlaid onto each other and to the adjusted reference squiggle obtained from the analysis. For each substrate, the corresponding reads were aligned to the reference squiggle for the unmodified substrate then the squiggle adjusted to yield a new adjusted reference squiggle for each substrate. Figure 124A plots the 3 adjusted squiggles for each mutation against the unmutated MBP-1, clearly showing the difference the mutation creates in the signal from aligned over a plurality of reads. FIG.125A shows the mutant substrates can be distinguished from the unmodified control and from each other, using electrophysiological reads. The adjusted squiggles for the four substrates are compared in FIG.125A and show long regions of agreement except for the area contained in the mutations denoted by the dashed box. FIG. 125B is zoomed into the area of FIG. 125A denoted by the dashed box and shows that the adjusted squiggle for each mutant is distinct from the others and from the unmodified control. Further, all reads in a plurality of reads for each substrate follow highly similar pathways (as indicated by the dot points spread from the read data overlaid). This demonstrates that the mutations can be resolved with very high accuracy from each other, and a number of analytical methods can be used to call the mutation at accuracies of greater than 90% and greater than 99% in some cases. Example 23. Protein isoform detection. [1503] This example demonstrates the ability to detect protein isoforms. Isoforms of the same protein typically differ over many residues, the median exon in the human genome being 120 nucleotides long, which translates into 40 residues, although some exons are much shorter and these are more challenging to -427- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 discriminate because fewer residues have changed. To demonstrate using the methods and systems of the invention’s ability to identify and quantify protein isoforms, three substrates with very challenging small differences were created from variants of the sequence with Uniprot name MALE_ECOLI. One substrate was wild-type MALE_ECOLI; the K residue at position 224 in the sequence were replaced by the sequences HNSVAEGNN (9 residues) and NGRRVSLGNNH (11 residues) for the second and third isoform/substrate respectively (see sequences in Table 40). [1504] Electrophysiological reads of ClpX controlled nanopore translocation were obtained for separate experiments on each of the substrates samples as described herein. Measurements were obtained from single or multiple experiments on the single chamber system described in the example herein, with MspA_D90N nanopores in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5 at -80 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM substrate and 2.5 mM ATP. Reads were extracted from the acquisition signals of these experiments, and denoised and segmented as described herein. Each read was scored by comparing against a database of reference squiggles containing one squiggle for each of the possible isoforms that was synthesized from their sequences using a trained model (trained as described herein; FIG. 135). The identity of isoform for each read was assigned as that of the best scoring reference squiggle. Table 40. Amino acid sequences for Isoforms Test. Underlined residues are charged residues. Peptide / Amino acid sequence SEQ Protein ID NO. MALE_E MGSSHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEEDTGIKVTVEHPD 168 COLI KLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTIN GPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEG LEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAA SGRQTVDEALKDAQTRITKGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHP DKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAAD GGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDE GLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINA ASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA Isoform 1 MGSSHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEEDTGIKVTVEHPD 169 KLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNHNSVAEGNNMNADTDYSIAEAAFNKG ETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLEN YLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVR TAVINAASGRQTVDEALKDAQTRITKGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGI KVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFT WPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNK GETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAV -428- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 RTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDE NYALAA Isoform 2 MGSSHHHHHHSSGLVPRGSHNKIEEGKLVIWINGDKGYNGLAEVGKKFEEDTGIKVTVEHPD 170 KLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNNGRRVSLGNNHMNADTDYSIAEAAFN KGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYA VRTAVINAASGRQTVDEALKDAQTRITKGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDT GIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFT WDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPY FTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAF NKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEF LENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWY AVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAAN DENYALAA [1505] The number of times that substrate is assigned to each of the three possible identities is shown in Table 41, and these counts demonstrate the correct isoform was identified more than 90% of the time on a single- molecule basis. As assignment was determined on a single-molecule basis and the acquisition conditions and database of reference squiggles were common to all substrates, the accurate assignment of identity also demonstrates isoform quantification and this is shown in the row and column sums and proportions in Table 41. Table 41. Identification analysis of protein isoforms results. Prediction MALE_ECOLI Isoform 1 Isoform 2 MALE_ECOLI 64 (64/69 = 93%) 2 3 Substrate Isoform 1 0 29 (29 / 31 = 94%) 2 Isoform 2 1 0 45 (45 / 46 = 98%) Example 24. Detection of protein post-translational modifications (PTMs). [1506] These examples demonstrate the ability of the nanopore system as herein disclosed to measure various types of natural and unnatural post-translational modifications of a target peptide/polypeptide/protein. Phosphorylation [1507] To demonstrate the ability to detect protein phosphorylation the target proteins MBP_KNK_PKA and MBP_ENG_PKA (see Table 5) were designed as mutants of MBP-1 that contain a recognition {RRXS/TY, Y hydrophobic residue} sequence for cAMP-dependent Protein Kinase A (PKA), which phosphorylates the 225 Ser amino acid of MBP_KNK_PKA (underlined in MBP_KNK_PKA in Table 29) and the 191 Ser amino acid of MBP_ENG_PKA (underlined in MBP_ENG_PKA in Table 29). To phosphorylate MBP_KNK_PKA -429- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 and MBP_ENG_PKA (substrates) 85 µL of each substrate (at approximately 50 µM concentration) was first mixed with 10 µl of 10x NEBuffer and 2µl of 10 mM ATP. This mixture was incubated while shaking at 30°C for 30 minutes. Afterwards, 2 µL of PKA (2500 units/µl) was added and the sample was incubated while shaking at 30°C for 2 hours. Table 29. Amino acid sequences of the mutant target protein used. Protein Amino acid sequence SEQ ID NO. MBP_KNK MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF 155 _PKA PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPI AVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAF KYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNNGGYGLRRASLGKYGNKHMNADTDYSIAE AAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQM SAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRR RRRAANDENYALAA MBP_ENG_ MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF 156 PKA PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPI AVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAF KYGLRRASLGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMT INGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLT DEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAV INAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYA LAA [1508] The target proteins of interest (MBP_KNK_PKA and MBP_ENG_PKA) were provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1509] In separate experiments, the control unmodified MBP_KNK_PKA and MBP_ENG_PKA target proteins of interest and phosphorylated MBP_KNK_PKA and MBP_ENG_PKA target proteins post- translationally modified by treatment with a kinase were analysed in the nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1510] After acquisition, multiple reads were extracted as described herein from the given experiment. The process described in “Analysis: Consensus Analysis (Mutations, Post-translational modifications, and variants )” was used to produce a consensus of the reads for each experiment. After alignment, the analysis outputs. The separate consensus squiggles obtained from the unmodified and modified proteins were then overlaid to -430- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 compare and check for any differences. When comparing unmodified and enzymatically phosphorylated MBP_KNK_PKA and MBP_ENG_PKA proteins, the individual and the consensus signals were determined to overlap closely through the majority of the signal, with the exception of differences in their ionic current signature at a specific location. FIG. 126A shows a portion of the full squiggle (top), and a further zoom (bottom) of the region where a deviation was detected for the analysis of the reads from the treated and untreated MBP_KNK_PKA proteins. Single-molecule alignments to the adjusted squiggles can be plotted on top of the squiggle to assess the extent to which the individual molecules match the average consensus. FIG. 126A plots the spread of the single-molecule alignments by the width of the band (1-standard deviation width), illustrating that the majority of the molecules closely cluster with the current levels of the consensus squiggle, thus all reads have a clear deviation as a result of phosphorylation, which was backed up by Mass Spectrometry data which indicated that the phosphorylation had proceeded to >95%. This demonstrates that the methods and systems of the invention are able to accurately detect phosphorylation PTMs on peptides/polypeptides/proteins. PTM Accuracy [1511] This example demonstrates that the deviations in signal here and throughout (e.g., for mutations and PTMs) that result from single point changes (inclusive of mutations, natural and unnatural post-translational modifications, conjugations, etc.) can be determined with high accuracy using the methods and systems of the invention. To determine the accuracy of correctly identifying the putative change or modification versus a reference it is necessary to know the ground truth of the change (for example by measuring the change with orthogonal measurements technologies). To this end, a phosphorylated and unmodified protein was measured with MALDI-TOF mass spectrometry to determine the extent of phosphorylation on both the original protein and the protein subjected to treatment. [1512] The target protein MBP_ENG_PKA (see Table 29) was a mutant of MBP-1 that contains a recognition {RRXS/TY, Y hydrophobic residue} sequence for cAMP-dependent Protein Kinase A (PKA), which phosphorylates the 191 Ser amino acid of MBP_ENG_PKA (underlined in MBP_ENG_PKA in Table 29). To phosphorylate MBP_ENG_PKA 85 µL of each substrate (at approximately 50 µM concentration) was first mixed with 10 µl of 10x NEBuffer and 2µl of 10 mM ATP. This mixture was incubated while shaking at 30°C for 30 minutes. Afterwards, 2 µL of PKA (2500 units/µl) was added and the sample was incubated while shaking at 30°C for 2 hours. Samples of both the unmodified precursor protein and the phosphorylated protein were measured by MALDI-TOF mass spectrometry showed that the starting protein had no measurable phosphorylation, and the treated sample was at least >98% phosphorylated. [1513] In separate experiments, the control unmodified MBP_ENG_PKA and phosphorylated MBP_ENG_PKA target proteins post-translationally modified by treatment with a kinase were analysed in the nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of -431- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1514] After acquisition, multiple reads were extracted as described herein from the given experiment. All reads were aligned to reference squiggles generated using the sequence of the unmodified protein (as described herein) and trained models. After alignment the analysis outputs an adjusted reference consensus squiggle that represents the consensus alignment of all reads. The separate consensus squiggles obtained from the unmodified and modified proteins were then overlaid to compare and check for any differences. When comparing unmodified and enzymatically phosphorylated MBP_ENG_PKA proteins, the individual and the consensus signals were determined to overlap closely through the majority of the signal, with the exception of differences in their ionic current signature at a specific location (FIG.126A). [1515] The adjusted consensus squiggles in this experiment where the treatment has gone to completion can be considered well resolved/representative to the majority population of molecules, and will in insensitive to a small percentage of unmodified reads or impurities for example (not skewed by a small percentage of alternative reads). The single-molecule reads can then be compared back against the consensus reference squiggles on a per read basis for each sample to determine the number of molecules that align better to one reference or the other. A selection of random reads for each sample (treated and untreated) were aligned back against the adjusted reference squiggles obtained from both the treated and untreated samples from the previous step. Table 42 shows the identity (highest scoring alignment) of how the reads in the samples aligned (filter criteria removed reads with poor scores that did not align to either reference). Based on the mass spectrometry data (showing the treated sample was almost fully phosphorylated and the untreated sample contained no phosphorylation), the electrophysiology results for the two samples show a 100% accuracy and greater than 80% accuracy of measuring the PTM presence/absence correctly. Table 42. Identity of how the reads in the samples aligned. Sample Number of Reads aligning Reads aligning Filtered out Accuracy reads to -phos to +phos (unaligned) Untreated 29 28 0 1 100% Treated 15 2 13 0 87% [1516] This example demonstrates the ability to accurately identify small changes in polypeptides, including for example point mutations and post-translational modifications, by the methods and systems of the invention from the deviations in ionic current. Glycosylation -432- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1517] To demonstrate the ability to detect protein glycosylation the target protein MBP_ENG[exPKA:C],KNK[exPKA:S] (see Table 30) was designed as a mutant of the double phosphorylation mutant of MBP-1 / MBP_ENG[exPKA],KNK[exPKA] that contains a cysteine at position S190C that can be modified by conjugation with a sugar. [1518] The MBP_ENG[exPKA:C],KNK[exPKA:S] protein was prepared in the same manner as described herein for MBP-1. Conjugation with the monosaccharide glucose was performed as follows. Reduction of the Cysteine of the protein was performed by mixing 80 ul of 20-100 uM of MBP- ENG[exPKA:C],KNK[exPKA:S] protein (in the experiment the exact concentration of MBP- ENG[exPKA:C],KNK[exPKA:S] was 68 uM) with 15 ul HEPES pH 8.0 and 4 ul 100 mM TCEP pH 7.0 and incubating for 15 min at 37°C. After the cysteine reduction step, cysteine capping was performed by adding 5 ul of 200 mM iodoacetamide-glucose and further incubating the mixture for 15 min at 37 °C. The final product was stored at -20 °C until further analysis. Table 30. Amino acid sequence of the target protein analyte. Protein Amino acid sequence SEQ ID NO. MBP- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL 157 ENG[exPKA:C], EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYN KNK[exPKA:S] GKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFT WPLIAADGGYAFKYGLRRACLGKYDIKDVGVDNAGAKAGLTFLVDLIKNNGGYGLRR ASLGKYGNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTF KGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSY EEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDA QTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA [1519] The target protein of interest (MBP_ENG[exPKA:C],KNK[exPKA:S]) was provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1520] In separate experiments, the control unmodified MBP_ENG[exPKA:C],KNK[exPKA: S] target protein of interest and glycosylated MBP_ENG[exPKA:C],KNK[exPKA:S]) target protein post-translationally modified by cysteine conjugation were analysed in the nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. -433- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1521] After acquisition, multiple reads were extracted as described herein from the given experiment. All reads were aligned to reference squiggles generated using the sequence of the unmodified protein (as described herein). After alignment, the analysis outputs an adjusted reference consensus squiggle that represents the consensus alignment of all reads. The separate consensus squiggles obtained from the unmodified and modified proteins were then overlaid to compare and check for any differences. When comparing unmodified and glycosylated MBP_ENG[exPKA:C],KNK[exPKA:S] proteins, the individual and the consensus signals overlap closely through the majority of the signal with the exception of differences in their ionic current at the expected location comprising the post-translational modification (FIG. 126B). This demonstrates that the glycosylation alters the ionic current signature to an extent that can be detected by the system. Further, the example demonstrates that cysteine reactions/modifications can also be detected by the system, further demonstrating the ability of the methods and systems of the invention to detect conjugations. Acetylation [1522] To demonstrate the ability to detect protein acetylation, MBP-1 protein was subjected to a treatment to post-translationally acetylate the protein. The MBP-1 protein was prepared in the same manner as described for MBP-1. Acetylation of MBP-1 was performed as follows. Briefly, 0.2 µL of acetic anhydride 10.5 M were added to 50 µL of MBP-110 µM. The mixture was then incubated overnight at room temperature. Table 31. Amino acid sequence of target protein analytes. Protein Amino acid sequence SEQ ID NO. MBP-1_Ac MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHP 162 DKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWD AVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFN LQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNNSV AEGNNHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTF KGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVD EALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALA A [1523] The target proteins of interest (MBP-1) were provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1524] In separate experiments, the control MBP-1 and the acetylated MBP-1 protein were analysed in the nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of -434- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1525] After acquisition, multiple reads were extracted as described herein from the given experiment. All reads were aligned to reference squiggles generated using the sequence of the unmodified protein (as described herein). After alignment, the analysis outputs an adjusted reference consensus squiggle that represents the consensus alignment of all reads. The separate consensus squiggles obtained from the unmodified and modified proteins were then overlaid to compare and check for any differences. When comparing unmodified and acetylated MBP proteins, the individual and the consensus signals overlap closely through the majority of the signal with the exception of differences in their ionic current at the locations where acetylation post- translational modifications are expected (FIG. 126C). This example demonstrates the ability of the methods and systems of the invention to accurately detect acetylation PTMs. Deamidation [1526] To demonstrate the ability to detect protein deamidation PTMs (a type of protein degradation) a number of target proteins (MBP_E174Q, MBP_N175D, MBP_N203D and MBP_Q255E) (see Table 32) were designed as mutants of MBP-1 that contain mutations to Asparagine (N) and Glutamine (Q) to replicate the change of the amino acid as a result of a deamidation (Aspartate (D) to Asparagine (N) and Glutamate (E) to Glutamine (Q)). [1527] The protein substrates were prepared in the same manner as described for MBP-1. Table 32. Amino acid sequence of target protein analytes. Protein Amino acid sequence SEQ ID NO. MBP- MHHHHHHSSGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVA 158 E174Q ATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYQN GKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSN IDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA MBP- MHHHHHHSSGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVA 159 N175D ATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYED GKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSN IDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA -435- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 MBP- MHHHHHHSSGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVA 160 N203D ATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYEN GKYDIKDVGVDNAGAKAGLTFLVDLIKDKHMNADTDYSIAEAAFNKGETAMTINGPWAWSN IDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA MBP- MHHHHHHSSGKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVA 161 Q255E ATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEA LSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYEN GKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSN IDTSKVNYGVTVLPTFKGEPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNK DKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQT VDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA [1528] The target proteins of interest (MBP_E174Q, MBP_N175D, MBP_N203D and MBP_Q255E) were provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1529] In separate experiments, the control MBP-1 and the four deamidated proteins (MBP_E174Q, MBP_N175D, MBP_N203D and MBP_Q255E) of interest were analysed in the nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1530] After acquisition, multiple reads were extracted as described herein from the given experiment. All reads were aligned to reference squiggles generated using the sequence of the unmodified protein (as described herein). After alignment, the analysis outputs an adjusted reference consensus squiggle that represents the consensus alignment of all reads. The separate consensus squiggles obtained from the unmodified and modified proteins were then overlaid to compare and check for any differences. When comparing unmodified and deamidated MBP proteins, the individual and the consensus signals overlap closely through the majority of the signal with the exception of differences in their ionic current at the expected locations where the deamidation post-translational modifications were introduced (FIG. 126D). This example demonstrates the ability of the methods and systems of the invention to accurately detect deamidation PTMs and protein degradation changes. Detection of Poly Post-translational Modifications -436- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1531] This example demonstrates the ability of the nanopore system as herein disclosed to measure a various multiple post-translational modifications at multiple positions along a protein molecule. [1532] To demonstrate the ability to detect multiple PTMs along target proteins the protein MBP_ENG_KNK_PKA was designed with two kinase sites to enable a double phosphorylation of the protein. The protein was designed as a mutant of MBP-1 that contains a recognition {RRXS/TY, Y hydrophobic residue} sequence for cAMP-dependent Protein Kinase A (PKA), which phosphorylates the 191 Ser and 230 Ser amino acid of MBP_ENG_KNK_PKA. To phosphorylate MBP_ENG_KNK_PKA 85 µL of MBP_ENG_KNK_PKA (at approximately 50 µM concentration) was first mixed with 10 µl of 10x NEBuffer and 2µl of 10 mM ATP. This mixture was incubated while shaking at 30°C for 30 minutes. Afterwards, 2 µL of PKA (2500 units/µl) was added and the sample was incubated while shaking at 30°C for 2 hours. The amino acid sequence of the target protein analyte is set forth in SEQ ID NO.155. [1533] The target protein of interest (MBP_ENG_KNK_PKA) was provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1534] In separate experiments, the control unmodified MBP_ENG_KNK_PKA target protein of interest and phosphorylated MBP_ENG_KNK_PKA target protein post-translationally modified by treatment with a kinase were analysed in the nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1535] After acquisition, multiple reads were extracted as described herein from the given experiment. All reads were aligned to reference squiggles generated using the sequence of the unmodified protein (as described herein). After alignment the analysis outputs an adjusted reference consensus squiggle that represents the consensus alignment of all reads. The separate consensus squiggles obtained from the unmodified and modified proteins were then overlaid to compare and check for any differences. When comparing unmodified and enzymatically phosphorylated MBP_ENG_KNK_PKA proteins, the individual and the consensus signals overlap closely through the majority of the signal (FIGs. 127A-127B), with the exception of differences in their ionic current signature at the two locations where the multiple phosphorylation PTMs were introduced. Furthermore, the differences at the two sites exactly matched the deviations in the signals detected from single phosphorylations. This example demonstrates the ability of the methods and systems of the invention to -437- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 accurately detect a plurality of PTMs and modifications along single-molecules of peptides/polypeptides/proteins. Post-translational Modifications: Detection of Variants and Sub-population Quantitation [1536] This example demonstrates the ability of the nanopore system as herein disclosed to accurately measure and quantitate differences in proteins from a protein mixture. [1537] In this example the protein MBP-1 (prepared in the same way as described in previous examples herein) was subjected to a stress that introduces deamidation PTMs. Briefly, MBP-1 was incubated at 40°C and pH 9 for different amounts of time (8, 16, 24 and 72 hours). Table 33. Amino acid sequence of target protein analyte. Protein Amino acid sequence SEQ ID NO. MBP- MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK 163 1_deamid FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAY ated PIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGG YAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLT DEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTA VINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDEN YALAA [1538] The target protein of interest (MBP-1) was provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1539] In separate experiments, the control unmodified MBP-1 target protein of interest and modified MBP- 1 proteins (samples extracted at different time points during the pH stress incubations) were analysed in the nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1540] After acquisition, multiple reads were extracted as described herein from the given experiment. All reads were aligned to reference squiggles generated using the sequence of the unmodified protein (as described herein). After alignment, the analysis outputs an adjusted reference consensus squiggle that represents the consensus alignment of all reads. The separate consensus squiggles obtained from the unmodified and modified -438- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 proteins were then overlaid to compare and check for any differences. When comparing unmodified and degraded MBP-1 proteins comprising a deamidation site, the individual and the consensus signals overlap closely through the majority of the signal, with the exception of differences in their ionic current signature at the location of the deamidation PTM (FIGs.128A-128C). FIGs.128A-128B shows a portion of the reads for all reads aligned to each other in index space against the reference squiggle. In this region FIGs.128A-128C shows that all the reads have the same pattern of current changes and align atop each other very closely. In FIG.128B (16 hour pH incubation point) a subset of the reads show a clear deviation in signal as a result the pH induced deamidation degradation. The deamidation can be classified by various means with almost 100% accuracy at the single-molecule level by measuring the current changes deviation vs. thresholds or by alignment. Furthermore, counting the number of reads that match the deamidated signal vs. the control signal enables quantitation of the percentage of molecules that are modified. FIG. 128C plots quantitation of the deamidated sub-population over time. This example demonstrates the ability to detect and quantify a small percentage of modified/different proteins in a mixture (with the ability to detect the sub-population at proportions of less than 1% in the same is achievable with high read count). The example also demonstrates the ability of the methods and systems of the invention to track changes in a sample over time. Example 25. Bi-directional reading of a protein: C-to-N and N-to-C translocation of target protein through nanopore. [1541] This example demonstrates the ability of a translocase to control the translocation of a target protein substrate through the nanopores both in the C-to-N direction and in the N-to-C direction relative to the target protein sequence. [1542] An MBP-1 substrate was provided with a long >30 amino acid tag at the C-termini that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation content to enable efficient capture into the nanopore and a domain to stall the ClpX translocase. [1543] The MBP-1 target protein was added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-90D nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein. [1544] FIGs. 129A-129B show an example of a C-to-N read (FIG. 129A) and an exemplary N-to-C read (FIG. 129B) obtained from ClpX processing of MBP-1 protein substrates through a nanopore. C-to-N reads are the result of ClpX loading onto the C-termini of the target protein, and passing the protein substrate through the nanopore C-to-N cis-to-trans when captured from the cis side of the nanopore. Conversely, N-to-C reads are the result of ClpX loading onto the N-termini of the target protein, and passing the protein substrate through the nanopore N-to-C cis-to-trans when captured from the cis side of the nanopore. -439- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1545] This example demonstrates the translocase controlled nanopore system of the invention is able to effectively capture and control the translocation of substrates through the nanopore either orientated C-N relative to the pore or N-C relative to the pore. The protein translocase can still control the movement of the protein under the EOF, resulting in a high quality ionic current signal (in terms of current range, noise, movement quality) from the read that is approximately reversed to the normal C-to-N direction (arrows marking key features in the reads are added to aid the eye). [1546] This example also demonstrates that measurement of both N-C and C-N signals can be obtained in the same system. The two (non-identical) signal orientations can then be combined informatically during analysis to derive further information from the molecules. For example, the signals can be aligned and combined to use better quality in signal in once part to compensate for poorer quality signal in the same region of the reversed molecule. A further advantage of having both types of read orientations is that information near the termini are different. For example, some information near the termini can be lost or otherwise compromised during the initial capture step or during impacted motor control when the motor is near a termini. By having both orientations in the sample the loss of information at one termini during translocation in one orientation can be recovered by gain of information during translocation at the second orientation. [1547] Further, since N-to-C reads obtained from this MBP-1 substrate were obtained from the translocase loading onto the N-termini of the substrate (where the N-termini does not comprise a specific recognition tag for ClpX binding) and controlling the translocation of the molecule into the nanopore in the cis to trans direction after capture in the nanopore, this example demonstrates the important ability to capture and translocate untagged native target proteins of interest. Example 26. Alternative leader designs. [1548] In this example, the presence of a stall motif (stalling domain) was tested to determine its effect on the capture and/or translocation of a target protein across the nanopore. To that end, a variant MBP target protein was used comprising a leader construct lacking the stalling region (poly-glycine) region to result in MBP-3 (Table 34) which corresponds to MBP-1 (see Table 24) but without the poly-glycine region. Table 34. Amino acid sequence of the target protein used in this example. Protein Amino acid sequence SEQ ID NO. MBP-3 MHHHHHHSSPWGAPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKF 164 (MBP-1 PQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPI without AVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAF poly- KYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPW glycine AWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLE region) AVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAAS GRQTVDEALKDAQTRITKHMGSRRRRRRRRRRRRRRRAANDENYALAA -440- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1549] The MBP-3 substrates was tested in the nanopore system as described. The adapted target protein was added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single CytK-4D2E nanopore in a membrane under –80 mV applied potential. The ionic current measurements are then performed using the standard methods described herein. [1550] FIG.131 shows a representative section of electrophysiology signal from measurements of the MBP- 3 substrate with shorter C-terminal tags. Measurements obtained with a CytK-4D2E (prepared as described previously herein) nanopore in 1 M potassium glutamate, 50 mM Tris, 25 mM MgCl2, 10 mM DTT and 1 mM EDTA, buffered to pH 7.5. The applied voltage is -80 mV. The cis compartment contained a final concentration of 0.2 µM ClpX, 0.1 µM MBP-3 and 2.5 mM ATP. [1551] The MBP-3 variant without poly-glycine region could still be captured into the pore (FIG. 132) and translocated through the nanopore under the control of the ClpX motor protein. Example 27. Other proteins. [1552] This section demonstrates translocations of a range of different protein substrates through nanopore systems of the present disclosure. Many proteins from organisms other than bacteria (e.g., from eukaryotes) can be more complex or have other structural or other properties. This example demonstrates the ability to measure a wide range of proteins. [1553] Six proteins of Eukaryotic origin (Alpha-synuclein, p53, TauF, Nanbody-1, Antibody light chain 1, Titin I27) were prepared by recombinant expression as described herein for MBP-1. The target proteins of interest were provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1554] In separate experiments, the target proteins of interest were analysed in the single chamber nanopore system. The (adapted) target proteins were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1555] FIGs.133A-133F shows representative examples of electrophysiology reads obtained for the proteins in this example. This demonstrated the ability to translocate and measure characteristic signals for a variety of eukaryotic proteins, including proteins with disulphides (e.g., antibodies) and proteins with highly stable and difficult to unfold structure (e.g., titin). Table 43. Amino acid sequences of analytes used in Example 27. -441- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Protein Sequence SEQ ID NO. aSyn- MHHHHHHSSGDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVA 173 MBP1 TVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILED MPVDPDNEAYEMPSEEGYQDYEPEAGGSGGGSLRSKIEEGKLVIWINGDKGYNGLAEVGKKF EKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKL YPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNL QEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIA EAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMS AFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALAA p53- MHHHHHHSSGEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQW 174 MBP1 FTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLH SGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCP HHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCN SSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPP GSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSR AHSSHLKSKKGQSTSRHKKLMFKTEGPDSDGSLRSKIEEGKLVIWINGDKGYNGLAEVGKKF EKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKL YPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNL QEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIA EAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMS AFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRR RAANDENYALAA TauF- MHHHHHHSSGAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTE 175 MBP1 DGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLE DEAAGHVTQARMVSKSKDGTGSDDKKAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPP APKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSA KSRLQTAPVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHVP GGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHV PGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQL ATLADEVSASLAKQGLGSLRSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPD KLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADG GYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTIN GPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEG LEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAA SGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA VHH01- MHHHHHHSSGMADVQLVESGGGLVQAGDSLRLSCAASGLTFSRYAMGWFRQAPGNEREFVAV 176 MBP1 ITASGRTTLYADSVKGRFTISRDNAKNTVALQMQSLKPEDTAVYYCAADYGTSRYTRRQSEY EYWGQGTQVTVSSEPKTPKPQPKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHP DKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGK LIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAAD GGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTI NGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDE GLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINA ASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA -442- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Titin_I27 MHHHHHHSSGSSLIEVEKPLYGVEPFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEII 177 {V13P}- EDGKKHILILHNCQLGMTGEVSFQAANAKSAANLKVKELVKIEEGKLVIWINGDKGYNGLAE MBP1 VGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKA FQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSA LMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADT DYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAAS PNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPN IPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRR RRRRRRAANDENYALAA Example 28. Long proteins. [1556] Three protein substrates, DNAK_ECOLI-MBP1, CH60_ECOLI-MBP1, and ODP2_ECOLI-MBP with masses greater than 100KDa and polypeptide lengths of about 1000 amino acids or greater were prepared as described in the Example 7 herein and measured in high EOF MspA nanopores using the C-terminus to N- terminus ClpX translocation methods described herein. FIGs. 134A-134C show representative examples of the ionic current vs. time signals obtained from the electrophysiology measurements of the translocation of these protein substrates. It was determined by length analysis and alignment of the translocation reads to a reference trained from these proteins (as described herein above in the methods section) that about greater than 80% of the observed translocation events were full-length complete molecules, demonstrating the ability of such a system and method to routinely identify very long polypeptides or proteins that are greater than 1000 amino acids in length and greater than 100KDa. [1557] These substrates were also formed by concatenating two proteins together (MBP with DNAK or CH60 or ODP2), demonstrating the ability of these methods and systems for measuring long polypeptides that were prepared in such a way (many means of concatenating polypeptides are known in the art) as to concatenate many smaller peptide/polypeptide/protein together into a longer molecule. In this manner the long read length capabilities of the system enable more efficient analysis of certain substrates by concatenating them together into a longer single molecule. Table 35. Amino acid sequences of proteins used in Example 28. Protein Amino acid sequence SEQ ID NO. DNAK_ MAWSHPWEFKGSSAWSHPQFEKGSSGKIIGIDLGTTNSCVAIMDGTTPRVLENAEGDRTTPSI 165 ECOLI- IAYTQDGETLVGQPAKRQAVTNPQNTLFAIKRLIGRRFQDEEVQRDVSIMPFKIIAADNGDAW MBP1 VEVKGQKMAPPQISAEVLKKMKKTAEDYLGEPVTEAVITVPAYFNDAQRQATKDAGRIAGLEV KRIINEPTAAALAYGLDKGTGNRTIAVYDLGGGTFDISIIEIDEVDGEKTFEVLATNGDTHLG GEDFDSRLINYLVEEFKKDQGIDLRNDPLAMQRLKEAAEKAKIELSSAQQTDVNLPYITADAT GPKHMNIKVTRAKLESLVEDLVNRSIEPLKVALQDAGLSVSDIDDVILVGGQTRMPMVQKKVA EFFGKEPRKDVNPDEAVAIGAAVQGGVLTGDVKDVLLLDVTPLSLGIETMGGVMTTLIAKNTT IPTKHSQVFSTAEDNQSAVTIHVLQGERKRAADNKSLGQFNLDGINPAPRGMPQIEVTFDIDA DGILHVSAKDKNSGKEQKITIKASSGLNEDEIQKMVRDAEANAEADRKFEELVQTRNQGDHLL HSTRKQVEEAGDKLPADDKTAIESALTALETALKGEDKAAIEAKMQELAQVSQKLMEIAQQQH AQQQTAGADASANNAKDDDVVDAEFEEVKDKKGSLRPSKIEEGKLVIWINGDKGYNGLAEVGK -443- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 KFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDK LYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNL QEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAE AAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAK EFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW YAVRTAVINAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAAN DENYALAA CH60_E MAWSHPWEFKGSSAWSHPQFEKGSSAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVV 166 COLI- LDKSFGAPTITKDGVSVAREIELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQAIITE MBP1 GLKAVAAGMNPMDLKRGIDKAVTAAVEELKALSVPCSDSKAIAQVGTISANSDETVGKLIAEA MDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGYLSPYFINKPETGAVELESPFILLADKKIS NIREMLPVLEAVAKAGKPLLIIAEDVEGEALATLVVNTMRGIVKVAAVKAPGFGDRRKAMLQD IATLTGGTVISEEIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIE EATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATRAAVEEGVVAGGGV ALIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQIVLNCGEEPSVVANTVKGGDGNYGYNA ATEEYGNMIDMGILDPTKVTRSALQYAASVAGLMITTECMVTDLPKNDAADLGAAGGMGGMGG MGGMMGSLRPSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSL IYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDI KDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVN YGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTR ITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA ODP2_E MAWSHPWEFKGSSAWSHPQFEKGSSAIEIKVPDIGADEVEITEILVKVGDKVEAEQSLITVEG 167 COLI- DKASMEVPSPQAGIVKEIKVSVGDKTQTGALIMIFDSADGAADAAPAQAEEKKEAAPAAAPAA MBP1 AAAKDVNVPDIGSDEVEVTEILVKVGDKVEAEQSLITVEGDKASMEVPAPFAGTVKEIKVNVG DKVSTGSLIMVFEVAGEAGAAAPAAKQEAAPAAAPAPAAGVKEVNVPDIGGDEVEVTEVMVKV GDKVAAEQSLITVEGDKASMEVPAPFAGVVKELKVNVGDKVKTGSLIMIFEVEGAAPAAAPAK QEAAAPAPAAKAEAPAAAPAAKAEGKSEFAENDAYVHATPLIRRLAREFGVNLAKVKGTGRKG RILREDVQAYVKEAIKRAEAAPAATGGGIPGMLPWPKVDFSKFGEIEEVELGRIQKISGANLS RNWVMIPHVTHFDKTDITELEAFRKQQNEEAAKRKLDVKITPVVFIMKAVAAALEQMPRFNSS LSEDGQRLTLKKYINIGVAVDTPNGLVVPVFKDVNKKGIIELSRELMTISKKARDGKLTAGEM QGGCFTISSIGGLGTTHFAPIVNAPEVAILGVSKSAMEPVWNGKEFVPRLMLPISLSFDHRVI DGADGARFITIINNTLSDIRRLVMGSLRPSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGI KVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDA VRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLL TDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVI NAASGRQTVDEALKDAQTRITKHMGGGGGGGGGGGGSRRRRRRRRRRRRRRRAANDENYALAA Example 29. Measurement of the kinetics of translocation. [1558] This example demonstrates detection of the kinetics of translocation, which can be used to inform on the properties of the translocating polypeptide. For example, changes in the kinetics of translocation can provide information on the secondary/tertiary/quaternary structure of the domains of the polypeptide, amino acid composition of the polypeptide, molecular entities bound to the polypeptide (e.g., small molecules, drugs, co-factors, peptides, proteins, nanoparticles, etc.), internal cross-links (e.g., cysteine-cysteine disulphide), and -444- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 conjugations (e.g., cysteine-cysteine disulphide crosslinks to other molecules or proteins, conjugate drugs, large PTMs such as ubiquitin or sumo, etc). [1559] The target protein of interest CH60_ECOLI (Table 35) was prepared by recombinant expression as described herein for MBP-1. The target protein of interest was provided with a long >30 amino acid tag that contains an AANDENYALAA capture motif to facilitate binding with a ClpX translocase, a domain with high cation (polyR) content to enable efficient capture into the nanopore and a domain (polyG) to stall the ClpX translocase. When added to the nanopore system the target protein are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1560] The target proteins of interest was analysed in the single chamber nanopore system. The (adapted) target protein were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1561] FIGs. 136A-136D illustrate some of the changes in the kinetics/speed of translocation that can be observed when substrates such as CH60_ECOLI translocate through nanopores of the invention. For example, FIG. 136A shows schematic examples of extended segments or pauses in the signal (marked with arrows) during translocation, after which the protein translocation proceeds at a similar speed as that prior to the translocation. FIG.136B illustrates and example a permanent pause/stall that terminates the translocation of the polypeptide (requiring the polypeptide to be ejected by a reversal of voltage to continue measurements on new molecules). FIG. 136C shows representative translocation reads for electrophysiology measurements of CH60_ECOLI. Similar pauses and other changes are kinetics are observed in many other proteins in the example described herein. The pauses or changes in speed are stochastic, and do not occur to the same extent in every molecule (either due to underlying probabilistic changes in speed through polypeptides that share the same properties, or due to an underlying difference in property between polypeptides (e.g., a protein domain that is either in a folded or unfolded state when processed by a motor protein and/or translocating through a nanopore). This stochastic difference molecule to molecule (or sample to sample) is illustrated by FIG.136C for CH60_ECOLI translocations extracted from a single experiment on the same sample, where read (i) shows no small or no reduced speed in the region of interest, while events (ii-iv) show clear pauses. [1562] For simplicity, a single pause is shown in FIG.136A-136B, but multiple pauses can occur during the translocation, relating to characteristics of the different portions of the polypeptide as it translocates for example. For simplicity of illustration, the pauses are also shown to be long relative to surrounding segments as is often observed, but pauses or changes in kinetics are relative to the average speed of translocation, and may only be marginally slower or marginally faster than the average for the rest of the polypeptide. -445- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 Furthermore, more subtle changes in kinetics cannot easily be detected in a single translocation due to the stochastic distribution of dwell times associated with each segment. However, sequence specific differences in translocation kinetics/speed versus the average can be determined by averaging the dwell time of the each segment over many translocation reads. For example, the alignment methods described herein can be employed to align multiple translocation reads to each other or to a reference squiggle, and then the dwell time meta data associated for specific segments that align to each other can be aggregated and analyzed together to better determine the average kinetics on a per segment basis. [1563] This example demonstrates the ability to measure characteristic changes in the speed or kinetics of translocation for nanopores of the invention that are related to differences in the structure of the protein or unknown crosslinks (e.g., internally or to another protein) since there are no known internal cysteine-cysteine crosslinks for this protein. The pauses and stalls in the in the CH60 measurements were counted on a per read basis, counting the number of stalls per molecule to inform if there were multiple regions containing a stall, it was determined that 100% of the molecule reads only showed one stall per read, indicating that a single region in the protein is affecting the translocation kinetics. The location of the stall along the read was determined to occur at approximately the same location in about 90% of all reads having a stall, indicating that the majority of the stalls are the result of the same feature in the protein affecting the translocation kinetics, for example a strongly folded protein domain. The proportion of reads was determined that contained a stall or pause to be 85%, (81% were permanent stalls that terminated translocation, 4% were pauses that resumed translocation after a period of time) and only 15% of events were complete or otherwise lacking detectable pauses or stalls, which can be used to determine the folding energy and/or the denaturation forces required to unfold the domain that gives rise to the stall. To test that the stalls arise from a strongly folded domain (and not a cysteine-cysteine crosslink for example), in a separate experiment a sample of CH60 proteins were subjected to a treatment with urea (the protein was pre-incubated in 8M Urea, 10 mM DTT at 37oC for 15 minutes before being diluted and added to an Axopatch electrophysiology system with ClpX and measured as described herein) before running electrophysiology measurements as described. In the urea treated sample it was observed that only 43% of the reads contained a stall or pause (27% were permanent stalls that terminated translocation, 16% were pauses that resumed translocation after a period of time) increasing the number of reads that were complete or otherwise lacking detectable pauses or stalls to 57%. This confirmed that a strongly folded protein domain was the most likely cause of the pausing and stalls, which can be quantified versus conditions (e.g. pretreatments, experimental temperature, etc) to determine the energetics of the molecular structure causing the stall. [1564] To demonstrate the effect of cross-links on kinetics an antibody target protein of interest was prepared. [1565] The antibody target protein of interest was analyzed in the single chamber nanopore system. Prior to measurement the protein was treated with DTT (10 mM for 1 hour, with or without heating to 50oC) to reduce -446- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 cysteine-cysteine disulfide crosslinks. The (adapted) target protein were added to the cis compartment in a final concentration of 0.1 µM together with 0.2 µM ClpX and 2.5 mM ATP in a system containing a single MspA-D90N nanopore in a membrane under –80 mV applied potential. The ionic current measurements were performed using the standard methods described herein above. [1566] FIG.136D shows representative examples of the two main types of reads with different translocation kinetics, where the presence of the cysteine-cysteine cross-link permanently stalls the progression of the translocation where expected. In this experiment, it was observed that approximately 20% of events are classified as complete FIG.136D (i) reads, and 80% as stalled FIG.136D (ii) reads. Separate experiments on the same Antibody protein that were not treated with the disulfide reducing steps showed 100% of reads were stalled type FIG.136D (ii) events (data not shown). This demonstrates the ability of the system of the invention to detection of cross-links. Example 30. Alternative capture methods. [1567] The methods and systems of the invention teach that A) motor proteins are able to control the movement of polypeptides (with or against the direction of the net EOF), and B) that high net EOF can be used to pull polypeptides into nanopores either to 1) initiate capture the polypeptide, and/or 2) keep the translocase bound to the polypeptide against the nanopore entrance and the polypeptide stretched within the nanopore. [1568] These following examples describe further variations of how the components of the system can be arranged to enable steps of: 1) capturing a polypeptides in a nanopore, 2) binding a motor protein to control the direction of the translocation, 3) set up a high net EOF to retain the translocase against the nanopore during translocation and to stretch the polypeptide in the nanopore. Without wishing to be bound by theory, cis and trans nomenclature as described in this example can be reversed and all descriptions here would be reversed in turn. Trans capture [1569] The target proteins of interest MBP-AMOV138A, MBP-AMOV138A, MBP-AMOV138B, MBP- AMOV140, MBP-AMOV142A MBP-AMOV142B MBP-AMOV144 were designed according to the sequences below in Table 44. MBP-AMOV138A was provided with a sequence RRRRRRRRRRRRRRRAANDENYALAA (SEQ ID NO.: 184) on the C-termini to promote capture in the nanopore and binding of the translocase, and a GFP stopper domain (underlined in Table 44) to prevent full translocation of the substrate during the initial nanopore capture step. MBP-AMOV138B has a similar design to MBP-AMOV138A, except that the stopper is replaced with an electrostatic stopper (underlined in Table 44). MBP-AMOV140 was provided with a sequence RRRRRRRRRRRRRRRASSSSSSSSSSSC on the C- termini. The purified protein was reacted with Maleimide-Biotin (Sigma Aldrich) to add a biotin to the final cysteine of the C-termini of the protein. MBP-AMOV142A was provided with a sequence -447- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 SGTYSSGGSGSAGSAGSASSGSDGSGASGAGSGSAGSKGSGASGSAGSGSSGSDDDDDDDDDDDD DDDAANDENYALAA (SEQ ID NO.: 185) on the C-termini to promote capture in the nanopore (from trans via electrophoresis against the EOF) and binding of the translocase. The leader also contains a GFP stopper (underlined in Table 44) to prevent full translocation of the substrate during the initial nanopore capture step. MBP-AMOV142B has a similar design to MBP-AMOV142A, except that the stopper is replaced with an electrostatic stopper (underlined in Table 44). MBP-AMOV144 was provided with a sequence SGSGSGRSRSRSAANDENYALAA (SEQ ID NO.: 186) on the C-termini to promote binding of the translocase. All proteins were prepared recombinantly from E.coli according to the same methods herein for MBP-1. Table 44. Amino acid sequences of target proteins. Protein Amino acid sequence SEQ ID NO. MBP- MHHHHHHSSASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP 178 AMOV1 VPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGD 38A TLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAD HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGISGSGSGSGPWKIEE GKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRF GGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWE EIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLT FLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSK PFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAA TMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITKHMSRRRRRRRRR RRRRRRAANDENYALAA MBP- MHHHHHHSSDDDDDDDDDDSDDDDDDDDDDSGSGSGSGPWKIEEGKLVIWINGDKGYNGLAEV 179 AMOV1 GKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQ 38B DKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMF NLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSI AEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSA FWYAVRTAVINAASGRQTVDEALKDAQTRITKHMSRRRRRRRRRRRRRRRAANDENYALAA MBP- MHHHHHHSSSGSGSGSGPWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE 180 AMOV1 EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYP 40 IAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFK YENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWS NIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKD KPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDE ALKDAQTRITKHMSRRRRRRRRRRRRRRRASSSSSSSSSSSC MBP- MHHHHHHSSPWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA 181 AMOV1 TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSL 42A IYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDI KDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVN YGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTR ITKHASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPT LVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN -448- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGISGTYSSGGSGSAGSAGSAS SGSDGSGASGAGSGSAGSKGSGASGSAGSGSSGSDDDDDDDDDDDDDDDAANDENYALAA MBP- MHHHHHHSSPWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA 182 AMOV1 TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSL 42B IYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDI KDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVN YGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTR ITKHRRRRRRRRRRRRRRRRSGTYSSGGSGSAGSAGSASSGSDGSGASGAGSGSAGSKGSGAS GSAGSGSSGSDDDDDDDDDDDDDDDAANDENYALAA MBP- MHHHHHHSSPWKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAA 183 AMOV1 TGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSL 44 IYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDI KDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVN YGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVAL KSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTR ITKHMSGSGSGRSRSRSAANDENYALAA [1570] When added to the nanopore system the target proteins are captured into the nanopore by a high EOF, in conjunction with a protein translocase motor that unfolds and transports the polypeptide through the MspA nanopore. [1571] In separate experiments, 0.1 µM of the MBP-AMOV138A and MBP-AMOV138B protein substrates was added the trans compartment of an electrophysiology system as described herein (and illustrated in FIGs. 138A-138E) containing a single CytK-4D nanopore inserted from the cis.0.2 µM ClpX and 2.5 mM ATP were added the cis compartment. Capture of the Protein substrate into the nanopore from the trans was promoted at +80 to +120 mV until a capture event was detected by the instantaneous change in current (state ii in FIG. 139). After holding the voltage for a further 3 seconds, the voltage was reversed to -80 mV. If a translocase had bound to the protein then the polypeptide was retained in the nanopore and proceeded to translocate through the nanopore trans-to-cis (state v in FIG. 139) (otherwise the protein was ejected back to the trans). When the translocase reaches the end of the strand (unfolding and/or pulling the stopper domain through the nanopore) the protein leaves the pore and the process is repeated by reversing the voltage back to +80 to +120 mV to start capture of the next protein. [1572] In another experiment, 0.1 µM MBP-AMOV140 protein substrate and 0.1 µM streptavidin (Sigma Aldrich) was added the trans compartment of an electrophysiology system as described herein (and illustrated in FIGs.140A-140E) containing a single CytK-4D nanopore inserted from the cis.0.2 µM ClpX and 2.5 mM ATP were added the cis compartment. Capture of the Protein substrate into the nanopore from the trans was promoted at +80 to +120 mV until a capture event was detected by the instantaneous change in current (state ii in FIG. 141). After holding the voltage for a further 30 seconds, the voltage was reversed to -80 mV. If translocases had bound to the protein then the polypeptide was retained in the nanopore and proceeded to -449- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 translocate through the nanopore cis-to-trans (state v in FIG. 141) (otherwise the protein was ejected back to the trans). When the translocase reaches the end of the protein the protein fully translocates the trans, and the process is repeated by reversing the voltage back to +80 to +120 mV to start capture of the next protein. [1573] In separate experiments, 0.1 µM of the MBP-AMOV142A and MBP-AMOV142B protein substrates was added the trans compartment of an electrophysiology system as described herein (and illustrated in FIGs. 142A-142E) containing a single CytK-4D nanopore inserted from the cis.0.2 µM ClpX and 2.5 mM ATP were added the cis compartment. Capture of the Protein substrate into the nanopore from the trans was promoted at -80 mV until a capture event was detected by the instantaneous change in current (state ii in FIG. 143). The system is able to capture the substrate under electrophoresis against the EOF due to the very high charge content of the leader (increased charge content can improve capture rates further). At the constant voltage of - 80 mV the signal is observed to pause temporarily, then begin a pattern of current changes indicative of a translocase binding to the portion of the leader in the cis and beginning to translocate along the polypeptide (trans-to-cis against the cis-to-trans EOF), in the process pulling the stopper through the nanopore followed by the rest of the protein, until the protein finally exits to cis (FIG.142E). [1574] In another experiment, 0.1 µM MBP-AMOV144 protein substrate, 0.2 µM ClpX and 2.5 mM ATP were added the cis compartment of an electrophysiology system as described herein (and illustrated in FIGs. 144A-144D) containing a single CytK-4D nanopore inserted from the cis. Capture of the Protein substrate into the nanopore from the cis was promoted at -80 mV until a capture event was detected by the instantaneous change in current (state ii in FIG. 145), which was immediately following by a pattern of changes in current levels (state iii in FIG.145) indicative of motor protein controlled translocation cis-to-trans, until the protein eventually fully translocates to the trans. Example 31. Array measurements. [1575] This example shows the use of a chip comprising an array of membranes and nanopores for measurement of peptides/polypeptides/proteins of the methods and systems of the present disclosure. [1576] MBP-1 target proteins of interest were prepared recombinantly as described herein. MspA_D90N nanopores were prepared as described herein. Separately Meca-16 chips (Nanion Technologies, GmbH) were prepared for measurement on the Orbit 16 TC instrument (Nanion Technologies, GmbH) according to the manufacturer instructions by painting a DPhPC membrane (using a 10 mg/ml solution of DPhPC in hexane) across the array to form membranes across the 16 trans-compartments thereon (buffer = 1 M potassium glutamate, 20 mM MgCl2 and 50 mM Tris, buffered to pH 7.5, temperature = 37oC). After determining that greater than five membranes were successfully formed by their characteristic capacitance values, MspA nanopores were added to the cis volume to insert single nanopores into the membranes. After obtaining more than 3 single nanopores 0.1 µM MBP-1 protein substrate, 0.2 µM ClpX and 2.5 mM ATP were added the cis -450- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 compartment of an electrophysiology system as described herein. Using the manufacturer’s Elements Data Reader (EDR) software, ClpX controlled protein translocations were recorded at -80mV. FIG. 146 shows representative data from experiments on the Nanion, showing 3 selected translocation signal reads for 3 of the active channels comprising good membrane seals and single MspA nanopores. Analysis of the signal reads by comparison to reads obtained from the Axopatch system described herein (and to predicted reference squiggles trained therefrom) showed that the reads from the Nanion array system (and the different nanopores and membranes thereon) are largely identical to reads from multiple nanopores from multiple experiments on the Axopatch. With the exception of some minor changes in noise profile of the raw signal (due to differences in the membrane, chip and the electronics), the current changes were highly correlated, and reads from the multiple nanopores and systems were mixed in analyses herein. EMBODIMENTS [1577] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention. [1578] Embodiment 1. A method, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; (2) a voltage or change thereof; or (3) a resistance or change thereof; or (4) any combination thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, (2) the voltage or change thereof, or (3) the resistance or change thereof, or (4) any combination thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. [1579] Embodiment 2. The method of embodiment 1, wherein (c) comprises using (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof to determine the characteristic of the at least the portion of the analyte with an accuracy of at least 70%. [1580] Embodiment 3. The method of embodiment 1 or 2, wherein (c) comprises using (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof to determine the characteristic of the at least the portion of the analyte with an accuracy of at least 80%. [1581] Embodiment 4. The method of any one of embodiments 1-3, wherein (c) comprises using (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof to determine the characteristic of the at least the portion of the analyte with an accuracy of at least 90%. -451- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1582] Embodiment 5. The method of any one of embodiments 1-4, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof. [1583] Embodiment 6. A method for determining a characteristic of an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof; (b) detecting (1) a current or change thereof; (2) a voltage or change thereof; or (3) a resistance or change thereof; or (4) any combination thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (iv) any combination thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte. [1584] Embodiment 7. The method of embodiment 6, wherein (c) comprises using (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof to determine the one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. [1585] Embodiment 8. The method of embodiment 6 or 7, wherein the average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second. [1586] Embodiment 9. The method of any one of embodiments 6-8, wherein the average rate of translocation is between about 1 amino acids per second to about 100 amino acids per second with a motor protein. [1587] Embodiment 10. The method of any one of embodiments 6-9, wherein the average rate of translocation is between about 500 amino acids per second to about 5000 amino acids per second without a motor protein. [1588] Embodiment 11. The method of any one of embodiments 6-10, wherein the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s. [1589] Embodiment 12. The method of any one of embodiments 6-11, wherein the average rate of translocation is between about 0.3 nm/s to about 30 nm/s. [1590] Embodiment 13. The method of any one of embodiments 6-12, further comprising translocating at least a portion of an additional analyte through the nanopore disposed within the membrane. -452- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1591] Embodiment 14. The method of embodiment 13, wherein the at least the portion of the additional analyte comprises at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or a combination thereof. [1592] Embodiment 15. The method of embodiment 13 or 14, wherein (i) the average rate of translocation is between about 0.1 amino acids per second to about at least 35000 amino acids per second, (ii) the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof. [1593] Embodiment 16. The method of any one of embodiments 13-15, further comprising detecting (1) an additional current or change thereof; (2) an additional voltage or change thereof; or (3) an additional resistance or change thereof; or (iv) any combination thereof while the at least the portion of the additional analyte is translocating through the nanopore. [1594] Embodiment 17. The method of embodiment 16, further comprising using (1) the additional current or change thereof; (2) the additional voltage or change thereof; or (3) the additional resistance or change thereof; or (4) any combination thereof to identify a characteristic of the at least the portion of the additional analyte. [1595] Embodiment 18. The method of any one of embodiments 6-12, further comprising translocating at least a portion of an additional analyte through an additional nanopore disposed within an additional membrane. [1596] Embodiment 19. The method of embodiments 18, wherein the at least the portion of the additional analyte comprises at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or at least additional fragments thereof, or a combination thereof. [1597] Embodiment 20. The method of embodiments 18 or 19, wherein (i) the average rate of translocation is between about 0.1 amino acids per second to about at least 35000 amino acids per second, (ii) the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof. [1598] Embodiment 21. The method of any one of embodiments 18-20, further comprising detecting (1) the additional current or change thereof; (2) the additional voltage or change thereof; or (3) the additional resistance or change thereof; or (4) any combination thereof while the at least the portion of the additional analyte is translocating through the additional nanopore. [1599] Embodiment 22. The method of embodiments 21, further comprising using (1) the additional current or change thereof; (2) the additional voltage or change thereof; or (3) the additional resistance or change thereof; or (4) any combination thereof to identify a characteristic of the at least the portion of the additional analyte. -453- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1600] Embodiment 23. The method of any one of embodiments 1-22, further comprising in (b) detecting the current or change thereof, and (c) comprises using the current or change thereof. [1601] Embodiment 24. The method of any one of embodiments 1-23, further comprising in (b) detecting the voltage or change thereof, and (c) comprises using the voltage or change thereof. [1602] Embodiment 25. The method of any one of embodiments 1-24, further comprising, in (c), determining/assigning the one or more characteristics of the at least the portion of the analyte based on (1) an electrical signal or change thereof of (i) the current or change thereof, (ii) the voltage or change thereof, or (iii) the resistance or change thereof, or (iv) any combination thereof, and (2) a database. [1603] Embodiment 26. The method of embodiment 25, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. [1604] Embodiment 27. The method of embodiment 25 or 26, wherein the database does not comprise a reference signal associated with the at least the portion of the analyte. [1605] Embodiment 28. The method of any one of embodiments 25-26, wherein the database does comprise a reference signal associated with the at least the portion of the analyte. [1606] Embodiment 29. The method of any one of embodiments 25-28, wherein the electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the electrical signal or change thereof. [1607] Embodiment 30. The method of any one of embodiments 25-29, wherein the one or more additional portions of the electrical signal or change thereof comprises one or more blocks of impurities. [1608] Embodiment 31. The method of any one of embodiments 25-30, wherein the electrical signal or change thereof comprises measurements of (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (iv) any combination thereof over a period of time. [1609] Embodiment 32. The method of embodiment 31, wherein the period of time comprises one or more portions associated with a measurement of concentration associated with a sample comprising the at least the portion of the analyte. [1610] Embodiment 33. The method of any one of embodiments 25-32, further comprising, in (c), pre- processing (e.g., denoising (smoothing, frequency manipulations, FFT, wavelets), segmenting (edge-detecting algorithms, wavelet transforms, filtering)) the electrical signal or change thereof, thereby generating a pre- processed electrical signal or change thereof. [1611] Embodiment 34. The method of embodiment 33, wherein the one or more characteristics are determined using the pre-processed electrical signal or change thereof. -454- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1612] Embodiment 35. The method of embodiment 33 or 34, further comprising extracting one or more reads from (1) the electrical signal or change thereof and/or (2) the pre-processed electrical signal or change thereof. [1613] Embodiment 36. The method of embodiment 35, further comprising pre-processing the one or more reads, thereby generating one or more pre-processed reads. [1614] Embodiment 37. The method of embodiment 36, wherein the pre-processing comprises denoising, filtering, segmenting, or scaling, or a combination thereof. [1615] Embodiment 38. The method of any one of embodiments 35-37, wherein the one or more characteristics are determined using (1) the one or more reads and/or (2) the one or more pre-processed reads (e.g., one or more segment). [1616] Embodiment 39. The method of embodiment 38, further comprising comparing (e.g., aligning) (1) the one or more reads and/or (2) the one or more pre-processed reads to one or more reference signals in the database. [1617] Embodiment 40. The method of embodiment 39, wherein the comparing comprises alignment (e.g., soft alignment). [1618] Embodiment 41. The method of embodiment 40, wherein the alignment comprises time warping. [1619] Embodiment 42. The method of any one of embodiments 38-41, further comprising scoring (1) the one or more reads and/or (2) the one or more pre-processed reads to the one or more reference signals, thereby determining the one or more characteristics of the at least the portion of the analyte. [1620] Embodiment 43. The method of embodiment 42, wherein scoring comprises aligning the at least the portion of the electrical signal or change thereof with the at least the portion of the one or more reference signals. [1621] Embodiment 44. The method of any one of embodiments 38-43, further comprising aggregating (1) the one or more reads or (2) the one or more pre-processed reads to determine the one or more characteristics of the at least the portion of the analyte. [1622] Embodiment 45. The method of any one of embodiments 25-44, wherein the one or more characteristics are determined using the electrical signal or change thereof. [1623] Embodiment 46. The method of any one of embodiments 25.45, wherein the database is generated from one or more reference sequences. [1624] Embodiment 47. The method of embodiment 46, wherein the one or more reference sequences are derived with genomic information and/or transcriptomic information of the sample. [1625] Embodiment 48. The method of embodiment 47, wherein the genomic information comprises genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of -455- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. [1626] Embodiment 49. The method of embodiment 47 or 48, wherein the transcriptomic information comprises genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. [1627] Embodiment 50. The method of any one of embodiments 25-49, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms. [1628] Embodiment 51. The method of any one of embodiments 25-50, wherein the database comprises one or more reference signals for the at least the portion of the analyte, or fragments thereof, or at least one proteoform thereof, or at least one variant thereof, or combination thereof. [1629] Embodiment 52. The method of any one of embodiments 26-51, wherein the one or more polypeptides comprise one or more expressible polypeptides. [1630] Embodiment 53. The method of any one of embodiments 26-52, wherein the one or more variants thereof comprise one or more post-translationally modified variants thereof. [1631] Embodiment 54. A method for characterizing an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. [1632] Embodiment 55. The method of embodiment 54, further comprising assigning the one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. [1633] Embodiment 56. The method of embodiment 54 or 55, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, (ii) the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof. [1634] Embodiment 57. The method of any one of embodiments 54-56, wherein the database does not comprise a reference signal associated with the at least the portion of the analyte. -456- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1635] Embodiment 58. The method of any one of embodiments 54-57, wherein the database comprises a reference signal associated with the at least the portion of the analyte. [1636] Embodiment 59. The method of any one of embodiments 54-58, wherein the electrical signal or change thereof may be a measurement of (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof. [1637] Embodiment 60. The method of any one of embodiments 54-59, wherein the electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the electrical signal or change thereof. [1638] Embodiment 61. The method of embodiment 60, wherein the one or more additional portions of the electrical signal or change thereof comprises one or more blocks of impurities. [1639] Embodiment 62. The method of any one of embodiments 54-58, wherein the electrical signal or change thereof comprises measurements of (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof over a period of time. [1640] Embodiment 63. The method of embodiment 62, wherein the period of time comprises one or more portions associated with a measurement of concentration associated with a sample comprising the at least the portion of the analyte. [1641] Embodiment 64. The method of any one of embodiments 54-63, further comprising, prior to (c), pre- processing (e.g., denoising, segmenting) the electrical signal or change thereof, thereby generating a pre- processed electrical signal or change thereof. [1642] Embodiment 65. The method of embodiment 64, wherein the one or more characteristics are assigned using the pre-processed electrical signal or change thereof. [1643] Embodiment 66. The method of embodiment 64 or 65, further comprising extracting one or more reads from (1) the electrical signal or change thereof or (2) the pre-processed electrical signal or change thereof. [1644] Embodiment 67. The method of embodiment 66, further comprising pre-processing the one or more reads, thereby generating one or more pre-processed reads. [1645] Embodiment 68. The method of embodiment 67, wherein the pre-processing comprises denoising, filtering, segmenting, or scaling, or a combination thereof. [1646] Embodiment 69. The method of any one of embodiments 66-68, wherein the one or more characteristics are assigned using (1) the one or more reads or (2) the one or more pre-processed reads (e.g., one or more segments). [1647] Embodiment 70. The method of any one of embodiments 66-69, further comprising comparing (e.g., aligning) (1) the one or more reads or (2) the one or more pre-processed reads to one or more reference signals in the database. -457- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1648] Embodiment 71. The method of embodiment 70, wherein the comparing comprises alignment (e.g., soft alignment). [1649] Embodiment 72. The method of embodiment 71, wherein the alignment comprises time warping [1650] Embodiment 73. The method of any one of embodiments 66-72, further comprising scoring (1) the one or more reads and/or (2) the one or more pre-processed reads to the one or more reference signals, thereby assigning the one or more characteristics to the at least the portion of the analyte. [1651] Embodiment 74. The method of embodiment 73, wherein scoring comprises aligning at least a portion of the electrical signal of change thereof with the at least the portion of the one or more reference signals. [1652] Embodiment 75. The method of any one of embodiments 67-74, further comprising aggregating (1) the one or more reads and/or (2) the one or more pre-processed reads to assignment the one or more characteristics to the at least the portion of the analyte. [1653] Embodiment 76. The method of any one of embodiments 54-75, wherein the one or more characteristics are assigned using the electrical signal or change thereof. [1654] Embodiment 77. The method of any one of embodiments 54-76, wherein the database is generated from one or more reference sequences. [1655] Embodiment 78. The method of embodiment 77, wherein the one or more reference sequences are derived with genomic information and/or transcriptomic information of the sample. [1656] Embodiment 79. The method of embodiment 78, wherein the genomic information comprises genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. [1657] Embodiment 80. The method of embodiment 78 or 79, wherein the transcriptomic information comprises genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. [1658] Embodiment 81. The method of any one of embodiments 77-80, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms. [1659] Embodiment 82. The method of any one of embodiments 54-81, wherein the database comprises one or more reference signals for the at least the portion of the analyte, or fragments thereof, or at least one proteoform thereof, or at least one variant thereof, or combination thereof. [1660] Embodiment 83. The method of any one of embodiments 54-82, wherein the one or more polypeptides comprise one or more expressible polypeptides. -458- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1661] Embodiment 84. The method of any one of embodiments 54-83, wherein the one or more variants thereof comprise one or more post-translationally modified variants thereof. [1662] Embodiment 85. The method of any one of embodiments 54-84, further comprising, prior to (b), translocating at least a portion of an additional analyte through an additional nanopore disposed within an additional membrane, wherein the at least the portion of the additional analyte comprises at least a portion of an additional polypeptide. [1663] Embodiment 86. The method of embodiment 85, wherein the at least the portion of the additional analyte comprises at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or fragments thereof, or a combination thereof. [1664] Embodiment 87. The method of embodiment 85 or 86, further comprising detecting an additional electrical signal or change thereof while the at least the portion of the additional analyte is translocating through the additional nanopore. [1665] Embodiment 88. The method of embodiment 87, further comprising assigning the one or more characteristics to the at least the portion of the additional analyte based on the additional electrical signal or change thereof and the database. [1666] Embodiment 89. The method of any one of embodiments 54-84, further comprising, prior to (b), translocating at least a portion of an additional analyte through the nanopore disposed within the membrane, wherein the at least the portion of the additional analyte comprises at least a portion of an additional polypeptide. [1667] Embodiment 90. The method of embodiment 89, wherein the at least the portion of the additional analyte comprises at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or fragments thereof, or a combination thereof. [1668] Embodiment 91. The method of embodiment 89 or 90, further comprising detecting an additional electrical signal or change thereof while the at least the portion of the additional analyte is translocating through the nanopore. [1669] Embodiment 92. The method of embodiment 91, further comprising assigning the one or more characteristics to the at least the portion of the additional analyte based on the additional electrical signal or change thereof and the database. [1670] Embodiment 93. The method of any one of embodiments 54-92, further comprising, repeating (a) and (c) for a plurality of analytes, thereby generating a plurality of electrical signals. [1671] Embodiment 94. The method of embodiment 93, wherein the plurality of analytes translocates through the plurality of nanopores disposed in a plurality of membranes. -459- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1672] Embodiment 95. The method of embodiment 93 or 94, further comprising assigning the one or more characteristics to the plurality of electrical signals. [1673] Embodiment 96. The method of any one of embodiments 93-95, further comprising determining a relative concentration or absolute concentration for one or more analytes in the plurality of analytes. [1674] Embodiment 97. The method of any one of embodiments 1-96, wherein translocating can comprise translocating the at least the portion of the analytes in the C-to-N direction and/or in the N-to-C direction relative to the at least the portion of the analytes sequence. [1675] Embodiment 98. The method of embodiment 97, wherein the determining the one or more characteristics of (c) comprises using (i) (1) the current or change thereof; (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof, and/or (ii) the electrical signal or change thereof associated with the C-to-N direction and/or the N-to-C direction. [1676] Embodiment 99. The method of any one of embodiments 1-98, further comprising, prior to (a), providing a sample comprising the at least the portion of the analyte. [1677] Embodiment 100. The method of embodiment 99, further comprising determining one or more properties of the sample using the one or more characteristics. [1678] Embodiment 101. The method of any one of embodiments 1-100, further comprising, prior to (a), providing a sample comprising the at least the portion of the analyte and at least a portion of an additional analyte. [1679] Embodiment 102. The method of embodiment 101, wherein the at least the portion of the additional analyte comprises at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or fragments thereof, or a combination thereof. [1680] Embodiment 103. The method of embodiments 101 or 102, wherein the at least the portion of the analyte and the at least the portion of the additional analyte are different analytes. [1681] Embodiment 104. The method of any one of embodiments 99-103, wherein the sample comprises one type of analyte. [1682] Embodiment 105. The method of embodiment 104, wherein the at least the portion of the analyte and the at least the portion of the additional analyte are from the same analyte type. [1683] Embodiment 106. The method of any one of embodiments 1-105, further comprising determining one or more of a number of analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample. [1684] Embodiment 107. The method of any one of embodiments 99-103, wherein the sample comprises a first type of analyte and a second type of analyte. -460- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1685] Embodiment 108. The method of embodiment 107, wherein the first type of analyte and the second type of analyte are different types. [1686] Embodiment 109. The method of any one of embodiments 107 or 108, wherein the first type of analyte comprises the at least the portion of the analyte and the second type of analyte comprises the at least the portion of the additional analyte. [1687] Embodiment 110. The method of any one of embodiments 107-109, further comprising determining in the first type of analyte or the second type of analyte one or more of a number of analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or a combination thereof. [1688] Embodiment 111. The method of embodiment 110, further comprising determining a presence or absence of the at least the portion of the analyte in the sample. [1689] Embodiment 112. The method of any one of embodiments 1-111, further comprising translocating at least a portion of an additional analyte through the nanopore disposed within the membrane. [1690] Embodiment 113. The method of embodiment 112, wherein the at least the portion of the additional analyte comprises at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or fragments thereof, or a combination thereof. [1691] Embodiment 114. The method of embodiment 112 or 113, further comprising detecting (i) (1) an additional current or change thereof, (2) an additional voltage or change thereof, or (3) an additional resistance or change thereof, or (4) any combination thereof, or (ii) an additional electrical signal or change thereof while the at least the portion of the additional analyte is translocating through the nanopore. [1692] Embodiment 115. The method of any one of embodiments 112-114, further comprising using (1) the additional current or change thereof, (2) the additional voltage or change thereof, or (3) the additional resistance or change thereof, or (4) any combination thereof to identify a characteristic of the at least the portion of the additional analyte with an accuracy of at least 60%. [1693] Embodiment 116. The method of any one of embodiments 1-115, further comprising translocating at least a portion of an additional analyte through an additional nanopore disposed within an additional membrane. [1694] Embodiment 117. The method of embodiment 116, wherein the at least the portion of the additional analyte comprises at least a portion of an additional protein, at least a portion of an additional polypeptide, or at least a portion of an additional peptide, or fragments thereof, or a combination thereof. [1695] Embodiment 118. The method of embodiment 116 or 117, further comprising detecting (i) (1) an additional current or change thereof, (2) an additional voltage or change thereof, or (3) an additional resistance -461- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 or change thereof, or (4) any combination thereof or (ii) an additional electrical signal or change thereof while the at least the portion of the additional analyte is translocating through the additional nanopore. [1696] Embodiment 119. The method of embodiment 118, further comprising using (1) the additional current or change thereof, (2) the additional voltage or change thereof, or (3) the additional resistance or change thereof, or (4) any combination thereof to identify a characteristic of the at least the portion of the additional analyte with an accuracy of at least 60%. [1697] Embodiment 120. The method of any one of embodiments 116-119, wherein the at least the portion of the analyte and the at least the portion of the additional analyte are different. [1698] Embodiment 121. The method of any one of embodiments 1-120, further comprising, prior to (b), translocating a plurality of analytes through (i) the nanopore disposed within the membrane, or (ii) a plurality of nanopores disposed in a plurality of membranes. [1699] Embodiment 122. The method of embodiment 121, wherein at least a portion of each analyte of the plurality of analytes translocates through a different nanopore of the plurality of nanopores. [1700] Embodiment 123. The method of embodiment 121 or 122, wherein each nanopore of the plurality of nanopores is disposed in a different membrane of the plurality of membranes. [1701] Embodiment 124. The method of any one of embodiments 121-123, wherein the plurality of analytes comprises the at least the portion of the analyte. [1702] Embodiment 125. The method of any one of embodiments 121-124, wherein the plurality of analytes comprises a plurality of proteins, a plurality of polypeptides, or a plurality of peptides, or fragments thereof, or a combination thereof. [1703] Embodiment 126. The method of any one of embodiments 121-125, further comprising repeating (b)- (c) for the plurality of analytes to generate a plurality of characteristics associated with the plurality of analytes. [1704] Embodiment 127. The method of embodiment 126, further comprising generating the plurality characteristics associated with the at least the portion of the analyte. [1705] Embodiment 128. The method of embodiments 126 or 127, further comprising using the plurality of characteristics to generate the one or more characteristics associated with the at least the portion of the analyte. [1706] Embodiment 129. The method of any one of embodiments 126-128, wherein the plurality of characteristics comprises differences between at least a subset of analytes of the plurality of analytes. [1707] Embodiment 130. The method of any one of embodiments 126-129, wherein the plurality of characteristics comprises differences in sequence (e.g., differences in sequence is of at most 10 amino acids, at most 5 amino acids etc.), of at most 10 units between at least a subset of analytes of the plurality of analytes. [1708] Embodiment 131. The method of any one of embodiments 126-130, further comprising determining a relative concentration or absolute concentration for one or more analytes in the plurality of analytes. -462- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1709] Embodiment 132. The method of any one of embodiments 126-131, further comprising determining a percentage of modified or different analytes in the plurality of analytes. [1710] Embodiment 133. The method of any one of embodiments 1-132, further comprising repeating (a)-(c) with one or more additional analytes. [1711] Embodiment 134. The method of embodiment 133, wherein the one or more additional analytes comprises one or more additional proteins, one or more additional polypeptides, or one or more additional peptides, or fragments thereof, or a combination thereof. [1712] Embodiment 135. The method of any one of embodiments 1-132, further comprising repeating (a)-(c) with one or more additional analytes and one or more additional nanopores disposed within one or more different membranes. [1713] Embodiment 136. The method of embodiment 135, wherein the one or more additional analytes comprises one or more additional proteins, one or more additional polypeptides, or one or more additional peptides, or fragments thereof, or a combination thereof. [1714] Embodiment 137. The method of embodiment 135 or 136, wherein each of the one or more additional analytes translocates through a different nanopore of the one or more additional nanopores. [1715] Embodiment 138. The method of any one of embodiments 135-137, wherein each of the one or more additional nanopores is disposed in a different membrane of the one or more different membranes. [1716] Embodiment 139. The method of any one of embodiments 1-138, wherein the one or more characteristics comprises natural or unnatural post-translational modifications. [1717] Embodiment 140. The method of any one of embodiments 1-139, wherein the one or more characteristics comprises a plurality of natural or unnatural post-translational modifications of the at least the portion of the analyte. [1718] Embodiment 141. The method of embodiment 139 or 140, wherein the natural or unnatural post- translation modifications is phosphorylation, glycosylation, deamidation, or acetylation, or any combination thereof. [1719] Embodiment 142. The method of any one of embodiments 1-141, wherein the one or more characteristics comprises a length of the at least the portion of the analyte. [1720] Embodiment 143. The method of any one of embodiments 1-142, wherein the one or more characteristics comprises a truncation associated with the at least the portion of the analyte. [1721] Embodiment 144. The method of any one of embodiments 1-143, wherein the one or more characteristics comprises an orientation of the at least the portion of the analyte in the nanopore. -463- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1722] Embodiment 145. The method of any one of embodiments 1-144, wherein the one or more characteristics comprises an average speed of translocation of the at least the portion of the analyte through the nanopore. [1723] Embodiment 146. The method of any one of embodiments 1-145, wherein the one or more characteristics comprises one or more translocation kinetics as a function along a sequence of the at least the portion of the analyte. [1724] Embodiment 147. The method of any one of embodiments 1-146, wherein the one or more characteristics comprises a volume or a mass of the at least the portion of the analyte. [1725] Embodiment 148. The method of any one of embodiments 1-147, wherein the one or more characteristics comprises determining whether the at least the portion of the analyte comprises one or more molecular entities. [1726] Embodiment 149. The method of any one of embodiments 1-148, wherein the at least the portion of the analyte comprises one or more molecular entities. [1727] Embodiment 150. The method of embodiment 149, wherein the one or more molecular entities is coupled to the at least the portion of the protein, the at least the portion of the polypeptide, or the at least the portion of the peptide. [1728] Embodiment 151. The method of embodiment 149 or 150, wherein the one or more characteristics comprises a property of a molecular entity of the one or more molecular entities coupled to the at least the portion of the analyte. [1729] Embodiment 152. The method of embodiments 151, wherein the property comprises a mass of the molecular entity, one or more charges of the molecular entity, one or more classes of the molecular entity, or identity of the molecular entity, or a combination thereof. [1730] Embodiment 153. The method of any one of embodiments 150-152, wherein the at least the portion of the analyte is coupled to two or more molecular entities. [1731] Embodiment 154. The method of embodiment 153, wherein the characteristic comprises a quantity of the two or more molecular entities. [1732] Embodiment 155. The method of any one of embodiments 149-154, wherein the at least the portion of the analyte is covalently coupled to a molecular entity. [1733] Embodiment 156. The method of embodiment 155, wherein the at least the portion of the analyte is non-covalently coupled to a molecular entity. [1734] Embodiment 157. The method of any one of embodiments 149-156, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof. -464- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1735] Embodiment 158. The method of any one of embodiments 1-157, wherein the one or more characteristics comprises at least one property of one or more intra cross-linkages within the at least the portion of the analyte, one or more inter cross-linkages with at least the portion of another analyte, or one or more covalent linkages with a molecular entity, or any combination thereof. [1736] Embodiment 159. The method of embodiment 158, wherein the at least one property comprises a position or a number of the one or more intra cross-linkages (e.g., disulfide bonds), the one or more inter cross- linkages, or the one or more covalent linkages, or any combination thereof. [1737] Embodiment 160. The method of embodiment 158 or 159, wherein the at least one property comprises a presence or absence of one or more linkers associated with the one or more intra cross-linkages, the one or more inter cross-linkages, or the one or more covalent linkages, or any combination thereof. [1738] Embodiment 161. The method of any one of embodiments 1-160, wherein the one or more characteristics comprises a category or identity associated with the at least the portion of the analyte. [1739] Embodiment 162. The method of embodiment 161, wherein the category or the identity comprises one or more of a type, class, gene ontology, sub-domains, functional domains, secondary structure elements, tertiary structural elements, quaternary structures, or protein binding cavities, or any combination thereof. [1740] Embodiment 163. The method of any one of embodiments 1-162, wherein the one or more characteristics comprises one or more of a secondary structure, tertiary structure, or quaternary structure, or a combination thereof associated with the at least the portion of the analyte. [1741] Embodiment 164. The method of any one of embodiments 1-163, wherein the one or more characteristics comprises a presence, absence, quantification, kinetics of the one or more of the secondary structure, tertiary structure, or quaternary structure, or a combination thereof associated with the at least the portion of the analyte. [1742] Embodiment 165. The method of any one of embodiments 1-164, wherein the one or more characteristics comprises a one or more of a folded portion, unfolded portion, or partially folded portion associated with the at least the portion of the analyte. [1743] Embodiment 166. The method of any one of embodiments 1-165, wherein the one or more characteristics comprises a percentage of unfolded portions associated with the at least the portion of the analyte. [1744] Embodiment 167. The method of any one of embodiments 1-166, wherein the one or more characteristics comprises a force, energy, or time, or combination thereof associated with unfolded portions associated with the at least the portion of the analyte. [1745] Embodiment 168. The method of any one of embodiments 1-167, wherein the one or more characteristics comprises a sequence associated with the at least the portion of the analyte. -465- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1746] Embodiment 169. The method of any one of embodiments 1-168, wherein the one or more characteristics comprises a one or more mutations associated with the at least the portion of the analyte. [1747] Embodiment 170. The method of any one of embodiments 1-169, wherein the one or more characteristics comprises a one or more isoforms associated with the at least the portion of the analyte. [1748] Embodiment 171. The method of any one of embodiments 1-170, wherein the one or more characteristics comprises a one or more translation errors associated with the at least the portion of the analyte. [1749] Embodiment 172. The method of any one of embodiments 1-171, wherein the one or more characteristics comprises a one or more degradations associated with the at least the portion of the analyte. [1750] Embodiment 173. The method of any one of embodiments 1-172, wherein the one or more characteristics comprises a one or more natural or unnatural modifications associated with the at least the portion of the analyte. [1751] Embodiment 174. The method of any one of embodiments 1-173, wherein the one or more characteristics comprises one or more variable regions associated with the at least the portion of the analyte. [1752] Embodiment 175. The method of any one of embodiments 1-174, wherein the one or more characteristics comprises one or more constant regions associated with the at least the portion of the analyte. [1753] Embodiment 176. The method of any one of embodiments 1-175, wherein the one or more characteristics comprises a one or more charges associated with the at least the portion of the analyte. [1754] Embodiment 177. The method of any one of embodiments 1-176, wherein the one or more characteristics comprises a hydrophobicity characteristic associated with the at least the portion of the analyte. [1755] Embodiment 178. The method of any one of embodiments 1-177, wherein the one or more characteristics comprises a polarity characteristic associated with the at least the portion of the analyte. [1756] Embodiment 179. The method of any one of embodiments 1-178, wherein the one or more characteristics comprises one or more buried and/or exposed amino acids associated with the at least the portion of the analyte. [1757] Embodiment 180. The method of any one of embodiments 1-179, further comprising, prior to (a), providing: (i) a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and; (2) a membrane comprising the nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side. [1758] Embodiment 181. The method of embodiment 180, wherein the nanopore system further comprises a pair of electrodes. [1759] Embodiment 182. The method of embodiment 181, wherein the pair of electrodes are configured to provide an applied voltage to generate an electrophoretic force (EPF). -466- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1760] Embodiment 183. The method of embodiment 182, wherein the applied voltage is a negative voltage on the second side. [1761] Embodiment 184. The method of embodiment 182, wherein the applied voltage is a positive voltage on the second side. [1762] Embodiment 185. The method of any one of embodiments 182-184, wherein a magnitude of the applied voltage is from about 20 mV to about 300 Mv. [1763] Embodiment 186. The method of any one of embodiments 182-185, wherein an absolute relative net electroosmotic current (e.g., through the nanopore and/or across the membrane) over the applied voltage is greater than about 0.10pA/mV. [1764] Embodiment 187. The method of any one of embodiments 180-186, further comprising, providing the electrophoretic force acting in an opposite direction to a first side to second side electro-osmotic force. [1765] Embodiment 188. The method of any one of embodiments 180-187, wherein the nanopore system further comprises a controller operatively coupled to the fluidic chamber and the nanopore, wherein the controller is configured for detecting (1) (a) the current or change thereof, (b) the voltage or change thereof, or (3) the resistance or change thereof, or (4) any combination thereof, or (2) the electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore or subsequent to translocation through the nanopore. [1766] Embodiment 189. The method of embodiment 188, wherein the controller uses the pair of electrodes to detect (1) (a) the current or change thereof, (b) the voltage or change thereof, or (3) the resistance or change thereof, or (4) any combination thereof, or (2) the electrical signal or change thereof. [1767] Embodiment 190. The method of any one of embodiments 180-189, wherein the first side comprises a first solution and the second side comprises a second solution, wherein the first solution and the second solution are configured to translocate the at least the portion of the analyte across the nanopore. [1768] Embodiment 191. The method of embodiment 190, wherein the first solution and the second solution are configured to generate an electro-osmotic force across the membrane. [1769] Embodiment 192. The method of embodiment 190 or 191, wherein the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of a solute. [1770] Embodiment 193. The method of embodiment 192, wherein a difference between the first concentration of the solute and the second concentration of the solute is configured to generate the electro- osmotic force. [1771] Embodiment 194. The method of embodiment 192 or 193, wherein a difference between the first concentration of the solute and the second concentration of the solute is configured to generate the electro- osmotic force in a presence of an applied potential. -467- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1772] Embodiment 195. The method of embodiment 194, wherein the electro-osmotic force comprises a net ionic current flow cis-to-trans or trans-to-cis. [1773] Embodiment 196. The method of any one of embodiments 191-195, wherein electro-osmotic force translocates the at least the portion of the analyte from the first side through the nanopore to the second side against an electrophoretic force acting in a direction opposite the electro-osmotic force. [1774] Embodiment 197. The method of embodiment 196, wherein the electro-osmotic force is at least 10% greater than the electrophoretic force. [1775] Embodiment 198. The method of any one of embodiments 190-197, wherein the first solution and the second solution are configured to generate a first side to second side electro-osmotic force (EOF). [1776] Embodiment 199. The method of embodiment 198, wherein the first side to second side electro- osmotic force maintains the translocase of the complex at a first side entrance of a channel of the nanopore. [1777] Embodiment 200. The method of embodiment 198 or 199, wherein the first side to second side electro- osmotic force comprises a net first side to second side ionic current flow. [1778] Embodiment 201. The method of any one of embodiments 198-200, wherein the first side to second side electro-osmotic force is modulated by a pH, a type of a salt, a concentration of a salt, an osmotic pressure across the membrane, or a modification of the nanopore, or any combination thereof. [1779] Embodiment 202. The method of embodiment 201, wherein the modification of the nanopore comprises a modification of a charge of the nanopore. [1780] Embodiment 203. The method of any one of embodiments 198-202, wherein the first side to second side electro-osmotic force is modulated by an asymmetric salt distribution between the first side and the second side of the fluidic chamber. [1781] Embodiment 204. The method of any one of embodiments 180-203, further comprising, prior to (a) contacting a complex comprising the at least the portion of the analyte and a translocase with the first side of the nanopore. [1782] Embodiment 205. The method of embodiment 204, further comprising contacting the at least the portion of the analyte with the translocase to generate the complex. [1783] Embodiment 206. The method of embodiment 204 or 205, wherein the translocase comprises an Adenosine triphosphate (ATP)-driven unfoldase and/or a Nucleotide triphosphate (NTP)-driven unfoldase. [1784] Embodiment 207. The method of any one of embodiments 204-206, wherein the translocase comprises an ATPases associated with various cellular activities (AAA+) enzyme. [1785] Embodiment 208. The method of any one of embodiments 180-207, wherein the nanopore system further comprises a preloading solution configured to interact with the at least the portion of the analyte. -468- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1786] Embodiment 209. The method of embodiment 208, wherein the preloading solution comprises one or more cofactors. [1787] Embodiment 210. The method of embodiment 209 or 210, wherein the preloading solution comprises a chemical that enhances a binding of the at least the portion of the analyte to a component of the preloading solution relative to binding in a solution of the first side of the fluidic chamber. [1788] Embodiment 211. The method of any one of embodiments 180-207, further comprising adding a combined solution to the first side of the fluidic chamber, wherein the combined solution comprises the at least the portion of the analyte and a preloading solution. [1789] Embodiment 212. The method of any one of embodiments 208-211, wherein the preloading solution comprises the translocase or a leader construct. [1790] Embodiment 213. The method of any one of embodiments 208-212, wherein the preloading solution comprises a chemical that enhances a binding of the at least the portion of the analyte to a component of the preloading solution. [1791] Embodiment 214. The method of any one of embodiments 1-213, wherein the nanopore is a biological nanopore. [1792] Embodiment 215. The method of embodiment 214, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide. [1793] Embodiment 216. The method of embodiment 215, wherein the alpha helical pore forming protein or peptide comprises a modification of one or more lumen facing amino acids into one or more natural and/or non-natural aromatic amino acids. [1794] Embodiment 217. The method of embodiment 214, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide. [1795] Embodiment 218. The method of embodiment 217, wherein the beta barrel pore forming protein or peptide comprises a modification of one or more lumen facing amino acids into one or more natural or non- natural aromatic amino acids . [1796] Embodiment 219. The method of any one of embodiments 214-218, wherein the biological nanopore comprises one or more monomers comprising one or more mutations. [1797] Embodiment 220. The method of any one of embodiments 214-219, wherein the biological nanopore comprises Aerolysin (Aer), Cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, or phage derived portal proteins, or fragments thereof, or modified variants thereof, or ion-selective mutants thereof. [1798] Embodiment 221. The method of any one of embodiments 1-220, wherein the nanopore comprises a engineered CytK nanopore. -469- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1799] Embodiment 222. The method of embodiment 221, wherein the engineered CytK nanopore comprises one or more amino acid substitutions. [1800] Embodiment 223. The method of embodiment 222, wherein the one or more amino acid substitutions comprises K128D, K128F, K115D, S120D, Q122D, or S151D, or any combination thereof. [1801] Embodiment 224. The method of embodiment 222, wherein the one or more amino acid substitutions comprises K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, or S151D, or any combination thereof. [1802] Embodiment 225. The method of any one of embodiments 1-220, wherein the nanopore comprises an engineered MspA nanopore or an engineered CsgG nanopore. [1803] Embodiment 226. The method of embodiment 225, wherein the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 171. [1804] Embodiment 227. The method of embodiment 226, wherein the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 171. [1805] Embodiment 228. The method of embodiment 226 or 227, wherein the monomer comprises a mutation corresponding to position T83, L88, I105, or N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 171. [1806] Embodiment 229. The method of any one of embodiments 1-228, wherein the nanopore comprises at least a portion of a proteasome. [1807] Embodiment 230. The method of any one of embodiments 214-229, wherein the biological nanopore comprises a recombinant nanopore. [1808] Embodiment 231. The method of any one of embodiments 214-230, wherein the biological nanopore comprises one or more point mutations. [1809] Embodiment 232. The method of embodiment 231, wherein the one or more point mutations affects a diameter of the biological nanopore. [1810] Embodiment 233. The method of embodiment 231 or 232, wherein the one or more point mutations create smaller openings on a first side or a second side of the biological nanopore. [1811] Embodiment 234. The method of any one of embodiments 231-233, wherein the one or more point mutations affects a charge of the biological nanopore. [1812] Embodiment 235. The method of any one of embodiments 231-234, wherein the one or more point mutations are one or more lumen facing mutations. -470- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1813] Embodiment 236. The method of any one of embodiments 231-235, wherein the one or more point mutations allow for conductance at a set pH. [1814] Embodiment 237. The method of embodiment 236, wherein the pH is from about 3 to about 11. [1815] Embodiment 238. The method of any one of embodiments 1-237, further comprising, prior to (a), unfolding the analyte with one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof. [1816] Embodiment 239. The method of embodiments 238, wherein the analyte is unfolded with prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, or Valosin-containing protein-like ATPase, or fragments thereof, or any combination thereof. [1817] Embodiment 240. The method of embodiment 238 or 239, wherein the one or more of unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are coupled to one or more monomers of the nanopore. [1818] Embodiment 241. The method of any one of embodiments 238-240, wherein the one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are suspended in an electrolyte solution on one side of the membrane. [1819] Embodiment 242. The method of any one of embodiments 1-241, further comprising, prior to (a), fragmenting the analyte before translocation. [1820] Embodiment 243. The method of any one of embodiments 1-240 and 242, wherein the at least the portion of the analyte is suspended in an electrolytic solution. [1821] Embodiment 244. The method of embodiment 243, wherein a concentration of one or more electrolytes in the electrolytic solution is from about 0.1 M to about 5 M. [1822] Embodiment 245. The method of any one of embodiments 1-244, wherein the at least the portion of the analyte comprises one or more post-translational modifications. [1823] Embodiment 246. The method of any one of embodiments 1-245, wherein the current or change thereof is from about 0.1 pA to about 150 pA. [1824] Embodiment 247. The method of any one of embodiments 238-241, wherein the one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are configured to position proximal to the nanopore upon a binding event with the polypeptide. [1825] Embodiment 248. The method of any one of embodiments 1-247, wherein the inner diameter of the nanopore is from about 0.5 nm to about 2 nm. -471- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1826] Embodiment 249. The method of any one of embodiments 1-248, wherein the nanopore comprises an increase in aromatic rings in the lumen of the nanopore as compared to another nanopore without a modification. [1827] Embodiment 250. The method of any one of embodiments 1-249, wherein the nanopore limits passage of one or more ions through the channel of the nanopore by modifying a charge of the channel of the nanopore. [1828] Embodiment 251. The method of any one of embodiments 1-250, wherein the at least the portion of the analyte comprises a linear length greater than a channel length of the nanopore [1829] Embodiment 252. The method of any one of embodiments 1-251, wherein the at least the portion of the analyte comprises a polypeptide of at least 30 peptide units. [1830] Embodiment 253. The method of any one of embodiments 1-252, wherein the at least the portion of the analyte comprises an elongated structure. [1831] Embodiment 254. The method of any one of embodiments 1-253, wherein the at least the portion of the analyte comprises one or more leader constructs at a N-terminus and/or a C-terminus. [1832] Embodiment 255. The method of embodiment 254, wherein a leader construct and another leader construct of the one or more leader constructs are configured to translocate the at least the portion of the analyte through the nanopore in a C-terminal to N-terminal direction and/or a N-terminal to C-terminal direction. [1833] Embodiment 256. The method of embodiment 254 or 255, wherein the leader construct is configured to couple one or more translocases to the polypeptide. [1834] Embodiment 257. The method of any one of embodiments 254-256, wherein the leader construct is configured to stall the one or more translocases. [1835] Embodiment 258. The method of any one of embodiments 254-257, wherein a coupling motif is configured to couple the leader construct to the at least the portion of the analyte. [1836] Embodiment 259. The method of embodiment 258, wherein the leader construct comprises a capture motif, a stall motif, a block motif, or recognition motif, or a combination thereof. [1837] Embodiment 260. The method of embodiment 259, wherein the stall motif is configured to disrupt interaction of a translocase with the at least the portion of the analyte. [1838] Embodiment 261. The method of embodiment 259 or 260, wherein the capture motif comprises a polycation tag or a polyanion tag. [1839] Embodiment 262. The method of any one of embodiments 259-261, wherein the recognition motif comprises a portion of ssrA, a Prokaryotic Ubiquitin-like Protein, SulA, or peroxisomal membrane protein (Pex15), or combinations thereof. -472- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1840] Embodiment 263. The method of any one of embodiments 259-262, wherein the block motif is configured to prevent a translocase from translocating the at least the portion of the analyte past the block motif or the leader construct. [1841] Embodiment 264. The method of any one of embodiments 259-263, wherein the block motif comprises a steric obstruction. [1842] Embodiment 265. The method of any one of embodiments 254-264, wherein the leader construct comprises one or more nucleic acids. [1843] Embodiment 266. The method of any one of embodiments 254-265, wherein the leader construct comprises one or more polypeptides or one or more peptides. [1844] Embodiment 267. The method of any one of embodiments 1-266, wherein the nanopore comprises an adaptor [1845] Embodiment 268. The method of embodiment 267, wherein at least a portion of the adaptor is within a channel of the nanopore. [1846] Embodiment 269. The method of embodiment 267 or 268, wherein the adaptor is configured to modify a geometry of the channel of the nanopore. [1847] Embodiment 270. The method of any one of embodiments 267-269, wherein the adaptor comprises a proteinaceous adapter and/or a chemical adaptor. [1848] Embodiment 271. The method of any one of embodiments 1-270, wherein the nanopore is coupled to one or more recognition elements. [1849] Embodiment 272. The method of embodiment 271, wherein the recognition element is a protein recognition element. [1850] Embodiment 273. The method of embodiments 271 or 272, prior to (a), contacting the one or more recognition elements with the at least the portion of the analyte. [1851] Embodiment 274. The method of any one of embodiments 271-273, wherein the recognition element is configured to move between an internal region of the nanopore and an external region of the nanopore. [1852] Embodiment 275. The method of any one of embodiments 271-274, wherein nanopore is coupled to the one or more recognition elements via a linker. [1853] Embodiment 276. The method of embodiment 275, wherein the nanopore is coupled to at least a portion of the linker. [1854] Embodiment 277. The method of any one of embodiments 271-276, wherein movement of the recognition element between an internal region of the nanopore and an external region of the nanopore effects a change in a current or voltage of the nanopore system. -473- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1855] Embodiment 278. The method of any one of embodiments 271-277, wherein the one or more recognition element is between about 5 kilodaltons to about 50 kilodaltons. [1856] Embodiment 279. The method of any one of embodiments 271-278, wherein the one or more recognition element coupled to the at least the portion of the analyte effects movement of the recognition element. [1857] Embodiment 280. The method of embodiment 279, wherein effecting the movement of the recognition element generates a change in (i) a frequency of the movement of the recognition element or (ii) a noise or a magnitude of a current or voltage of the nanopore system. [1858] Embodiment 281. The method of any one of embodiments 271-280, wherein a change in (i) a frequency of the movement of the recognition element or (ii) a noise or a magnitude of a current or voltage block decreases when the recognition element is coupled to the at least the portion of the analyte. [1859] Embodiment 282. The method of any one of embodiments 271-281, wherein the recognition element and another recognition element are configured to bind to different regions of the at least the portion of the analyte. [1860] Embodiment 283. The method of any one of embodiments 1-282, wherein the nanopore comprises an inner pore constriction from about 0.5 nanometers (nm) to about 2 nm. [1861] Embodiment 284. The method of any one of embodiments 1-283, wherein the nanopore has an ion- selectivity P(+)/P(-) of greater than 2.0. [1862] Embodiment 285. The method of any one of embodiments 1-283, wherein the nanopore has an ion- selectivity P(+)/P(-) of less than 0.50. [1863] Embodiment 286. The method of any one of embodiments 1-285, wherein the detecting comprises: measuring a signal for states of (i) an open channel of the nanopore; (ii) capture of the at least the portion of the analyte by the nanopore; or (iii) passage of the at least the portion of the analyte through the nanopore. [1864] Embodiment 287. The method of any one of embodiments 1-286, wherein the at least the portion of the analyte is in a folded state. [1865] Embodiment 288. The method of any one of embodiments 1-287, wherein the at least the portion of the analyte is in a denatured state. [1866] Embodiment 289. The method of any one of embodiments 1-288, wherein the at least the portion of the analyte is at least 1 kDa. [1867] Embodiment 290. The method of any one of embodiments 1-289, wherein the at least the portion of the analyte is at least 100 amino acids. -474- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1868] Embodiment 291. The method of any one of embodiments 1-290, wherein the analyte comprises two or more of a protein, a polypeptide, a peptide. [1869] Embodiment 292. The method of any one of embodiments 1-291, wherein prior to (a), coupling one or more barcodes to the at least the portion of the analyte. [1870] Embodiment 293. The method of any one of embodiments 1-292, wherein the at least the portion of the analyte comprises an elongated structure. [1871] Embodiment 294. The method of any one of embodiments 1-293, wherein the at least the portion of the analyte is from a sample. [1872] Embodiment 295. The method of embodiment 294, wherein the sample is a complex sample. [1873] Embodiment 296. The method of embodiment 295, wherein the complex sample comprises a mixture of proteins. [1874] Embodiment 297. The method of any one of embodiments 294-296, wherein the sample is a clinical sample. [1875] Embodiment 298. The method of embodiment 297, wherein the clinical sample comprises a bodily fluid. [1876] Embodiment 299. The method of embodiment 298, wherein the bodily fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, or sputum, or any combination thereof. [1877] Embodiment 300. A method for sample analysis, comprising: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or first fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or second fragments thereof, or a combination thereof; (c) detecting (i) (1) a first current or change thereof, (2) a first voltage or change thereof, or (3) a first resistance or change thereof, or (4) any combination thereof while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (5) a second current or change thereof, (6) a second voltage or change thereof, or (7) a second resistance or change thereof, or (8) any combination thereof while the at least the portion of the second analyte is translocating through the second nanopore; (d) using (i) (1) the first current or change thereof, (2) the first voltage or change thereof, or (3) the first resistance or change thereof, or (4) any combination thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (5) the second current or change thereof, (6) the second voltage or change thereof, or (7) the -475- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 second resistance or change thereof, or (8) any combination thereof to determine a second characteristic of the at least the portion of the second analyte; and (e) characterizing one or more properties of the sample using the first characteristic and/or the second characteristic determined in (d). [1878] Embodiment 301. The method of embodiment 300, wherein (c) comprises detecting (1) the first current or change thereof and (2) the second current or change thereof, and (d) comprises using (1) the first current or change thereof and (2) the second current or change thereof. [1879] Embodiment 302. The method of embodiment 300 or 301, wherein (c) comprises detecting (1) the first voltage or change thereof and (2) the second voltage or change thereof, and (d) comprises using (1) the first voltage or change thereof and (2) the second voltage or change thereof. [1880] Embodiment 303. The method of any one of embodiments 300-302, wherein (c) comprises detecting (1) the first resistance or change thereof and (2) the second resistance or change thereof, and (d) comprises using (1) the first resistance or change thereof and (2) the second resistance or change thereof. [1881] Embodiment 304. The method of any one of embodiments 300-303, wherein the translocating can comprise translocating the at least the portion of the first analyte or the at least the portion of the second analyte in the C-to-N direction and/or in the N-to-C direction relative to the at least the portion of the analytes sequence. [1882] Embodiment 305. The method of embodiment 304, wherein the determining the first characteristic or the second characteristic of (d) comprises using the current or change thereof, the voltage or change thereof, or the resistance or change thereof, or any combination thereof associated with the C-to-N direction and/or the N-to-C direction. [1883] Embodiment 306. The method of any one of embodiments 300-305, further comprising, in (d), determining (A) the first characteristic based on (1) a first electrical signal or change thereof of the first current or change thereof, the first voltage or change thereof, or the first resistance or change thereof, or any combination thereof and (2) a database, or (B) the second characteristic based on (1) a second electrical signal or change thereof of the second current or change thereof, the second voltage or change thereof, or the second resistance or change thereof, or any combination thereof and (2) the database. [1884] Embodiment 307. The method of embodiment 306, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. [1885] Embodiment 308. The method of embodiment 307 or 308, wherein the database comprises a reference signal associated with the at least the portion of the first analyte or a reference signal associated with the at least the portion of the second analyte. -476- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1886] Embodiment 309. The method of embodiment 308 or 309, wherein the database does not comprise a reference signal associated with the at least the portion of the first analyte or a reference signal associated with the at least the portion of the second analyte. [1887] Embodiment 310. The method of any one of embodiments 306-309, wherein the first electrical signal or change thereof or the second electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the signal or change thereof. [1888] Embodiment 311. The method of embodiment 310, wherein the one or more additional portions of the electrical signal or change thereof comprises one or more blocks of impurities. [1889] Embodiment 312. The method of any one of embodiments 306-311, wherein (1) the first electrical signal or change thereof comprises measurements of the first current or change thereof, the first voltage or change thereof, or the first resistance or change thereof, or any combination thereof over a first period of time; or (2) the second electrical signal or change thereof comprises measurements of the second current or change thereof, the second voltage or change thereof, or the second resistance or change thereof, or any combination thereof over a second period of time. [1890] Embodiment 313. The method of embodiment 312, wherein the first period of time or the second period of time comprises one or more portions associated with a measurement of concentration associated with a sample comprising the at least the portion of the first analyte and the at least the portion of the second analyte. [1891] Embodiment 314. The method of any one of embodiments 306-313, further comprising, in (d), pre- processing (e.g., denoising, segmenting) the first electrical signal or change thereof or the second electrical signal or change thereof, thereby generating (1) a pre-processed first electrical signal or change thereof and/or (2) a pre-processed second electrical signal or change thereof. [1892] Embodiment 315. The method of embodiment 314, wherein the first characteristic or the second characteristic are determined using (1) the pre-processed first electrical signal or change thereof, and/or the (2) the pre-processed second electrical signal or change thereof, respectively. [1893] Embodiment 316. The method of embodiment 314 or 315, further comprising extracting (A) one or more first reads from (1) the first electrical signal or change thereof or (2) the pre-processed first electrical signal or change thereof; and/or (B) one or more second reads from (1) the second electrical signal or change thereof or (2) the pre-processed second electrical signal or change thereof. [1894] Embodiment 317. The method of embodiment 316, further comprising pre-processing the one or more first reads or one or more second reads, thereby generating one or more pre-processed first reads or one or more pre-processed second reads, respectively. [1895] Embodiment 318. The method of embodiment 317, wherein the pre-processing comprises denoising, filtering, segmenting, or scaling, or a combination thereof. -477- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1896] Embodiment 319. The method of embodiment 317 or 318, wherein the first characteristic or the second characteristic is determined using (1) the one or more first reads or the one or more pre-processed first reads (e.g., one or more first segments); and/or (2) the one or more second reads or the one or more pre-processed second reads (e.g., one or more second segments). [1897] Embodiment 320. The method of any one of embodiments 317-319, further comprising comparing (e.g., aligning) (1) the one or more first reads, the one or more pre-processed reads, the one or more second reads, the one or more pre-processed second reads to (2) one or more reference signals in the database. [1898] Embodiment 321. The method of embodiment 320, wherein the comparing comprises alignment. [1899] Embodiment 322. The method of embodiment 321, wherein the alignment comprises time warping. [1900] Embodiment 323. The method of any one of embodiments 317-322, further comprising scoring (1) the one or more first reads, the one or more pre-processed first reads, the one or more second reads, and/or the one or more pre-processed second reads to (2) the one or more reference signals, thereby determining the first characteristic or the second characteristic. [1901] Embodiment 324. The method of any one of embodiments 317-322, further comprising scoring (1) the one or more first reads, the one or more pre-processed first reads, the one or more second reads, and/or the one or more pre-processed second reads to (2) a reference signal of the one or more reference signals, thereby determining the first characteristic or the second characteristic. [1902] Embodiment 325. The method of embodiment 323 or 324, wherein scoring comprises aligning at least a portion of the first electrical signal of change thereof or at least a portion of the second electrical signal or change thereof with the at least the portion of the one or more reference signals, thereby determining the first characteristic or the second characteristic. [1903] Embodiment 326. The method of embodiment 325, wherein the comparing comprises soft alignment. [1904] Embodiment 327. The method of any one of embodiments 317-326, further comprising (1) aggregating the one or more first reads or the one or more pre-processed first reads to determine the first characteristic, or (2) aggregating the one or more second reads, or the one or more pre-processed second reads to determine the second characteristic. [1905] Embodiment 328. The method of any one of embodiments 306-327, wherein the first characteristic or the second characteristic is determined using the first electrical signal or change thereof, or the second electrical signal or change thereof. [1906] Embodiment 329. The method of any one of embodiments 306-328, wherein the database is generated from one or more reference sequences. [1907] Embodiment 330. The method of embodiment 329, wherein the one or more reference sequences are derived with genomic information and/or transcriptomic information of the sample. -478- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1908] Embodiment 331. The method of embodiment 330, wherein the genomic information comprises genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. [1909] Embodiment 332. The method of embodiment 330 or 331, wherein the transcriptomic information comprises genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. [1910] Embodiment 333. The method of any one of embodiments 329-332, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms. [1911] Embodiment 334. The method of any one of embodiments 306-333, wherein the database comprises one or more reference signals for the at least the portion of the analyte, or at least one fragment thereof, or at least one proteoform thereof, or at least one variant thereof, or combination thereof. [1912] Embodiment 335. The method of any one of embodiments 307-334, wherein the one or more polypeptides comprise one or more expressible polypeptides. [1913] Embodiment 336. The method of any one of embodiments 307-335, wherein the one or more variants thereof comprise one or more post-translationally modified variants thereof. [1914] Embodiment 337. The method of any one of embodiments 300-336, further comprising determining the first characteristic or the second characteristic with an accuracy of at least 60%. [1915] Embodiment 338. The method of any one of embodiments 300-337, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, (ii) the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof. [1916] Embodiment 339. The method of any one of embodiments 300-338, wherein the characterizing the one or more properties comprises determining at least one feature of a proteome associated with the sample. [1917] Embodiment 340. The method of embodiment 339, wherein (i) proteome coverage is at least 1% or (ii) sequence coverage of the at least the portion of the first polypeptide or the at least the portion of the second polypeptide is at least 1%. [1918] Embodiment 341. The method of embodiment 340, wherein the proteome coverage is at least 1%. [1919] Embodiment 342. The method of embodiment 340 or 341, wherein the proteome coverage is at least 20%. -479- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1920] Embodiment 343. The method of any one of embodiments 340-342, wherein the sequence coverage of the at least the portion of the first polypeptide and/or the at least the portion of the second polypeptide is at least 1%. [1921] Embodiment 344. The method of any one of embodiments 340-343, wherein the sequence coverage of the at least the portion of the first polypeptide and/or the at least the portion of the second polypeptide is at least 20%. [1922] Embodiment 345. The method of any one of embodiments 300-344, wherein the first nanopore and the second nanopore are the same nanopore. [1923] Embodiment 346. The method of any one of embodiments 300-344, wherein the first nanopore and the second nanopore are different nanopores. [1924] Embodiment 347. The method of any one of embodiments 300-346, wherein the first membrane and the second membrane are the same membranes. [1925] Embodiment 348. The method of any one of embodiments 300-346, wherein the first membrane and the second membrane are different membranes. [1926] Embodiment 349. The method of any one of embodiments 300-348, wherein the sample comprises a first type of analyte and a second type of analyte. [1927] Embodiment 350. The method of embodiment 349, wherein the first type of analyte and the second type of analyte are different types. [1928] Embodiment 351. The method of embodiment 349 or 350, wherein the first type of analyte comprises the at least the portion of the first analyte and the second type of analyte comprises the at least the portion of the second analyte. [1929] Embodiment 352. The method of any one of embodiments 349-351, wherein the characterizing the one or more properties comprises determining the number of analytes in the first type of analyte and determining the number of analytes in the second type of analyte. [1930] Embodiment 353. The method of any one of embodiments 349-352, further comprising determining in the first type of analyte or the second type of analyte one or more of a number of analytes, relative abundance of analytes in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or a combination thereof. [1931] Embodiment 354. The method of any one of embodiments 300-348, wherein the sample comprises one type of analyte. [1932] Embodiment 355. The method of embodiment 354, wherein the at least the portion of the first analyte and the at least the portion of the second analyte are from the same analyte type. -480- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1933] Embodiment 356. The method of any one of embodiments 300-355, further comprising determining one or more of a number of analytes in the sample, relative abundance of analytes in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or a combination thereof. [1934] Embodiment 357. The method of any one of embodiments 300-356, further comprising repeating (b)- (e) for the plurality of analytes. [1935] Embodiment 358. The method of any one of embodiments 300-357, wherein the plurality of analytes comprises a plurality of different types of analytes. [1936] Embodiment 359. The method of any one of embodiments 300-357, wherein the plurality of analytes are the same type. [1937] Embodiment 360. The method of any one of embodiments 300-359, wherein the plurality of analytes comprises a plurality of proteins, a plurality of polypeptides, or a plurality of peptides, or fragments thereof, or any combination thereof. [1938] Embodiment 361. The method of any one of embodiments 300-360, further comprising repeating (b)- (e) with the plurality of analytes and a plurality of nanopores disposed within a plurality of membranes. [1939] Embodiment 362. The method of embodiment 361, wherein each of the plurality of analytes translocates through a different nanopore of the plurality of nanopores. [1940] Embodiment 363. The method of embodiment 361 or 362, wherein each of the plurality of nanopores is disposed in a different membrane of the plurality of membranes. [1941] Embodiment 364. The method of any one of embodiments 300-363, further comprising characterizing one or more properties of the sample using a plurality of characteristics associated with the plurality of analytes. [1942] Embodiment 365. The method of any one of embodiments 300-364, wherein the one or more properties comprises an absolute or relative abundance, absolute concentration, relative concentration, or origin of one or more analyte types in the sample. [1943] Embodiment 366. The method of any one of embodiments 300-365, wherein the one or more properties comprises an absolute or relative abundance, absolute concentration, relative concentration, and/or origin of one or more analytes in the sample. [1944] Embodiment 367. The method of any one of embodiments 300-366, wherein the one or more properties comprises differences between at least a subset of analytes of the plurality of analytes. [1945] Embodiment 368. The method of any one of embodiments 300-367, wherein the one or more properties comprises differences in sequence (e.g., differences in sequence is of at most 10 amino acids, at most 5 amino acids etc.) of at most 10 units between at least a subset of analytes of the plurality of analytes. -481- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1946] Embodiment 369. The method of any one of embodiments 300-368, wherein the one or more properties comprises a percentage of modified or different analytes in the plurality of analytes. [1947] Embodiment 370. The method of any one of embodiments 300-369, wherein the first analyte and the second analyte are different analytes. [1948] Embodiment 371. The method of any one of embodiments 300-370, wherein the first analyte and the second analyte are the same analytes. [1949] Embodiment 372. The method of any one of embodiments 300-371, wherein the one or more properties comprises a presence or an absence of one or more proteins, polypeptides or peptides in the sample. [1950] Embodiment 373. The method of any one of embodiments 300-372, wherein the one or more properties comprises a quantification of one or more proteins, polypeptides or peptides in the sample. [1951] Embodiment 374. The method of any one of embodiments 300-373, wherein the one or more properties comprises a quantification of one or more protein, polypeptide or peptide types in the sample. [1952] Embodiment 375. The method of any one of embodiments 300-374, wherein the one or more properties comprises identification of a type associated with the sample or with an origin of the sample. [1953] Embodiment 376. The method of any one of embodiments 300-375, wherein the one or more properties comprises determining an interaction of the first analyte or the second analyte with one or more molecules in the sample. [1954] Embodiment 377. The method of any one of embodiments 300-376, wherein the one or more properties comprises determining an interaction of one or more analytes of the plurality of analytes with one or more molecules in the sample. [1955] Embodiment 378. The method of any one of embodiments 300-377, wherein the one or more molecules comprises one or more polypeptides, one or more proteins, one or more peptides, one or more nucleic acids, or one or more small molecules, or fragments thereof, or any combination thereof. [1956] Embodiment 379. The method of any one of embodiments 300-378, wherein the first characteristic or second characteristic comprises natural and/or unnatural post-translational modifications [1957] Embodiment 380. The method of any one of embodiments 300-379, wherein the first characteristic or second characteristic comprises a plurality of natural and/or unnatural post-translational modifications of the at least the portion of the analyte. [1958] Embodiment 381. The method of embodiment 379 or 380, wherein the natural and/or unnatural post translation modification is phosphorylation, glycosylation, deamidation, or acetylation, or any combination thereof. -482- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1959] Embodiment 382. The method of any one of embodiments 300-381, wherein the first characteristic or second characteristic comprises a length of the at least the portion of the analyte. [1960] Embodiment 383. The method of any one of embodiments 300-382, wherein the first characteristic or second characteristic comprises an orientation of the at least the portion of the analyte in the nanopore. [1961] Embodiment 384. The method of any one of embodiments 300-383, wherein the first characteristic or second characteristic comprises an average speed of translocation of the at least the portion of the analyte through the nanopore. [1962] Embodiment 385. The method of any one of embodiments 300-384, wherein the first characteristic or second characteristic comprises one or more translocation kinetics as a function along a sequence of the at least the portion of the analyte. [1963] Embodiment 386. The method of any one of embodiments 300-385, wherein the first characteristic or second characteristic comprises a volume or a mass of the at least the portion of the analyte. [1964] Embodiment 387. The method of any one of embodiments 300-386, wherein the first characteristic or second characteristic comprises determining whether the at least the portion of the analyte comprises one or more molecular entities. [1965] Embodiment 388. The method of any one of embodiments 300-387, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more molecular entities. [1966] Embodiment 389. The method of embodiment 388, wherein the one or more molecular entities is coupled to (1) the at least the portion of the first protein, the at least the portion of the first polypeptide, or the at least the portion of the first peptide; and/or (2) the at least the portion of the second protein, the at least the portion of the second polypeptide, or the at least the portion of the second peptide. [1967] Embodiment 390. The method of embodiment 388 or 389, wherein the first characteristic or second characteristic comprises a property of the molecular entity coupled to the at least the portion of the first analyte or the at least the portion of the second analyte. [1968] Embodiment 391. The method of embodiment 390, wherein the property comprises a mass of the molecular entity, one or more charges of the molecular entity, one or more classes of the molecular entity, or identity of the molecular entity, or a combination thereof. [1969] Embodiment 392. The method of any one of embodiments 300-391, wherein the at least the portion of the analyte is coupled to two or more molecular entities. [1970] Embodiment 393. The method of embodiment 392, wherein the characteristic comprises a quantity of the two or more molecular entities. [1971] Embodiment 394. The method of any one of embodiments 300-393, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is covalently coupled to a molecular entity. -483- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1972] Embodiment 395. The method of any one of embodiments 300-393, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is non-covalently coupled to a molecular entity. [1973] Embodiment 396. The method of any one of embodiments 300-395, Wherein the first characteristic or second characteristic comprises at least one property of one or more intra cross-linkages within the at least the portion of the analyte, one or more inter cross-linkages with at least the portion of another analyte, or one or more covalent linkages with a molecular entity, or any combination thereof. [1974] Embodiment 397. The method of embodiment 396, wherein the at least one property comprises a position or a number of the one or more intra cross-linkages (e.g., disulfide bonds), the one or more inter cross- linkages, or the one or more covalent linkages, or any combination thereof. [1975] Embodiment 398. The method of embodiments 396 or 397, wherein the at least one property comprises a presence or absence of one or more linkers associated with the one or more intra cross-linkages, the one or more inter cross-linkages, or the one or more covalent linkages, or any combination thereof. [1976] Embodiment 399. The method of any one of embodiments 388-390, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof. [1977] Embodiment 400. The method of any one of embodiments 300-399, wherein the first characteristic or second characteristic comprises a category or identity associated with the at least the portion of the first analyte or the at least the portion of the second analyte. [1978] Embodiment 401. The method of embodiment 400, wherein the category or the identity comprises one or more of a type, class, gene ontology, sub-domains, functional domains, secondary structure elements, tertiary structural elements, quaternary structures, or protein binding cavities, or any combination thereof. [1979] Embodiment 402. The method of any one of embodiments 300-401, wherein the first characteristic or second characteristic comprises one or more of a secondary structure, tertiary structure, quaternary structure, or a combination thereof associated with the at least the portion of the first analyte or the at least the portion of the second analyte. [1980] Embodiment 403. The method of any one of embodiments 300-402, wherein the first characteristic or second characteristic comprises a presence, absence, quantification, kinetics of the one or more of the secondary structure, tertiary structure, or quaternary structure, or a combination thereof associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1981] Embodiment 404. The method of any one of embodiments 300-403, wherein the first characteristic or second characteristic comprises a one or more of a folded portion, unfolded portion, or partially folded portion associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. -484- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1982] Embodiment 405. The method of any one of embodiments 300-404, wherein the first characteristic or second characteristic comprises a percentage of unfolded portions associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1983] Embodiment 406. The method of any one of embodiments 300-405, wherein the first characteristic or second characteristic comprises a force, energy, or time, or combination thereof associated with unfolded portions associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1984] Embodiment 407. The method of any one of embodiments 300-406, wherein the first characteristic or second characteristic comprises a sequence associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1985] Embodiment 408. The method of any one of embodiments 300-407, wherein the first characteristic or second characteristic comprises a one or more mutations associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1986] Embodiment 409. The method of any one of embodiments 300-408, wherein the first characteristic or second characteristic comprises a one or more isoforms associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1987] Embodiment 410. The method of any one of embodiments 300-409, wherein the first characteristic or second characteristic comprises a one or more translation errors associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1988] Embodiment 411. The method of any one of embodiments 300-410, wherein the first characteristic or second characteristic comprises a one or more degradations associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1989] Embodiment 412. The method of any one of embodiments 300-411, wherein the first characteristic or second characteristic comprises a one or more natural or unnatural modifications associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1990] Embodiment 413. The method of any one of embodiments 300-412, wherein the first characteristic or second characteristic comprises one or more variable regions associated with the at least the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1991] Embodiment 414. The method of any one of embodiments 300-413, wherein the first characteristic or second characteristic comprises one or more constant regions associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. -485- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [1992] Embodiment 415. The method of any one of embodiments 300-414, wherein the first characteristic or second characteristic comprises a one or more charges associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1993] Embodiment 416. The method of any one of embodiments 300-415, wherein the first characteristic or second characteristic comprises a hydrophobicity characteristic associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1994] Embodiment 417. The method of any one of embodiments 300-416, wherein the first characteristic or second characteristic comprises a polarity characteristic associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1995] Embodiment 418. The method of any one of embodiments 300-417, wherein the first characteristic or second characteristic comprises one or more buried and/or exposed amino acids associated with the at least the portion of the first analyte and/or the at least the portion of the second analyte. [1996] Embodiment 419. The method of any one of embodiments 300-418, further comprising, prior to (b), providing: (i) a first nanopore system, wherein the first nanopore system comprises (1) a first fluidic chamber and (2) the first membrane comprising the first nanopore, wherein the first membrane separates the first fluidic chamber into a first side and a second side, and (ii) a second nanopore system, wherein the second nanopore system comprises (1) a second fluidic chamber and; (2) the second membrane comprising the second nanopore, wherein the second membrane separates the second fluidic chamber into a side and an additional side. [1997] Embodiment 420. The method of embodiment 419, wherein the first nanopore system and the second nanopore system are different nanopore systems. [1998] Embodiment 421. The method of embodiment 419, wherein the first nanopore system and the second nanopore system are the same nanopore system. [1999] Embodiment 422. The method of any one of embodiments 419-421, wherein the first nanopore system further comprises a first pair of electrodes. [2000] Embodiment 423. The method of embodiment 422, wherein the second nanopore system further comprises a second pair of electrodes. [2001] Embodiment 424. The method of any one of embodiments 419-423, wherein the first nanopore system and the second nanopore system share one or more common electrodes. [2002] Embodiment 425. The method of embodiment 423, wherein the first pair of electrodes and/or the second pair of electrodes are configured to provide a first applied voltage and/or a second applied voltage to generate a first electrophoretic force (EPF) and/or a second EPF. [2003] Embodiment 426. The method of embodiment 425, wherein the first applied voltage or the second applied voltage is a negative voltage on the second side and/or the additional side. -486- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2004] Embodiment 427. The method of embodiment 425, wherein the first applied voltage or the second applied voltage is a positive voltage on the second side and/or the additional side. [2005] Embodiment 428. The method of any one of embodiments 425-427, wherein a magnitude of the first applied voltage or the second applied voltage is from about 20 mV to about 300 mV. [2006] Embodiment 429. The method of any one of embodiments 425-428, wherein a first absolute relative net electroosmotic current and/or a second absolute relative net electroosmotic current over the first applied voltage or the second applied voltage is greater than about 0.10pA/mV. [2007] Embodiment 430. The method of any one of embodiments 425-429, further comprising, providing (i) the first EPF acting in an opposite direction to a first side to second side electro-osmotic force, or (ii) the second EPF acting in an opposite direction to a side to additional side electro-osmotic force. [2008] Embodiment 431. The method of any one of embodiments 419-430, wherein (i) the first nanopore system further comprises a first controller operatively coupled to the first fluidic chamber and the first nanopore; or (ii) wherein the second nanopore system further comprises a second controller operatively coupled to the second fluidic chamber and the second nanopore. [2009] Embodiment 432. The method of embodiment 431, wherein (i) the first controller is configured for detecting the first current or change thereof, the first voltage or change thereof, or the first resistance or change thereof, or any combination thereof while the at least the portion of the first analyte is translocating through the first nanopore or subsequent to translocation through the first nanopore; or (ii) the second controller is configured for detecting the second current or change thereof, or the second voltage of change thereof while the at least the portion of the second analyte is translocating through the second nanopore or subsequent to translocation through the second nanopore. [2010] Embodiment 433. The method of embodiment 431 or 432, wherein (i) the first controller uses a first pair of electrodes to detect the first current or change thereof, the first voltage or change thereof, or the first resistance or change thereof, or any combination thereof or (ii) the second controller uses a second pair of electrodes to detect the second current or change thereof, the second voltage or change thereof, or the second resistance or change thereof, or any combination thereof. [2011] Embodiment 434. The method of any one of embodiments 431-433, wherein the first controller and the second controller are the same controller. [2012] Embodiment 435. The method of any one of embodiments 431-433, wherein the first controller and the second controller are different controllers. [2013] Embodiment 436. The method of any one of embodiments 419-435 wherein (i) the first side comprises a first solution and the second side comprises a second solution, and/or (ii) the side comprises a solution and the additional side comprises an additional solution. -487- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2014] Embodiment 437. The method of embodiment 436, wherein (i) the first solution and the second solution are configured to translocate the at least the portion of the first analyte across the first nanopore, and/or (ii) the solution and the additional solution are configured to translocate the at least the portion of the second analyte across the second nanopore. [2015] Embodiment 438. The method of embodiment 436 or 437, wherein (i) the first solution and the second solution are configured to generate a first electro-osmotic force across the first membrane; (i) the solution and the additional solution are configured to generate a second electro-osmotic force across the second membrane. [2016] Embodiment 439. The method of any one of embodiments 436-438, wherein (i) the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of a solute; and/or (ii) the solution comprises a concentration of a solute and the additional solution comprises an additional concentration of a solute. [2017] Embodiment 440. The method of embodiment 439, wherein (i) a difference between the first concentration of the solute and the second concentration of the solute is configured to generate the first electro- osmotic force; and/or (ii) a difference between the concentration of the solute and the additional concentration of the solute is configured to generate the second electro-osmotic force. [2018] Embodiment 441. The method of embodiment 439 or 440, wherein (i) a difference between the first concentration of the solute and the second concentration of the solute is configured to generate the first electro- osmotic force in a presence of a first applied potential; and/or (ii) wherein a difference between the concentration of the solute and the additional concentration of the solute is configured to generate the second electro-osmotic force in a presence of a second applied potential. [2019] Embodiment 442. The method of any one of embodiments 438-441, wherein the first electro-osmotic force or the second electro-osmotic force comprises a net ionic current flow cis-to-trans. [2020] Embodiment 443. The method of any one of embodiments 438-442, wherein (i) the first electro- osmotic force translocates the at least the portion of the first analyte from the first side through the first nanopore to the second side against a first electrophoretic force acting in a direction opposite the first electro- osmotic force; and/or (ii) a second electro-osmotic force translocates the at least the portion of the second analyte from the side through the second nanopore to the additional side against a second electrophoretic force acting in a direction opposite the second electro-osmotic force. [2021] Embodiment 444. The method of any one of embodiments 438-443, wherein (i) the first electro- osmotic force is at least 10% greater than the first electrophoretic force; or (ii) the second electro-osmotic force is at least 10% greater than the second electrophoretic force. [2022] Embodiment 445. The method of any one of embodiments 438-444, wherein (i) the first solution and the second solution are configured to generate a first side to second side electro-osmotic force (EOF) or (ii) -488- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 the solution and the additional solution are configured to generate a side to an additional side electro-osmotic force (EOF). [2023] Embodiment 446. The method of embodiment 445, wherein (i) the first side to second side electro- osmotic force maintains the first translocase of the first complex at a first side entrance of a channel of the first nanopore; or (ii) the side to additional side electro-osmotic force maintains the second translocase of the second complex at the side entrance of a channel of the second nanopore. [2024] Embodiment 447. The method of embodiment 445 or 446, wherein the first side to second side electro- osmotic force comprises a net first side to second side ionic current flow; or (ii) the side to additional side electro-osmotic force comprises a net side to additional side ionic current flow. [2025] Embodiment 448. The method of any one of embodiments 445-447, wherein (i) the first side to second side electro-osmotic force; or (ii) the side to additional side electro-osmotic force is modulated by a pH, a type of a salt, a concentration of a salt, an osmotic pressure across the membrane, a modification of the nanopore, or any combination thereof. [2026] Embodiment 449. The method of embodiment 448, wherein the modification of the nanopore comprises a modification of a charge of the nanopore. [2027] Embodiment 450. The method of any one of embodiments 445-449, wherein (i) the first side to second side electro-osmotic force is modulated by an asymmetric salt distribution between the first side and the second side of the first fluidic chamber; and/or (ii) the side to additional side electro-osmotic force is modulated by an asymmetric salt distribution between the side and the additional side of the second fluidic chamber. [2028] Embodiment 451. The method of any one of embodiments 419-450, further comprising, prior to (a) (i) contacting a first complex comprising the at least the portion of the first analyte and a first translocase with the first side of the first nanopore; and/or (ii) contacting a second complex comprising the at least the portion of the second analyte and a second translocase with the side of the second nanopore. [2029] Embodiment 452. The method of embodiment 451, further comprising contacting (i) the at least the portion of the first analyte with the first translocase to generate the first complex; and/or (ii) the at least the portion of the second analyte with the second translocase to generate the second complex. [2030] Embodiment 453. The method of embodiments 451 or 452, wherein the first translocase and/or the second translocase comprises an Adenosine triphosphate (ATP)-driven unfoldase and/or a Nucleotide triphosphate (NTP)-driven unfoldase. [2031] Embodiment 454. The method of any one of embodiments 451-453, wherein the first translocase and/or the second translocase comprises an ATPases associated with various cellular activities (AAA+) enzyme. -489- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2032] Embodiment 455. The method of any one of embodiments 419-454, wherein (i) the first nanopore system further comprises a first preloading solution configured to interact with the at least the portion of the first analyte; and/or (ii) the second nanopore system further comprises a second preloading solution configured to interact with the at least the portion of the second analyte. [2033] Embodiment 456. The method of embodiment 455, wherein the first preloading solution and/or the second preloading solution comprises one or more cofactors. [2034] Embodiment 457. The method of embodiment 455 or 456, wherein the first preloading solution or the second preloading solution comprises a chemical that enhances a binding of (i) the at least the portion of the first analyte to a component of the first preloading solution relative to binding in a solution of the first side of the first fluidic chamber; and/or (ii) the at least the portion of the second analyte to a component of the second preloading solution relative to binding in a solution of the second side of the second fluidic chamber. [2035] Embodiment 458. The method of any one of embodiments 419-454, further comprising adding (i) a first combined solution to the first side of the first fluidic chamber, wherein the first combined solution comprises the at least the portion of the first analyte and a first preloading solution; or (ii) a second combined solution to the second side of the second fluidic chamber, wherein the second combined solution comprises the at least the portion of the second analyte and a second preloading solution. [2036] Embodiment 459. The method of embodiment 458, wherein (i) the first preloading solution comprises the first translocase or a first leader construct; and/or (ii) the second preloading solution comprises the second translocase or a second leader construct. [2037] Embodiment 460. The method of embodiment 458 or 459, wherein (i) the first preloading solution comprises a chemical that enhances a binding of the at least the portion of the first analyte to a component of the first preloading solution; and/or (ii) the second preloading solution comprises a chemical that enhances a binding of the at least the portion of the second analyte to a component of the second preloading solution. [2038] Embodiment 461. The method of any one of embodiments 300-460, wherein the first nanopore or the second nanopore is a biological nanopore. [2039] Embodiment 462. The method of embodiment 461, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide. [2040] Embodiment 463. The method of embodiment 462, wherein the alpha helical pore forming protein or peptide comprises a modification of one or more lumen facing amino acids into one or more natural and/or non-natural aromatic amino acids. [2041] Embodiment 464. The method of embodiment 461, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide. -490- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2042] Embodiment 465. The method of embodiment 464, wherein the beta barrel pore forming protein or peptide comprises a modification of one or more lumen facing amino acids into one or more natural and/or non-natural aromatic amino acids. [2043] Embodiment 466. The method of any one of embodiments 461-465, wherein the biological nanopore comprises one or more monomers comprising one or more mutations. [2044] Embodiment 467. The method of any one of embodiments 461-466, wherein the biological nanopore comprises Aerolysin (Aer), Cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, or phage derived portal proteins, or fragments thereof, or modified variants thereof, or ion-selective mutants thereof. [2045] Embodiment 468. The method of any one of embodiments 300-467, wherein the first nanopore and/or the second nanopore comprises a mutant CytK nanopore. [2046] Embodiment 469. The method of embodiment 468, wherein the mutant CytK nanopore comprises one or more amino acid substitutions. [2047] Embodiment 470. The method of embodiment 469, wherein the one or more amino acid substitutions comprises K128D, K128F, K115D, S120D, Q122D, or S151D, or any combination thereof. [2048] Embodiment 471. The method of embodiment 469, wherein the one or more amino acid substitutions comprises K128D, K155Q, T116D, S120D, Q122D, S126D, T143D, Q145D, T147D, or S151D, or any combination thereof. [2049] Embodiment 472. The method of any one of embodiments 300-471, wherein the first nanopore and/or the second nanopore comprises at least a portion of a proteasome. [2050] Embodiment 473. The method of any one of embodiments 300-467, wherein the first nanopore and/or the second nanopore comprises an engineered MspA nanopore or an engineered CsgG nanopore. [2051] Embodiment 474. The method of embodiment 473, wherein the engineered MspA nanopore comprises a monomer with an amino acid sequence with at least about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 171. [2052] Embodiment 475. The method of embodiments 473 or 474, wherein the monomer comprises a mutation corresponding to position D90 or D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 171. [2053] Embodiment 476. The method of any one of embodiments 473-475, wherein the monomer comprises a mutation corresponding to position T83, L88, I105, or N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 171. [2054] Embodiment 477. The method of any one of embodiments 300-476, wherein the first nanopore and/or the second nanopore comprises a recombinant nanopore. -491- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2055] Embodiment 478. The method of any one of embodiments 300-477, wherein the first nanopore or the second nanopore comprises one or more point mutations. [2056] Embodiment 479. The method of embodiment 478, wherein the one or more point mutations affects a diameter of the first nanopore and/or the second nanopore. [2057] Embodiment 480. The method of embodiment 478 or 479, wherein the one or more point mutations create smaller openings on a first side and/or a second side of the first nanopore and/or the second nanopore. [2058] Embodiment 481. The method of any one of embodiments 478-480, wherein the one or more point mutations affects a charge of the first nanopore and/or the second nanopore. [2059] Embodiment 482. The method of any one of embodiments 478-481, wherein the one or more point mutations are one or more lumen facing mutations. [2060] Embodiment 483. The method of any one of embodiments 478-482, wherein the one or more point mutations allow for conductance at a set pH. [2061] Embodiment 484. The method of embodiment 483, wherein the pH is from about 5 to about 10. [2062] Embodiment 485. The method of any one of embodiments 300-484, further comprising, prior to (a), unfolding the first analyte and/or second analyte with one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof. [2063] Embodiment 486. The method of embodiment 485, wherein the first analyte and/or the second analyte is unfolded with prokaryotic AAA+ unfoldase, ClpX, PAN unfoldase, or Valosin-containing protein-like ATPase, or any combination thereof. [2064] Embodiment 487. The method of embodiment 485 or 486, wherein the one or more of unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are coupled to one or more monomers of the first nanopore and/or the second nanopore. [2065] Embodiment 488. The method of any one of embodiments 485-487, wherein the one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are suspended in an electrolyte solution on one side of the membrane. [2066] Embodiment 489. The method of any one of embodiments 300-484, further comprising, prior to (a), fragmenting the at least the portion of the first analyte and/or the at least the portion of the second analyte before translocation. [2067] Embodiment 490. The method of any one of embodiments 300-489, wherein the at least the portion of the first analyte or the at least the portion of the second analyte is suspended in an electrolytic solution [2068] Embodiment 491. The method of embodiment 490, wherein a concentration of one or more electrolytes in the electrolytic solution is from about 0.1 M to about 5 M. -492- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2069] Embodiment 492. The method of any one of embodiments 300-491, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte comprises one or more post-translational modifications. [2070] Embodiment 493. The method of any one of embodiments 300-492, wherein the current or change thereof is from about 0.1 pA to about 150 pA. [2071] Embodiment 494. The method of any one of embodiments 485-488, wherein the one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are configured to position proximal to the nanopore upon a binding event with the polypeptide. [2072] Embodiment 495. The method of any one of embodiments 300-494, wherein the inner diameter of the first nanopore and/or the second nanopore is from about 0.5 nm to about 2 nm. [2073] Embodiment 496. The method of any one of embodiments 300-495, wherein the first nanopore and/or the second nanopore comprises an increase in aromatic rings in the lumen of the first nanopore and/or the second nanopore as compared to another nanopore without a modification. [2074] Embodiment 497. The method of any one of embodiments 300-496, wherein the first nanopore and/or the second nanopore limits passage of one or more ions through the channel of the first nanopore and/or the second nanopore by modifying a charge of the channel of the first nanopore and/or the second nanopore. [2075] Embodiment 498. The method of any one of embodiments 300-497, wherein the at least the portion of the first analyte and/or the second analyte comprises a linear length greater than a channel length of the first nanopore and/or the second nanopore. [2076] Embodiment 499. The method of any one of embodiments 300-498, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte comprises a polypeptide of at least 30 peptide units. [2077] Embodiment 500. The method of any one of embodiments 300-499, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte comprises an elongated structure. [2078] Embodiment 501. The method of any one of embodiments 300-500, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte comprises one or more leader constructs at a N-terminus and/or a C- terminus. [2079] Embodiment 502. The method of embodiment 501, wherein a leader construct and another leader construct of the one or more leader constructs are configured to translocate the at least the portion of the first analyte and/or the at least the portion of the second analyte through the nanopore in a C-terminal to N-terminal direction and/or a N-terminal to C-terminal direction. [2080] Embodiment 503. The method of embodiment 501 or 502, wherein the one or more leader constructs is configured to couple one or more translocases to the polypeptide. -493- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2081] Embodiment 504. The method of any one of embodiments 501-503, wherein the one or more leader constructs is configured to stall the one or more translocases. [2082] Embodiment 505. The method of any one of embodiments 501-504, wherein a coupling motif is configured to couple the one or more leader constructs to the at least the portion of the analyte. [2083] Embodiment 506. The method of any one of embodiments 501-505, wherein the one or more leader constructs comprises a capture motif, a stall motif, a block motif, or recognition motif, or a combination thereof. [2084] Embodiment 507. The method of embodiment 506, wherein the stall motif is configured to disrupt interaction of a translocase with the at least the portion of the analyte. [2085] Embodiment 508. The method of embodiment 506 or 507, wherein the capture motif comprises a polycation tag and/or a polyanion tag. [2086] Embodiment 509. The method of any one of embodiments 506-508, wherein the recognition motif comprises a portion of ssrA, a Prokaryotic Ubiquitin-like Protein, SulA, or peroxisomal membrane protein (Pex15), or combinations thereof. [2087] Embodiment 510. The method of any one of embodiments 506-509, wherein the block motif is configured to prevent a translocase from translocating the at least the portion of the analyte past the block motif and/or the leader construct. [2088] Embodiment 511. The method of any one of embodiments 506-510, wherein the block motif comprises a steric obstruction. [2089] Embodiment 512. The method of any one of embodiments 501-511, wherein the one or more leader constructs comprises one or more nucleic acids. [2090] Embodiment 513. The method of any one of embodiments 501-512, wherein the one or more leader constructs comprises one or more polypeptides or one or more peptides. [2091] Embodiment 514. The method of any one of embodiments 300-513, wherein the first nanopore or the second nanopore comprises an adaptor. [2092] Embodiment 515. The method of embodiment 514, wherein at least a portion of the adaptor is within a channel of the nanopore. [2093] Embodiment 516. The method of embodiment 514 or 515, wherein the adaptor is configured to modify a geometry of the channel of the nanopore. [2094] Embodiment 517. The method of any one of embodiments 514-516, wherein the adaptor comprises a proteinaceous adapter or a chemical adaptor. -494- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2095] Embodiment 518. The method of any one of embodiments 300-518, wherein the first nanopore or the second nanopore is coupled to one or more recognition elements. [2096] Embodiment 519. The method of embodiment 518, wherein the one or more recognition elements is a protein recognition element. [2097] Embodiment 520. The method of embodiment 518 or 519, prior to (a), contacting the one or more recognition elements with the at least the portion of the first analyte or the at least the portion of the second analyte. [2098] Embodiment 521. The method of any one of embodiments 518-520, wherein the one or more recognition elements is configured to move between (i) an internal region of the first nanopore and an external region of the first nanopore; (ii) an internal region of the second nanopore and an external region of the second nanopore. [2099] Embodiment 522. The method of any one of embodiments 518-521, wherein the first nanopore and/or the second nanopore is coupled to the one or more recognition elements via a linker [2100] Embodiment 523. The method of embodiment 522, wherein the first nanopore and/or the second nanopore is coupled to at least a portion of the linker. [2101] Embodiment 524. The method of any one of embodiments 518-523, wherein (i) movement of the one or more recognition element between an internal region of the first nanopore and an external region of the first nanopore effects a change in a current of the first nanopore system; and/or (ii) movement of the one or more recognition element between an internal region of the second nanopore and an external region of the second nanopore effects a change in a current of the second nanopore system. [2102] Embodiment 525. The method of any one of embodiments 518-524, wherein the one or more recognition element is between about 5 kilodaltons to about 50 kilodaltons. [2103] Embodiment 526. The method of any one of embodiments 518-525, wherein the one or more recognition element coupled to the at least the portion of the first analyte and/or the at least the portion of the second analyte effects movement of the recognition element. [2104] Embodiment 527. The method of embodiment 526, wherein effecting the movement of the recognition element generates a change in (i) a frequency of the movement of the recognition element and/or (ii) a noise or a magnitude of a current of the first nanopore system and/or the second nanopore system. [2105] Embodiment 528. The method of any one of embodiments 524-527, wherein a change in (i) a frequency of the movement of the recognition element and/or (ii) a noise or a magnitude of a current block decreases when the recognition element is coupled to the at least the portion of the first analyte and/or the at least the portion of the second analyte. -495- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2106] Embodiment 529. The method of any one of embodiments 518-528, wherein the recognition element and another recognition element are configured to bind to different regions of the at least the portion of the first analyte and/or the at least the portion of the second analyte. [2107] Embodiment 530. The method of any one of embodiments 300-529, wherein the first nanopore and/or the second nanopore comprises an inner pore constriction from about 0.5 nanometers (nm) to about 2 nm. [2108] Embodiment 531. The method of any one of embodiments 300-530, wherein the first nanopore and/or the second nanopore has an ion-selectivity P(+)/P(-) of greater than 2.0. [2109] Embodiment 532. The method of any one of embodiments 300-530, wherein the first nanopore and/or the second nanopore has an ion-selectivity P(+)/P(-) of less than 0.50. [2110] Embodiment 533. The method of any one of embodiments 300-532, wherein the detecting comprises: measuring a signal for states of (i) an open channel of v; (ii) capture of the at least the portion of the first analyte by the first nanopore or the the at least the portion of the second analyte by second nanopore; and/or (iii) passage of the at least the portion of the first analyte through the first nanopore and/or the at least the portion of the second analyte through the second nanopore. [2111] Embodiment 534. The method of any one of embodiments 300-533, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is in a folded state. [2112] Embodiment 535. The method of any one of embodiments 300-534, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is in a denatured state. [2113] Embodiment 536. The method of any one of embodiments 300-535, wherein the the at least the portion of the first analyte and/or the at least the portion of the second analyte is at least 1 kDa. [2114] Embodiment 537. The method of any one of embodiments 300-536, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is at least 100 amino acids. [2115] Embodiment 538. The method of any one of embodiments 300-537, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte comprises two or more of a protein, a polypeptide, or a peptide, or fragments thereof, or any combination thereof. [2116] Embodiment 539. The method of any one of embodiments 300-538, wherein prior to (a), coupling one or more barcodes to the at least the portion of the first analyte or the at least the portion of the second analyte. [2117] Embodiment 540. The method of any one of embodiments 300-539, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte comprises an elongated structure. [2118] Embodiment 541. The method of any one of embodiments 300-540, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is from a sample. [2119] Embodiment 542. The method of embodiment 541, wherein the sample is a complex sample. -496- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2120] Embodiment 543. The method of embodiment 542, wherein the complex sample comprises a mixture of proteins. [2121] Embodiment 544. The method of any one of embodiments 541-543, wherein the sample is a clinical sample. [2122] Embodiment 545. The method of embodiment 544, wherein the clinical sample comprises a bodily fluid. [2123] Embodiment 546. The method of embodiment 545, wherein the bodily fluid comprises whole blood, plasma, serum, urine, feces, saliva, cerebrospinal fluid, breast milk, or sputum, or any combination thereof. [2124] Embodiment 547. A system, comprising: a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side; one or more controllers operatively coupled to the nanopore system, wherein the one or more controllers are individually or collectively configured to: (a) translocate at least a portion of an analyte through the nanopore, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or at least a fragment thereof, or a combination thereof, (b) detect (1) a current or change thereof; (2) a voltage or change thereof; or (3) a resistance or change thereof; or (4) any combination thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) use (1) the current or change thereof, (2) the voltage or change thereof; or (3) the resistance or change thereof; or (4) any combination thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. [2125] Embodiment 548. A system, comprising: a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side; and one or more controllers operatively coupled to the nanopore system, wherein the one or more controllers are individually or collectively configured to: (a) translocate at least a portion of an analyte through the nanopore, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or at least a fragment thereof, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof, (b) detect (1) a current or change thereof, (2) a voltage or change thereof, or (3) a resistance or change thereof, or (4) any combination thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) use (1) the current or change thereof, (2) the voltage or change thereof, or (3) the resistance or change thereof, or (4) any combination thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte. -497- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2126] Embodiment 549. The system of embodiment 547 or 548, wherein the average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second. [2127] Embodiment 550. The system of embodiment 547 or 548, wherein the average rate of translocation is between about 1 amino acids per second to about 100 amino acids per second with a motor protein. [2128] Embodiment 551. The system of embodiment 547 or 548, wherein the average rate of translocation is between about 500 amino acids per second to about 5000 amino acids per second without a motor protein. [2129] Embodiment 552. The system of embodiment 547 or 548, wherein the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s. [2130] Embodiment 553. The system of embodiment 547 or 548, wherein the step size is from about 0.5 amino acids to about 5 amino acids. [2131] Embodiment 554. The system of embodiment 547 or 548, wherein the step size is from about 1 amino acid to about 3 amino acids. [2132] Embodiment 555. The system of any one of embodiments 547-554, wherein the one or more controllers are individually or collectively further configured to repeat (a)-(c) with at least a portion of an additional analyte. [2133] Embodiment 556. A system, comprising: a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising a nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side; and one or more controllers operatively coupled to the nanopore system, wherein the one or more controllers are individually or collectively configured to: (a) translocate at least a portion of an analyte through the nanopore, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or at least fragments thereof, or a combination thereof, (b) detect an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assign one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more fragments thereof, or one or more proteoforms thereof, or one or more variants thereof, or a combination thereof. [2134] Embodiment 557. The system of embodiment 556, wherein the one or more controllers are individually or collectively further configured to repeat (a)-(c) with at least a portion of an additional analyte. [2135] Embodiment 558. The system of embodiment 556 or 557, wherein the database does not comprise a reference signal associated with the at least the portion of the analyte. [2136] Embodiment 559. The system of embodiment 556 or 557, wherein the database comprises a reference signal associated with the at least the portion of the analyte. -498- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2137] Embodiment 560. The system of any one of embodiments 556-559, wherein the electrical signal or change thereof comprises (1) one or more reads; and/or (2) one or more additional portions of the electrical signal or change thereof. [2138] Embodiment 561. The system of any one of embodiments 556-560, wherein the electrical signal or change thereof comprises measurements of (1) a current or change thereof, (2) a voltage or change thereof, or (3) a resistance or change thereof, or (4) any combination thereof over a period of time. [2139] Embodiment 562. The system of any one of embodiments 556-561, wherein the one or more controllers are individually or collectively further configured to, prior to (c), pre-process (e.g., denoising, segmenting) the electrical signal or change thereof, thereby generate a pre-processed electrical signal or change thereof. [2140] Embodiment 563. The system of embodiment 562, wherein the one or more characteristics are assigned using the pre-processed electrical signal or change thereof. [2141] Embodiment 564. The system of embodiment 562 or 563, wherein the one or more controllers are individually or collectively further configured to extract one or more reads from (1) the electrical signal or change thereof or (2) the pre-processed electrical signal or change thereof. [2142] Embodiment 565. The system of any one of embodiments 562-564, wherein the one or more characteristics are assigned using (1) the one or more reads or (2) the one or more pre-processed reads (e.g., one or more segments). [2143] Embodiment 566. The system of any one of embodiments 562-566, wherein the one or more controllers are individually or collectively further configured to compare (e.g., align) (1) the one or more reads or (2) the one or more pre-processed reads to one or more reference signals in the database. [2144] Embodiment 567. The system of any one of embodiments 556-566, wherein the one or more characteristics are assigned using the electrical signal or change thereof. [2145] Embodiment 568. The system of any one of embodiments 556-567, wherein the database is generated from one or more reference sequences. [2146] Embodiment 569. The system of embodiment 568, wherein the one or more reference sequences are derived with genomic information or transcriptomic information of the sample. [2147] Embodiment 570. The system of embodiment 569, wherein the genomic information comprises genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. [2148] Embodiment 571. The system of embodiment 569 or 570, wherein the transcriptomic information comprises genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, -499- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. [2149] Embodiment 572. The system of any one of embodiments 556-571, wherein the database comprises one or more reference signals for the at least the portion of the analyte, at least one proteoform thereof, or at least one variant thereof, or fragments thereof, or combination thereof. [2150] Embodiment 573. The system of any one of embodiments 556-572, wherein the one or more polypeptides comprise one or more expressible polypeptides. [2151] Embodiment 574. The system of any one of embodiments 556-573, wherein the one or more controllers are individually or collectively further configured to, prior to (c), translocate at least a portion of an additional analyte through an additional nanopore disposed within an additional membrane, wherein the at least the portion of the additional analyte comprises at least a portion of an additional polypeptide, at least a portion of an additional protein, or at least a portion of an additional peptide, or fragments thereof, or combination thereof. [2152] Embodiment 575. The system of any one of embodiments 556-574, wherein the one or more controllers are individually or collectively further configured to, repeat (a) and (c) for a plurality of analytes, thereby generating a plurality of electrical signals. [2153] Embodiment 576. The system of any one of embodiments 547-575, wherein the one or more controllers are individually or collectively further configured to, in (b), detect the current or change thereof, and (c) comprises using the current or change thereof. [2154] Embodiment 577. The system of any one of embodiments 547-576, wherein the one or more controllers are individually or collectively further configured to, in (b), detect the voltage or change thereof, and (c) comprises using the voltage or change thereof. [2155] Embodiment 578. The system of any one of embodiments 547-577, wherein the one or more controllers are individually or collectively further configured to, in (b), detect the resistance or change thereof, and (c) comprises using the resistance or change thereof. [2156] Embodiment 579. The system of any one of embodiments 547-578, wherein the one or more controllers are individually or collectively further configured to, in (c), determine or assign the one or more characteristics of the at least the portion of the analyte based on (1) an electrical signal or change thereof of (i) the current or change thereof, (ii) the voltage or change thereof, or (iii) the resistance or change thereof, or (iv) any combination thereof, and (2) a database. [2157] Embodiment 580. The system of embodiment 579, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more -500- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 fragments thereof, or one or more proteoforms thereof, or one or more variants thereof, or a combination thereof. [2158] Embodiment 581. The system of any one of embodiments 547-580, wherein the electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the signal or change thereof. [2159] Embodiment 582. The system of any one of embodiments 547-581, wherein the electrical signal or change thereof comprises measurements of (1) the current or change thereof, (2) the voltage or change thereof, or (3) the resistance or change thereof, or (4) any combination thereof over a period of time. [2160] Embodiment 583. The system of embodiment 582, wherein the period of time comprises one or more portions associated with a measurement of concentration associated with a sample comprising the at least the portion of the analyte. [2161] Embodiment 584. The system of any one of embodiments 547-583, wherein the one or more controllers are individually or collectively further configured to in (c), pre-process (e.g., denoising (smoothing, frequency manipulations, FFT, wavelets), segment (edge-detecting algorithms, wavelet transforms, filtering) the electrical signal or change thereof, thereby generating a pre-processed electrical signal or change thereof. [2162] Embodiment 585. The system of embodiment 584, wherein the one or more controllers are individually or collectively further configured to extract one or more reads from (1) the electrical signal or change thereof or (2) the pre-processed electrical signal or change thereof. [2163] Embodiment 586. The system of embodiment 584 or 585, wherein the one or more characteristics is determined using (1) the one or more reads or (2) one or more pre-processed reads (e.g., one or more segment). [2164] Embodiment 587. The system of any one of embodiments 584-586, wherein the one or more controllers are individually or collectively further configured to compare (e.g., aligning) (1) the one or more reads or (2) the one or more pre-processed reads to one or more reference signals in the database. [2165] Embodiment 588. The system of any one of embodiments 547-587, wherein the database is generated from one or more reference sequences. [2166] Embodiment 589. The system of embodiment 588, wherein the one or more reference sequences are derived with genomic information and/or transcriptomic information of the sample [2167] Embodiment 590. The system of embodiment 589, wherein the genomic information comprises genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. [2168] Embodiment 591. The system of embodiment 589 or 590, Wherein the transcriptomic information comprises genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, -501- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, cellular origin information, or any combination thereof. [2169] Embodiment 592. The system of any one of embodiments 547-591, Wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms. [2170] Embodiment 593. The system of any one of embodiments 547-592, wherein the one or more controllers are individually or collectively further configured to, apply a voltage to translocate the at least the portion of the analyte. [2171] Embodiment 594. The system of any one of embodiments 547-593, wherein the first side is larger in size than the second side. [2172] Embodiment 595. The system of any one of embodiments 547-593, wherein the first side is the same size as the second side. [2173] Embodiment 596. The system of any one of embodiments 547-595, wherein the first side and/or the second side comprise an enclosed compartment. [2174] Embodiment 597. The system of any one of embodiments 547-596, wherein the first side and/or the second side comprise an open compartment. [2175] Embodiment 598. The system of any one of embodiments 547-597, wherein the first side is in an open compartment and the second side comprise an enclosed compartment. [2176] Embodiment 599. The system of embodiment 598, wherein the open compartment comprises a continuous aqueous phase. [2177] Embodiment 600. The system of any one of embodiments 547-599, wherein the nanopore system further comprises a pair of electrodes comprising a first electrode and a second electrode. [2178] Embodiment 601. The system of any one of embodiments 547-600, wherein a first solution of the first side and a second solution of the second side are configured to translocate the at least the portion of the analyte using an electro-osmotic flow. [2179] Embodiment 602. The system of embodiment 601, wherein translocating can comprise translocating the at least the portion of the analytes in the C-to-N direction and/or in the N-to-C direction relative to the at least the portion of the analytes sequence. [2180] Embodiment 603. The system of any one of embodiments 547-602, wherein the one or more controllers are individually or collectively further configured to, translocate at least a portion of an additional analyte through an additional nanopore disposed within an additional membrane. [2181] Embodiment 604. The system of any one of embodiments 547-603, wherein the one or more controllers are individually or collectively further configured to detect (i) (1) an additional current or change thereof, (2) an additional voltage or change thereof, or (3) an additional resistance or change thereof, or (4) -502- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 any combination thereof, or (ii) an additional electrical signal or change thereof while the at least the portion of the additional analyte is translocating through the additional nanopore. [2182] Embodiment 605. The system of embodiment 604, wherein the at least the portion of the analyte and the at least the portion of the additional analyte is among a sample. [2183] Embodiment 606. The system of embodiment 604 or 605, wherein the at least the portion of the analyte and the at least the portion of the additional analyte are different. [2184] Embodiment 607. The system of any one of embodiments 604-606, wherein the one or more controllers are individually or collectively further configured to determine a presence or absence of the at least the portion of the analyte in the sample. [2185] Embodiment 608. The system of any one of embodiments 604-607, wherein the sample comprises one or more types of analytes. [2186] Embodiment 609. The system of any one of embodiments 547-608, wherein the one or more controllers are individually or collectively further configured to, prior to (b), translocate a plurality of analytes through (i) the nanopore disposed within the membrane, and/or (ii) a plurality of nanopores disposed in a plurality of membranes. [2187] Embodiment 610. The system of embodiment 609, wherein the one or more controllers are individually or collectively further configured to repeat (b)-(c) for the plurality of analytes to generate a plurality of characteristics associated with the plurality of analytes. [2188] Embodiment 611. The system of embodiment 610, wherein the one or more controllers are individually or collectively further configured to generate the one or more characteristics associated with the at least the portion of the analyte. [2189] Embodiment 612. The system of embodiment 610 or 611, wherein the one or more controllers are individually or collectively further configured to use the plurality of characteristics to generate the one or more characteristics associated with the at least the portion of the analyte. [2190] Embodiment 613. The system of any one of embodiments 609-612, wherein the one or more controllers are individually or collectively further configured to determine a relative concentration or absolute concentration for one or more analytes in the plurality of analytes. [2191] Embodiment 614. The system of any one of embodiments 609-613, wherein the one or more controllers are individually or collectively further configured to determine a percentage of modified or different analytes in the plurality of analytes. [2192] Embodiment 615. The system of any one of embodiments 547-614, wherein the one or more characteristics comprises identity, natural or unnatural post-translational modifications, a length, an orientation, one or more translocation kinetics, a sequence, one or more mutations associated, a one or more -503- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 isoforms, one or more natural or unnatural modifications, or one or more charges, or any combination thereof associated with the at least the portion of the analyte. [2193] Embodiment 616. The system of any one of embodiments 547-615, wherein the one or more characteristics comprises determining whether the at least the portion of the analyte comprises one or more molecular entities. [2194] Embodiment 617. The system of embodiment 616, wherein the at least the portion of the analyte comprises one or more molecular entities. [2195] Embodiment 618. The system of embodiment 616 or 617, wherein the one or more characteristics comprises a property of the molecular entity coupled to the at least the portion of the analyte. [2196] Embodiment 619. The system of any one of embodiments 616-618, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, protein, or any combination thereof. [2197] Embodiment 620. The system of any one of embodiments 547-619, wherein the one or more characteristics comprises at least one property of one or more intra cross-linkages within the at least the portion of the analyte, one or more inter cross-linkages with at least the portion of another analyte, one or more covalent linkages with a molecular entity, or any combination thereof. [2198] Embodiment 621. The system of any one of embodiments 547-620, wherein the one or more characteristics comprises a category or identity associated with the at least the portion of the analyte. [2199] Embodiment 622. The system of any one of embodiments 547-621, wherein the one or more characteristics comprises one or more of a secondary structure, tertiary structure, quaternary structure, or a combination thereof associated with the at least the portion of the analyte. [2200] Embodiment 623. The system of any one of embodiments 547-622, wherein the nanopore system further comprises a pair of electrodes. [2201] Embodiment 624. The system of embodiment 623, wherein the pair of electrodes are configured to provide an applied voltage to generate an electrophoretic force (EPF). [2202] Embodiment 625. The system of embodiment 624, wherein a magnitude of the applied voltage is from about 20 mV to about 300 mV. [2203] Embodiment 626. The system of embodiment 624 or 625, wherein an absolute relative net electroosmotic current (e.g., through the nanopore or across the membrane) over the applied voltage is greater than about 0.10pA/mV. [2204] Embodiment 627. The system of any one of embodiments 547-626, wherein the first side comprises a first solution and the second side comprises a second solution, wherein the first solution and the second solution are configured to translocate the at least the portion of the analyte across the nanopore. -504- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2205] Embodiment 628. The system of embodiment 627, wherein the first solution and the second solution are configured to generate an electro-osmotic force across the membrane. [2206] Embodiment 629. The system of embodiment 628, wherein electro-osmotic force translocates the at least the portion of the analyte from the first side through the nanopore to the second side against an electrophoretic force acting in a direction opposite the electro-osmotic force. [2207] Embodiment 630. The system of embodiment 628 or 629, wherein the first solution and the second solution are configured to generate a first side to second side electro-osmotic force (EOF). [2208] Embodiment 631. The system of embodiment 630, wherein the first side to second side electro-osmotic force maintains the translocase of the complex at a first side entrance of a channel of the nanopore. [2209] Embodiment 632. The system of any one of embodiments 547-631, wherein the system further comprises a preloading solution configured to interact with the at least the portion of the analyte. [2210] Embodiment 633. The system of embodiment 632, wherein the preloading solution comprises one or more cofactors. [2211] Embodiment 634. The system of embodiment 632 or 633, wherein the preloading solution comprises a chemical that enhances a binding of the at least the portion of the analyte to a component of the preloading solution relative to binding in a solution of the first side of the fluidic chamber. [2212] Embodiment 635. The system of any one of embodiments 632-634, wherein the preloading solution comprises the translocase and/or a leader construct. [2213] Embodiment 636. The system of any one of embodiments 547-635, wherein the nanopore is a biological nanopore. [2214] Embodiment 637. The system of embodiment 636, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide. [2215] Embodiment 638. The system of embodiment 636, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide. [2216] Embodiment 639. The system of any one of embodiments 636-638, wherein the biological nanopore comprises one or more monomers comprising one or more mutations. [2217] Embodiment 640. The system of any one of embodiments 636-639, wherein the biological nanopore comprises Aerolysin (Aer), Cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, phage derived portal proteins, modified variants thereof, or ion- selective mutants thereof. [2218] Embodiment 641. The system of any one of embodiments 547-640, wherein the nanopore comprises an engineered CytK nanopore, MspA nanopore, or engineered CsgG nanopore. -505- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2219] Embodiment 642. The system of any one of embodiments 547-641, wherein the nanopore comprises at least a portion of a proteasome. [2220] Embodiment 643. The system of any one of embodiments 547-642, wherein the nanopore comprises one or more point mutations. [2221] Embodiment 644. The system of embodiment 643, wherein the one or more point mutations comprises affects a diameter of the biological nanopore, creates smaller openings on a first side or a second side of the biological nanopore, affects a charge of the biological nanopore, are one or more lumen facing mutations, or allow for conductance at a set pH, or any combination thereof. [2222] Embodiment 645. The system of any one of embodiments 547-644, wherein the current or change thereof is from about 0.1 pA to about 150 pA. [2223] Embodiment 646. The system of any one of embodiments 547-645, further comprising: one or more unfoldases, translocases, unfoldase domains, or translocase domains, or any combination thereof. [2224] Embodiment 647. The system of any one of embodiments 547-646, wherein the inner diameter of the nanopore is from about 0.5 nm to about 2 nm. [2225] Embodiment 648. The system of any one of embodiments 547-647, wherein the nanopore comprises an increase in aromatic rings in the lumen of the nanopore as compared to another nanopore without a modification. [2226] Embodiment 649. The system of any one of embodiments 547-648, wherein the at least the portion of the analyte comprises a linear length greater than a channel length of the nanopore. [2227] Embodiment 650. The system of any one of embodiments 547-649, wherein the at least the portion of the analyte comprises one or more leader constructs at a N-terminus or a C- terminus. [2228] Embodiment 651. The system of any one of embodiments 547-650, wherein the nanopore comprises an adaptor. [2229] Embodiment 652. The system of any one of embodiments 547-651, wherein the nanopore is coupled to one or more recognition elements. [2230] Embodiment 653. The system of any one of embodiments 547-652, wherein the nanopore comprises an inner pore constriction from about 0.5 nanometers (nm) to about 2 nm. [2231] Embodiment 654. The system of any one of embodiments 547-653, wherein the nanopore has an ion- selectivity P(+)/P(-) of greater than 2.0. [2232] Embodiment 655. The system of any one of embodiments 547-653, wherein the nanopore has an ion- selectivity P(+)/P(-) of less than 0.50. -506- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2233] Embodiment 656. The system of any one of embodiments 547-655, wherein the at least the portion of the analyte is at least 1 kDa. [2234] Embodiment 657. The system of any one of embodiments 547-656, wherein the at least the portion of the analyte is at least 100 amino acids. [2235] Embodiment 658. A system, comprising: a first nanopore system, wherein the first nanopore system comprises (1) a first fluidic chamber, and (2) a first membrane comprising a first nanopore, wherein the first membrane separates the first fluidic chamber into a first side and a second side; a second nanopore system, wherein the second nanopore system comprises (1) a second fluidic chamber, and (2) a second membrane comprising a second nanopore, wherein the second membrane separates the second fluidic chamber into a third side and a fourth side; and one or more controllers operatively coupled to the first nanopore system or the second nanopore system, wherein the one or more controllers are individually or collectively configured to: (a) translocate at least a portion of the first analyte through the first nanopore disposed within the first membrane and at least a portion of the second analyte through the second nanopore disposed within the second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or fragments thereof, or a combination thereof, wherein the first analyte and the second analyte are among a sample, (b) detect (i) (1) a first current or change thereof, (2) a first voltage or change thereof, or (3) a first resistance or change thereof, or (4) any combination thereof, while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (5) a second current or change thereof, (6) a second voltage or change thereof, or (7) a second resistance or change thereof, or (8) any combination thereof, while the at least the portion of the second analyte is translocating through the second nanopore; (c) use (i) (1) the first current or change thereof, (2) the first voltage or change thereof, or (3) the first resistance or change thereof, or (4) any combination thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (5) the second current or change thereof, (6) the second voltage or change thereof, or (7) the second resistance or change thereof, or (8) any combination thereof to determine a second characteristic of the at least the portion of the second analyte; and (d) characterize one or more properties of the sample using the first characteristic or the second characteristic determined in (c). [2236] Embodiment 659. The system of embodiment 658, wherein the first side and the third side are larger in size than the second side and the fourth side, respectively. [2237] Embodiment 660. The system of embodiment 658, wherein the first side and the third side the same size as the second side and the fourth side, respectively. -507- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2238] Embodiment 661. The system of any one of embodiments 658-660, wherein the first side, the second side, the third side, the fourth side, or any combination thereof comprise an enclosed compartment. [2239] Embodiment 662. The system of any one of embodiments 658-661, wherein the first side, the second side, the third side, the fourth side, or any combination thereof comprise an open compartment. [2240] Embodiment 663. The system of any one of embodiments 658-662, wherein the first side and/or the third side comprise an open compartment and the second side and/or fourth side comprise an enclosed compartment. [2241] Embodiment 664. The system of embodiment 662, wherein the open compartment comprises a continuous aqueous phase. [2242] Embodiment 665. The system of any one of embodiments 658-664, wherein the one or more controllers are individually or collectively further configured to, in (b), detect (1) the first current or change thereof and (2) the second current or change thereof, and (d) comprises using (1) the first current or change thereof and (2) the second current or change thereof. [2243] Embodiment 666. The system of any one of embodiments 658-665, wherein the one or more controllers are individually or collectively further configured to, in (c), use (1) the first voltage or change thereof and (2) the second voltage or change thereof, and (d) comprises using (1) the first voltage or change thereof and (2) the second voltage or change thereof. [2244] Embodiment 667. The system of any one of embodiments 658-666, wherein the one or more controllers are individually or collectively further configured to, in (b) translocate the at least the portion of the first analyte or the at least the portion of the second analyte in the C-to-N direction and/or in the N-to-C direction relative to the at least the portion of the analytes sequence. [2245] Embodiment 668. The system of any one of embodiments 658-667, wherein the one or more controllers are individually or collectively further configured to, in (c), determine (A) the first characteristic based on (1) a first electrical signal or change thereof of the first current or change thereof, the first voltage or change thereof, or the first resistance or change thereof, or any combination thereof, and (2) a database, or (B) the second characteristic based on (1) a second electrical signal or change thereof of the second current or change thereof, the second voltage or change thereof, or the second resistance or change thereof, or any combination thereof, and (2) the database. [2246] Embodiment 669. The system of embodiment 668, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. -508- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2247] Embodiment 670. The system of embodiment 668 or 669, wherein the first electrical signal or change thereof or the second electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the signal or change thereof. [2248] Embodiment 671. The system of any one of embodiments 668-670, wherein (1) the first electrical signal or change thereof comprises measurements of the first current or change thereof, the first voltage or change thereof, or the first resistance or change thereof, or any combination thereof over a first period of time; or (2) the second electrical signal or change thereof comprises measurements of the second current or change thereof, the second voltage or change thereof, or the second resistance or change thereof, or any combination thereof over a second period of time. [2249] Embodiment 672. The system of any one of embodiments 668-671, wherein the one or more controllers are individually or collectively further configured to, in (c), pre-process (e.g., denoising, segmenting) the first electrical signal or change thereof or the second electrical signal or change thereof, thereby generating (1) a pre-processed first electrical signal or change thereof or (2) a pre-processed second electrical signal or change thereof. [2250] Embodiment 673. The system of embodiment 672, wherein the first characteristic or the second characteristic are determined using (1) the pre-processed first electrical signal or change thereof, or the (2) the pre-processed second electrical signal or change thereof, respectively. [2251] Embodiment 674. The system of embodiment 672 or 673, wherein the one or more controllers are individually or collectively further configured to extract (A) one or more first reads from (1) the first electrical signal or change thereof or (2) the pre-processed first electrical signal or change thereof; or (B) one or more second reads from (1) the second electrical signal or change thereof or (2) the pre-processed second electrical signal or change thereof. [2252] Embodiment 675. The system of any one of embodiments 672-674, wherein the first characteristic or the second characteristic is determined using (1) the one or more first reads or the one or more pre-processed first reads (e.g., one or more first segments); or (2) the one or more second reads or the one or more pre- processed second reads (e.g., one or more second segments). [2253] Embodiment 676. The system of embodiment 675, wherein the one or more controllers are individually or collectively further configured to compare (e.g., aligning) (1) the one or more first reads, the one or more pre-processed first reads, the one or more second reads, the one or more pre-processed second reads to (2) one or more reference signals in the database. [2254] Embodiment 677. The system of any one of embodiments 668-676, wherein the first characteristic or the second characteristic is determined using the first electrical signal or change thereof, or the second electrical signal or change thereof. -509- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2255] Embodiment 678. The system of any one of embodiments 668-677, wherein the database is generated from one or more reference sequences. [2256] Embodiment 679. The system of embodiment 678, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms. [2257] Embodiment 680. The system of any one of embodiments 668-679, wherein the database comprises one or more reference signals for the at least the portion of the analyte, at least one proteoform thereof, at least one variant thereof, or combination. [2258] Embodiment 681. The system of any one of embodiments 678-680, Wherein the one or more reference sequences are derived with genomic information or transcriptomic information of the sample. [2259] Embodiment 682. The system of any one of embodiments 658-681, wherein the one or more controllers are individually or collectively further configured to determine the first characteristic or the second characteristic with an accuracy of at least 60%. [2260] Embodiment 683. The system of any one of embodiments 658-682, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, (ii) the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s, or (iii) a step size is from about 0.5 amino acids to about 5 amino acids, (iv) or any combination thereof. [2261] Embodiment 684. The system of any one of embodiments 658-683, wherein the characterizing the one or more properties comprises determining at least one feature of a proteome associated with the sample. [2262] Embodiment 685. The system of embodiments 684, wherein (i) proteome coverage is at least 1% or (ii) sequence coverage of the at least the portion of the first polypeptide or the at least the portion of the second polypeptide is at least 1%. [2263] Embodiment 686. The system of any one of embodiments 658-685, wherein the first nanopore and the second nanopore are the same nanopore. [2264] Embodiment 687. The system of any one of embodiments 658-685, wherein the first nanopore and the second nanopore are different nanopores. [2265] Embodiment 688. The system of any one of embodiments 658-687, wherein the first membrane and the second membrane are the same membranes. [2266] Embodiment 689. The system of any one of embodiments 658-687, wherein the first membrane and the second membrane are different membranes. [2267] Embodiment 690. The system of any one of embodiments 658-689, wherein the sample comprises a first type of analyte and a second type of analyte. -510- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2268] Embodiment 691. The system of embodiment 690, wherein the first type of analyte and the second type of analyte are different types. [2269] Embodiment 692. The system of embodiments 690 or 691, wherein the first type of analyte comprises the at least the portion of the first analyte and the second type of analyte comprises the at least the portion of the second analyte. [2270] Embodiment 693. The system of any one of embodiments 690-692, wherein the characterizing the one or more properties comprises determining the number of analytes in the first type of analyte and determining the number of analytes in the second type of analyte. [2271] Embodiment 694. The system of any one of embodiments 690-693, wherein the one or more controllers are individually or collectively further configured to determine in the first type of analyte or the second type of analyte one or more of a number of analytes, relative abundance of analytes in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, one or more impurities in the sample, or a combination thereof. [2272] Embodiment 695. The system of any one of embodiments 658-689, wherein the sample comprises one type of analyte. [2273] Embodiment 696. The system of any one of embodiments 690-695, wherein the at least the portion of the first analyte and the at least the portion of the second analyte are from the same analyte type. [2274] Embodiment 697. The system of any one of embodiments 658-696, wherein the one or more controllers are individually or collectively further configured to determine one or more of a number of analytes in the sample, relative abundance of analytes in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, one or more impurities in the sample, or a combination thereof. [2275] Embodiment 698. The system of any one of embodiments 658-697, wherein the one or more controllers are individually or collectively further configured to repeat (a)-(d) for a plurality of analytes. [2276] Embodiment 699. The system of embodiment 698, wherein the one or more controllers are individually or collectively further configured to repeat (a)-(d) with the plurality of analytes and a plurality of nanopores disposed within a plurality of membranes. [2277] Embodiment 700. The system of embodiments 698 or 699, wherein the one or more controllers are individually or collectively further configured to characterize one or more properties of the sample using a plurality of characteristics associated with the plurality of analytes. -511- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2278] Embodiment 701. The system of embodiment 700, wherein the one or more properties comprises an absolute or relative abundance, absolute concentration, relative concentration, or origin of one or more analyte types in the sample. [2279] Embodiment 702. The system of embodiment 700 or 701, wherein the one or more properties comprises differences between at least a subset of analytes of the plurality of analytes. [2280] Embodiment 703. The system of any one of embodiments 700-702, wherein the one or more properties comprises a presence, an absence, quantitation, or identification of one or more proteins, polypeptides, or peptides, or fragments thereof, or any combination thereof in the sample. [2281] Embodiment 704. The system of any one of embodiments 658-703, wherein the first characteristic or second characteristic comprises natural and/or unnatural post-translational modifications. [2282] Embodiment 705. The system of embodiment 704, wherein the first characteristic or second characteristic comprises identity, natural or unnatural post-translational modifications, a length, an orientation, one or more translocation kinetics, a sequence, one or more mutations associated, a one or more isoforms, one or more natural or unnatural modifications, or one or more charges, or any combination thereof associated with the at least the portion of the analyte. [2283] Embodiment 706. The system of any one of embodiments 658-705, wherein the first characteristic or second characteristic comprises determining whether the at least the portion of the analyte comprises one or more molecular entities. [2284] Embodiment 707. The system of embodiment 706, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more molecular entities. [2285] Embodiment 708. The system of embodiment 706 or 707, wherein the first characteristic or second characteristic comprises a property of the molecular entity coupled to the at least the portion of the analyte. [2286] Embodiment 709. The system of any one of embodiments 706-708, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, protein, or any combination thereof. [2287] Embodiment 710. The system of any one of embodiments 658-709, wherein the first characteristic or second characteristic comprises at least one property of one or more intra cross-linkages within the at least the portion of the analyte, one or more inter cross-linkages with at least the portion of another analyte, one or more covalent linkages with a molecular entity, or any combination thereof. [2288] Embodiment 711. The system of any one of embodiments 658-710, wherein the first characteristic or second characteristic comprises a category or identity associated with the at least the portion of the analyte. -512- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2289] Embodiment 712. The system of any one of embodiments 658-711, wherein the first characteristic or second characteristic comprises one or more of a secondary structure, tertiary structure, quaternary structure, or a combination thereof associated with the at least the portion of the analyte. [2290] Embodiment 713. The system of any one of embodiments 658-712, wherein the first nanopore system further comprises a first pair of electrodes or the second nanopore system further comprises a second pair of electrodes. [2291] Embodiment 714. The system of any one of embodiments 658-713, wherein the first nanopore system and the second nanopore system share one or more common electrodes. [2292] Embodiment 715. The system of embodiment 713, wherein the first pair of electrodes or the second pair of electrodes are configured to provide a first applied voltage or a second applied voltage to generate a first electrophoretic force (EPF) or a second EPF. [2293] Embodiment 716. The system of embodiment 713 or 715, wherein a first absolute relative net electroosmotic current or a second absolute relative net electroosmotic current over the first applied voltage or the second applied voltage is greater than about 0.10pA/mV. [2294] Embodiment 717. The system of embodiment 715, wherein the one or more controllers are individually or collectively further configured to provide (i) the first EPF acting in an opposite direction to a first side to second side electro-osmotic force, or (ii) the second EPF acting in an opposite direction to a side to additional side electro-osmotic force. [2295] Embodiment 718. The system of any one of embodiments 658-717, wherein (i) the first nanopore system further comprises a first controller operatively coupled to the first fluidic chamber and the first nanopore; or (ii) wherein the second nanopore system further comprises a second controller operatively coupled to the second fluidic chamber and the second nanopore. [2296] Embodiment 719. The system of any one of embodiments 658-718, wherein (i) the first side comprises a first solution and the second side comprises a second solution, or (ii) the side comprises a solution and the additional side comprises an additional solution. [2297] Embodiment 720. The system of embodiment 719, wherein (i) the first solution and the second solution are configured to translocate the at least the portion of the first analyte across the first nanopore, or (ii) the solution and the additional solution are configured to translocate the at least the portion of the second analyte across the second nanopore. [2298] Embodiment 721. The system of any one of embodiments 658-720, wherein (i) a first electro-osmotic force translocates the at least the portion of the first analyte from the first side through the first nanopore to the second side against a first electrophoretic force acting in a direction opposite the first electro-osmotic force; or (ii) a second electro-osmotic force translocates the at least the portion of the second analyte from the side -513- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 through the second nanopore to the additional side against a second electrophoretic force acting in a direction opposite the second electro-osmotic force. [2299] Embodiment 722. The system of any one of embodiments 658-721, wherein (i) the first nanopore system further comprises a first preloading solution configured to interact with the at least the portion of the first analyte; or (ii) the second nanopore system further comprises a second preloading solution configured to interact with the at least the portion of the second analyte. [2300] Embodiment 723. The system of embodiment 722, wherein (i) the first preloading solution comprises the first translocase or a first leader construct; or (ii) the second preloading solution comprises the second translocase or a second leader construct. [2301] Embodiment 724. The system of any one of embodiments 658-723, wherein the first nanopore or the second nanopore is a biological nanopore. [2302] Embodiment 725. The system of embodiment 724, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide. [2303] Embodiment 726. The system of embodiment 724, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide. [2304] Embodiment 727. The system of any one of embodiments 724-726, wherein the biological nanopore comprises one or more monomers comprising one or more mutations. [2305] Embodiment 728. The system of any one of embodiments 724-727, wherein the biological nanopore comprises Aerolysin (Aer), Cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, phage derived portal proteins, modified variants thereof, or ion- selective mutants thereof. [2306] Embodiment 729. The system of any one of embodiments 724-728, wherein the first nanopore or the second nanopore comprises a mutant CytK nanopore, MspA nanopore, or an engineered CsgG nanopore. [2307] Embodiment 730. The system of any one of embodiments 658-729, wherein the first nanopore or the second nanopore comprises at least a portion of a proteasome. [2308] Embodiment 731. The system of any one of embodiments 724-727, wherein the nanopore comprises one or more point mutations. [2309] Embodiment 732. The system of embodiment 731, wherein the one or more point mutations comprises affects a diameter of the biological nanopore, creates smaller openings on any side of the biological nanopore, affects a charge of the biological nanopore, are one or more lumen facing mutations, or allow for conductance at a set pH, or any combination thereof. [2310] Embodiment 733. The system of any one of embodiments 658-732, wherein the first current or change thereof or the second current or change thereof is from about 0.1 pA to about 150 pA. -514- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2311] Embodiment 734. The system of any one of embodiments 658-733, further comprising: one or more unfoldases, translocases, unfoldase domains, or translocase domains, or any combination thereof. [2312] Embodiment 735. The system of any one of embodiments 658-734, wherein the inner diameter of the first nanopore and/or the second nanopore is from about 0.5 nm to about 2 nm. [2313] Embodiment 736. The system of any one of embodiments 658-735, wherein the first nanopore and/or the second nanopore comprises an increase in aromatic rings in the lumen of the first nanopore and/or the second nanopore as compared to another nanopore without a modification. [2314] Embodiment 737. The system of any one of embodiments 658-736, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises a linear length greater than a channel length of the first nanopore and/or the second nanopore, respectively. [2315] Embodiment 738. The system of any one of embodiments 658-737, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte comprises one or more leader constructs at a N-terminus or a C- terminus. [2316] Embodiment 739. The system of any one of embodiments 658-738, wherein the first nanopore and/or the second nanopore comprises an adaptor. [2317] Embodiment 740. The system of any one of embodiments 658-739, wherein the first nanopore and/or the second nanopore is coupled to one or more recognition elements. [2318] Embodiment 741. The system of any one of embodiments 658-740, wherein the first nanopore and/or the second nanopore comprises an inner pore constriction from about 0.5 nanometers (nm) to about 2 nm. [2319] Embodiment 742. The system of any one of embodiments 658-741, wherein the first nanopore and/or the second nanopore has an ion-selectivity P(+)/P(-) of greater than 2.0. [2320] Embodiment 743. The system of any one of embodiments 658-741, wherein the first nanopore and/or the second nanopore has an ion-selectivity P(+)/P(-) of less than 0.50. [2321] Embodiment 744. The system of any one of embodiments 658-743, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is at least 1 kDa. [2322] Embodiment 745. The system of any one of embodiments 658-744, wherein the at least the portion of the first analyte and/or the at least the portion of the second analyte is at least 100 amino acids. [2323] Embodiment 746. A device comprising an array of a system comprising the system according to any one of embodiments 547-745. [2324] Embodiment 747. The use of the method of any one of embodiments 1-546 for detection and analysis of one or more analytes at a single molecule level. -515- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 [2325] Embodiment 748. The use of the system of any one of embodiments 547-745 for detection and analysis of one or more analytes at a single molecule level. [2326] Embodiment 749. The method of any one of embodiments 1-546, wherein the at least the portion of the analyte is coupled to a stopper domain on one end of the at least the portion analyte and/or a leader construct on the other end of the at least the portion analyte. [2327] Embodiment 750. The system of any one of embodiments 547-745, wherein the at least the portion of the analyte is coupled to a stopper domain on one end of the at least the portion analyte and/or a leader construct on the other end of the at least the portion analyte. [2328] Embodiment 751. The method of any one of embodiments 1-546, further comprising translocating the analyte from a first side to a second side of the nanopore and/or from a second side to a first side of the nanopore. [2329] Embodiment 752. The method of any one of embodiments 1-546, further comprising translocating the at least the portion of the analyte from the first side to the second side. [2330] Embodiment 753. The method of any one of embodiments 1-546, wherein the at least the portion of the analyte is coupled to leader construct, wherein the leader construct comprises a stopper domain. [2331] Embodiment 754. The system of any one of embodiments 547-745, wherein the at least the portion of the analyte is coupled to leader construct, wherein the leader construct comprises a stopper domain. [2332] Embodiment 755. The method of any one of embodiments 1-546, further comprising translocating the analyte from a first side to a second side of the nanopore and/or from a second side to a first side of the nanopore. [2333] Embodiment 756. The method of any one of embodiments 1-546, wherein the at least the portion of the analyte is coupled to a plurality of translocases. [2334] Embodiment 757. The system of any one of embodiments 547-745, wherein the at least the portion of the analyte is coupled to a plurality of translocases. [2335] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. -516- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. -517- WSGR Docket No.64828-710.601

Claims

WSGR Docket Number: 64828-710.601 CLAIMS WHAT IS CLAIMED IS: 1. A method, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte with an accuracy of at least 60%. 2. The method of claim 1, wherein (c) comprises using (1) the current or change thereof, or (2) the voltage or change thereof to determine the characteristic of the at least the portion of the analyte with an accuracy of at least 80%. 3. A method for determining a characteristic of an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof, wherein (i) an average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second, or (ii) an average rate of translocation is between about 0.1 nm/s to about 10000 nm/s; (b) detecting (1) a current or change thereof; or (2) a voltage or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) using (1) the current or change thereof, or (2) the voltage or change thereof detected in (b) to determine one or more characteristics of the at least the portion of the analyte. 4. The method of claim 3, wherein the average rate of translocation is between about 0.1 amino acids per second to about 35000 amino acids per second. 5. The method of claim 4, wherein the average rate of translocation is between about 1 amino acids per second to about 100 amino acids per second with a motor protein. 6. The method of claim 4, wherein the average rate of translocation is between about 500 amino acids per second to about 5000 amino acids per second without a motor protein. -518- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 7. The method of any one of claims 3-6, wherein the average rate of translocation is between about 0.1 nm/s to about 10000 nm/s. 8. The method of claim 7, wherein the average rate of translocation is between about 0.3 nm/s to about 30 nm/s. 9. The method of any one of claims 1-8, further comprising in (b) detecting the current or change thereof, and (c) comprises using the current or change thereof. 10. The method of any one of claims 1-9, further comprising in (b) detecting the voltage or change thereof, and (c) comprises using the voltage or change thereof. 11. The method of any one of claims 1-10, further comprising, in (c), determining/assigning the one or more characteristics of the at least the portion of the analyte based on (1) an electrical signal or change thereof of (i) the current or change thereof, or (ii) the voltage or change thereof, and (2) a database. 12. The method of claim 11, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. 13. A method for characterizing an analyte, comprising: (a) translocating at least a portion of an analyte through a nanopore disposed within a membrane, wherein the at least the portion of the analyte comprises at least a portion of a protein, at least a portion of a polypeptide, or at least a portion of a peptide, or fragments thereof, or a combination thereof; (b) detecting an electrical signal or change thereof while the at least the portion of the analyte is translocating through the nanopore; and (c) assigning one or more characteristics to the at least the portion of the analyte based on the electrical signal and a database, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. 14. The method of claim 13, wherein the database does not comprise a reference signal associated with the at least the portion of the analyte. 15. The method of claim 13, wherein the database comprises a reference signal associated with the at least the portion of the analyte. 16. The method of any one of claims 13-15, wherein the electrical signal or change thereof may be a measurement of (1) the current or change thereof or (2) the voltage or change thereof. -519- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 17. The method of any one of claims 13-16, wherein the electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the electrical signal or change thereof. 18. The method of claims 17, wherein the one or more additional portions of the electrical signal or change thereof comprises one or more blocks of impurities. 19. The method of any one of claims 13-18, wherein the electrical signal or change thereof comprises measurements of (1) the current or change thereof or (2) the voltage or change thereof over a period of time. 20. The method of claims 19, wherein the period of time comprises one or more portions associated with a measurement of concentration associated with a sample comprising the at least the portion of the analyte. 21. The method of any one of claims 13-20, further comprising, prior to (c), pre-processing the electrical signal or change thereof, thereby generating a pre-processed electrical signal or change thereof. 22. The method of claims 21, wherein the one or more characteristics are assigned using the pre- processed electrical signal or change thereof. 23. The method of claims 21 or 22, further comprising extracting one or more reads from (1) the electrical signal or change thereof or (2) the pre-processed electrical signal or change thereof. 24. The method of claim 23, further comprising pre-processing the one or more reads, thereby generating one or more pre-processed reads. 25. The method of claim 24, wherein the pre-processing comprises denoising, filtering, segmenting, or scaling, or a combination thereof. 26. The method of any one of claims 22-25, wherein the one or more characteristics are assigned using (1) the one or more reads or (2) the one or more pre-processed reads (e.g., one or more segments). 27. The method of claim 26, further comprising comparing (1) the one or more reads or (2) the one or more pre-processed reads to one or more reference signals in the database. 28. The method of claim 27, wherein the comparing comprises alignment. 29. The method of claim 28, wherein the alignment comprises time warping 30. The method of any one of claims 24-29, further comprising scoring (1) the one or more reads or (2) the one or more pre-processed reads to the one or more reference signals, thereby assigning the one or more characteristics to the at least the portion of the analyte. 31. The method of claim 30, wherein scoring comprises aligning at least a portion of the electrical signal of change thereof with the at least the portion of the one or more reference signals. -520- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 32. The method of any one of claims 24-31, further comprising aggregating (1) the one or more reads or (2) the one or more pre-processed reads to assignment the one or more characteristics to the at least the portion of the analyte. 33. The method of any one of claims 13-32, wherein the one or more characteristics are assigned using the electrical signal or change thereof. 34. The method of any one of claims 13-32, wherein the database is generated from one or more reference sequences. 35. The method of claim 77, wherein the one or more reference sequences are derived with genomic information or transcriptomic information of the sample. 36. The method of claim 35, wherein the genomic information comprises genome sequencing information (e.g., DNA) related to polynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. 37. The method of claim 35 or 36, wherein the transcriptomic information comprises genome sequencing information (e.g., RNA) related to ribopolynucleic acid sequences, abundance, number of copies of sequences, base modifications of sequences, 3D structural representations of sequences, or cellular origin information, or any combination thereof. 38. The method of any one of claims 34-37, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms. 39. The method of any one of claims 15-38, wherein the database comprises one or more reference signals for the at least the portion of the analyte, or fragments thereof, or at least one proteoform thereof, or at least one variant thereof, or combination thereof. 40. The method of any one of claims 13-39, wherein the one or more polypeptides comprise one or more expressible polypeptides. 41. The method of any one of claims 13-40, wherein the one or more variants thereof comprise one or more post-translationally modified variants thereof. 42. The method of any one of claims 1-41, wherein translocating can comprise translocating the at least the portion of the analytes in the C-to-N direction or in the N-to-C direction relative to the at least the portion of the analytes sequence. 43. The method of any one of claims 1-42, wherein the one or more characteristics comprises a plurality of natural or unnatural post-translational modifications of the at least the portion of the analyte. 44. The method of claim 43, wherein the natural or unnatural post-translation modifications is phosphorylation, glycosylation, deamidation, or acetylation, or any combination thereof. -521- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 45. The method of any one of claims 1-44, wherein the one or more characteristics comprises a length of the at least the portion of the analyte. 46. The method of any one of claims 1-45, wherein the one or more characteristics comprises an average speed of translocation of the at least the portion of the analyte through the nanopore 47. The method of any one of claims 1-46, wherein the one or more characteristics comprises determining whether the at least the portion of the analyte comprises one or more molecular entities 48. The method of claim 47, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof. 49. The method of any one of claims 1-48, wherein the one or more characteristics comprises a category or identity associated with the at least the portion of the analyte. 50. The method of any one of claims 1-49, wherein the one or more characteristics comprises one or more of a secondary structure, tertiary structure, or quaternary structure, or a combination thereof associated with the at least the portion of the analyte. 51. The method of any one of claims 1-50, wherein the one or more characteristics comprises a sequence associated with the at least the portion of the analyte. 52. The method of any one of claims 1-51, wherein the one or more characteristics comprises a one or more mutations associated with the at least the portion of the analyte. 53. The method of any one of claims 1-52, wherein the one or more characteristics comprises a one or more isoforms associated with the at least the portion of the analyte. 54. The method of any one of claims 1-53, further comprising, prior to (a), providing: (i) a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and; (2) a membrane comprising the nanopore, wherein the membrane separates the fluidic chamber into a first side and a second side. 55. The method claim 54, wherein the first side comprises a first solution and the second side comprises a second solution, wherein the first solution and the second solution are configured to translocate the at least the portion of the analyte across the nanopore. 56. The method of claim 55, wherein the first solution and the second solution are configured to generate an electro-osmotic force across the membrane. 57. The method of claim 56, wherein electro-osmotic force translocates the at least the portion of the analyte from the first side through the nanopore to the second side against an electrophoretic force acting in a direction opposite the electro-osmotic force. -522- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 58. The method of any one of claims 54-57, further comprising, prior to (a) contacting a complex comprising the at least the portion of the analyte and a translocase with the first side of the nanopore. 59. The method of any one of claims 1-58, wherein the nanopore is a biological nanopore. 60. The method of claim 59, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide. 61. The method of claim 59, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide. 62. The method of any one of claims 59-61, wherein the biological nanopore comprises Aerolysin (Aer), Cytolysin K (CytK), MspA, alpha-hemolysin (aHL), CsgG, Fragaceatoxin C (FraC), Lysenin, OmpF, OmpG, FhuA, or phage derived portal proteins, or fragments thereof, or modified variants thereof, or ion-selective mutants thereof. 63. The method of any one of claims 1-62, wherein the nanopore comprises a engineered CytK nanopore. 64. The method of any one of claims 1-62, wherein the nanopore comprises an engineered MspA nanopore or an engineered CsgG nanopore. 65. The method of any one of claims 1-64, further comprising, prior to (a), unfolding the analyte with one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof. 66. The method of any one of claims 1-65, wherein the inner diameter of the nanopore is from about 0.5 nm to about 2 nm. 67. The method of any one of claims 1-66, wherein the at least the portion of the analyte comprises a linear length greater than a channel length of the nanopore. 68. The method of any one of claims 1-67, wherein the at least the portion of the analyte comprises one or more leader constructs at a N-terminus or a C-terminus. 69. The method of any one of claims 1-68, wherein the nanopore comprises an adaptor. 70. The method of any one of claims 1-69, wherein the nanopore is coupled to one or more recognition elements 71. The method of any one of claims 1-70, wherein the at least the portion of the analyte is at least 100 amino acids 72. A method for sample analysis, comprising: (a) providing a sample comprising a plurality of analytes, wherein the plurality of analytes comprises a first analyte and a second analyte; -523- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 (b) translocating at least a portion of the first analyte through a first nanopore disposed within a first membrane and at least a portion of the second analyte through a second nanopore disposed within a second membrane, wherein the at least a portion of the first analyte comprises at least a portion of a first protein, at least a portion of a first polypeptide, or at least a portion of a first peptide, or first fragments thereof, or a combination thereof, wherein the at least a portion of the second analyte comprises at least a portion of a second protein, at least a portion of a second polypeptide, or at least a portion of a second peptide, or second fragments thereof, or a combination thereof; (c) detecting (i) (1) a first current or change thereof or (2) a first voltage or change thereof while the at least the portion of the first analyte is translocating through the first nanopore, and (ii) (3) a second current or change thereof or (4) a second voltage or change thereof while the at least the portion of the second analyte is translocating through the second nanopore; (d) using (i) (1) the first current or change thereof or (2) the first voltage or change thereof to determine a first characteristic of the at least the portion of the first analyte and (ii) (3) the second current or change thereof or (4) the second voltage or change thereof to determine a second characteristic of the at least the portion of the second analyte; and (e) characterizing one or more properties of the sample using the first characteristic or the second characteristic determined in (d). 73. The method of claim 72, wherein the at least the portion of the first analyte or the at least the portion of the second analyte is at least 100 amino acids. 74. The method of claim 72 or 73, further comprising, in (d), determining (A) the first characteristic based on (1) a first electrical signal or change thereof of the first current or change thereof or the first voltage or change thereof and (2) a database, or (B) the second characteristic based on (1) a second electrical signal or change thereof of the second current or change thereof or the second voltage or change thereof and (2) the database. 75. The method of claim 74, wherein the database comprises one or more reference signals for one or more polypeptides, one or more proteins, or one or more peptides, or one or more proteoforms thereof, or one or more variants thereof, or one or more fragments thereof, or a combination thereof. 76. The method of claim 74 or 75, wherein the first electrical signal or change thereof or the second electrical signal or change thereof comprises (1) one or more reads; or (2) one or more additional portions of the signal or change thereof. 77. The method of any one of claims 74-76, wherein (1) the first electrical signal or change thereof comprises measurements of the first current or change thereof or the first voltage or change thereof -524- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 over a first period of time; or (2) the second electrical signal or change thereof comprises measurements of the second current or change thereof or the second voltage or change thereof over a second period of time. 78. The method of any one of claims 74-77, further comprising, in (d), pre-processing the first electrical signal or change thereof or the second electrical signal or change thereof, thereby generating (1) a pre-processed first electrical signal or change thereof or (2) a pre-processed second electrical signal or change thereof. 79. The method of claim 78, further comprising extracting (A) one or more first reads from (1) the first electrical signal or change thereof or (2) the pre-processed first electrical signal or change thereof; or (B) one or more second reads from (1) the second electrical signal or change thereof or (2) the pre- processed second electrical signal or change thereof. 80. The method of claim 78 or 79, wherein the first characteristic or the second characteristic is determined using (1) the one or more first reads or one or more pre-processed first reads; or (2) the one or more second reads or one or more pre-processed second reads. 81. The method of claim 80, further comprising comparing (1) the one or more first reads, the one or more pre-processed reads, the one or more second reads, the one or more pre-processed second reads to (2) one or more reference signals in the database 82. The method of any one of claims 72-81, wherein the database is generated from one or more reference sequences. 83. The method of claim 82, wherein the one or more reference sequences are derived with genomic information or transcriptomic information of the sample. 84. The method of claim 82 or 83, wherein the database is generated from the one or more reference sequences using one or more machine learning algorithms. 85. The method of any one of claims 72-84, wherein the database comprises one or more reference signals for the at least the portion of the analyte, or at least one fragment thereof, or at least one proteoform thereof, or at least one variant thereof, or combination thereof. 86. The method of any one of claims 72-85, wherein the characterizing the one or more properties comprises determining at least one feature of a proteome associated with the sample. 87. The method of claim 86, wherein (i) proteome coverage is at least 1% or (ii) sequence coverage of the at least the portion of the first polypeptide or the at least the portion of the second polypeptide is at least 1%. 88. The method of any one of claims 72-87, wherein the first nanopore and the second nanopore are different nanopores. -525- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 89. The method of any one of claims 72-88, wherein the first membrane and the second membrane are different membranes 90. The method of any one of claims 72-89, wherein the sample comprises a first type of analyte and a second type of analyte. 91. The method of claim 90, wherein the first type of analyte and the second type of analyte are different types. 92. The method of claim 90 or 91, wherein the first type of analyte comprises the at least the portion of the first analyte and the second type of analyte comprises the at least the portion of the second analyte. 93. The method of any one of claims 72-92, wherein the characterizing the one or more properties comprises determining the number of analytes in the first type of analyte and determining the number of analytes in the second type of analyte. 94. The method of any one of claims 72-93, wherein the sample comprises one type of analyte. 95. The method of claim 94, wherein the at least the portion of the first analyte and the at least the portion of the second analyte are from the same analyte type. 96. The method of claim 94 or 95, further comprising determining one or more of a number of analytes in the sample, relative abundance of analytes in the sample, an absolute abundance of analytes in the sample, identification of origins of the analytes in the sample, analytes with secondary structures, analytes with tertiary structures, analytes with quaternary structures, or one or more impurities in the sample, or a combination thereof. 97. The method of any one of claims 72-96, further comprising characterizing one or more properties of the sample using a plurality of characteristics associated with the plurality of analytes. 98. The method of any one of claims 72-97, wherein the one or more properties comprises an absolute or relative abundance, absolute concentration, relative concentration, or origin of one or more analyte types in the sample. 99. The method of any one of claims 72-98, wherein the one or more properties comprises an absolute or relative abundance, absolute concentration, relative concentration, or origin of one or more analytes in the sample. 100. The method of any one of claims 72-99, wherein the one or more properties comprises differences between at least a subset of analytes of the plurality of analytes. 101. The method of any one of claims 72-100, wherein the one or more properties comprises differences in sequence of at most 10 units between at least a subset of analytes of the plurality of analytes. -526- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 102. The method of any one of claims 72-101, wherein the one or more properties comprises a percentage of modified or different analytes in the plurality of analytes. 103. The method of any one of claims 72-102, wherein the one or more properties comprises a quantification of one or more proteins, polypeptides or peptides in the sample. 104. The method of any one of claims 72-103, wherein the one or more properties comprises a quantification of one or more protein, polypeptide or peptide types in the sample. 105. The method of any one of claims 72-104, wherein the one or more properties comprises identification of a type associated with the sample or with an origin of the sample 106. The method of any one of claims 72-105, wherein the first characteristic or second characteristic comprises a plurality of natural or unnatural post-translational modifications of the at least the portion of the analyte. 107. The method of claim 106, wherein the natural or unnatural post translational modification is phosphorylation, glycosylation, deamidation, or acetylation, or any combination thereof. 108. The method of any one of claims 72-107, wherein the first characteristic or second characteristic comprises a length of the at least the portion of the analyte 109. The method of any one of claims 72-108, wherein the first characteristic or second characteristic comprises an average speed of translocation of the at least the portion of the analyte through the nanopore 110. The method of any one of claims 72-109, wherein the first characteristic or second characteristic comprises determining whether the at least the portion of the analyte comprises one or more molecular entities. 111. The method of claim 110, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more molecular entities. 112. The method of claim 110 or 111, wherein the one or more molecular entities is a compound (e.g., drug, small molecule), particle, nucleic acid, polynucleic acid, peptide, polynucleotide, or protein, or fragments thereof, or any combination thereof. 113. The method of any one of claims 72-112, wherein the first characteristic or second characteristic comprises a category or identity associated with the at least the portion of the first analyte or the at least the portion of the second analyte. 114. The method of any one of claims 72-113, further comprising, prior to (b), providing: (i) a first nanopore system, wherein the first nanopore system comprises (1) a first fluidic chamber and (2) the first membrane comprising the first nanopore, wherein the first membrane separates the first fluidic chamber into a first side and a second side, and (ii) a second nanopore system, wherein the second -527- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 nanopore system comprises (1) a second fluidic chamber and; (2) the second membrane comprising the second nanopore, wherein the second membrane separates the second fluidic chamber into a side and an additional side. 115. The method of claim 114, further comprising, providing (i) a first electrophoretic force (EPF) acting in an opposite direction to a first side to second side electro-osmotic force, or (ii) a second electrophoretic force (EPF) acting in an opposite direction to a side to additional side electro-osmotic force. 116. The method of claim 114 or 115, wherein (i) the first side comprises a first solution and the second side comprises a second solution, or (ii) the side comprises a solution and the additional side comprises an additional solution. 117. The method of claim 116, wherein (i) the first solution and the second solution are configured to translocate the at least the portion of the first analyte across the first nanopore, or (ii) the solution and the additional solution are configured to translocate the at least the portion of the second analyte across the second nanopore. 118. The method of claim 116 or 117, wherein (i) the first solution and the second solution are configured to generate a first electro-osmotic force across the first membrane; (i) the solution and the additional solution are configured to generate a second electro-osmotic force across the second membrane. 119. The method of claim 118, wherein (i) the first electro-osmotic force translocates the at least the portion of the first analyte from the first side through the first nanopore to the second side against a first electrophoretic force acting in a direction opposite the first electro-osmotic force; or (ii) a second electro-osmotic force translocates the at least the portion of the second analyte from the side through the second nanopore to the additional side against a second electrophoretic force acting in a direction opposite the second electro-osmotic force. 120. The method of any one of claims 114-119, further comprising, prior to (a) (i) contacting a first complex comprising the at least the portion of the first analyte and a first translocase with the first side of the first nanopore; or (ii) contacting a second complex comprising the at least the portion of the second analyte and a second translocase with the side of the second nanopore. 121. The method of any one of claims 72-120, wherein the first nanopore or the second nanopore is a biological nanopore. 122. The method of claim 121, wherein the biological nanopore comprises at least a portion of an alpha helical pore forming protein or peptide. -528- WSGR Docket No.64828-710.601 WSGR Docket Number: 64828-710.601 123. The method of claim 121, wherein the biological nanopore comprises at least a portion of a beta barrel pore forming protein or peptide. 124. The method of any one of claims 72-123, further comprising, prior to (a), unfolding the first analyte or second analyte with one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof. 125. The method of claim 124, wherein the one or more unfoldases, translocases, unfoldase domains, or translocase domains, or fragments thereof, or any combination thereof are configured to position proximal to the nanopore upon a binding event with the polypeptide. 126. The method of any one of claims 72-125, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more post-translational modifications. 127. The method of any one of claims 72-126, wherein the at least the portion of the first analyte or the at least the portion of the second analyte comprises one or more leader constructs at a N-terminus or a C-terminus. 128. The method of any one of claims 72-127, wherein the first nanopore or the second nanopore comprises an adaptor. 129. The method of any one of claims 72-128, wherein the first nanopore or the second nanopore is coupled to one or more recognition elements. 130. The method of any one of claims 72-129, wherein the first nanopore or the second nanopore comprises an inner pore constriction from about 0.5 nanometers (nm) to about 2 nm. 131. The method of any one of claims 72-130, wherein the first nanopore or the second nanopore has an ion-selectivity P(+)/P(-) of greater than 2.0. 132. The method of any one of claims 72-130, wherein the first nanopore or the second nanopore has an ion-selectivity P(+)/P(-) of less than 0.50. 133. The method of claim 72-132, wherein the sample is a complex sample. -529- WSGR Docket No.64828-710.601
PCT/GB2024/052258 2023-08-30 2024-08-29 Methods for nanopore-based analyte detection Pending WO2025046236A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB2313202.0A GB202313202D0 (en) 2023-08-30 2023-08-30 Systems and methods for nanopore-based analyte detection
GB2313202.0 2023-08-30

Publications (1)

Publication Number Publication Date
WO2025046236A1 true WO2025046236A1 (en) 2025-03-06

Family

ID=88237176

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2024/052258 Pending WO2025046236A1 (en) 2023-08-30 2024-08-29 Methods for nanopore-based analyte detection

Country Status (2)

Country Link
GB (1) GB202313202D0 (en)
WO (1) WO2025046236A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114981450A (en) * 2019-11-19 2022-08-30 格罗宁根大学 Artificial nanopores and related uses and methods

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120366293B (en) * 2025-06-27 2025-10-24 杭州华大序风科技有限公司 Sequencing adapter for nanopore sequencing and its use

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005124888A1 (en) 2004-06-08 2005-12-29 President And Fellows Of Harvard College Suspended carbon nanotube field effect transistor
WO2013123379A2 (en) * 2012-02-16 2013-08-22 The Regents Of The University Of California Nanopore sensor for enzyme-mediated protein translocation
WO2020152563A1 (en) * 2019-01-21 2020-07-30 Fondazione Istituto Italiano Di Tecnologia Method and device for nanopore-based optical recognition of molecules
NL2023062B1 (en) * 2019-05-03 2020-11-30 Univ Delft Tech Protein current trace signal acquisition using a nanopore
WO2023050633A1 (en) 2021-09-30 2023-04-06 合肥美的电冰箱有限公司 Shelf and storage cabinet
WO2023050568A1 (en) 2021-09-30 2023-04-06 西门子股份公司 Data query method, data service and electronic device
WO2023050570A1 (en) 2021-09-30 2023-04-06 明度智云(浙江)科技有限公司 User-defined examination and approval method and apparatus for medical electronic experiment records and storage medium

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005124888A1 (en) 2004-06-08 2005-12-29 President And Fellows Of Harvard College Suspended carbon nanotube field effect transistor
WO2013123379A2 (en) * 2012-02-16 2013-08-22 The Regents Of The University Of California Nanopore sensor for enzyme-mediated protein translocation
WO2020152563A1 (en) * 2019-01-21 2020-07-30 Fondazione Istituto Italiano Di Tecnologia Method and device for nanopore-based optical recognition of molecules
NL2023062B1 (en) * 2019-05-03 2020-11-30 Univ Delft Tech Protein current trace signal acquisition using a nanopore
WO2023050633A1 (en) 2021-09-30 2023-04-06 合肥美的电冰箱有限公司 Shelf and storage cabinet
WO2023050568A1 (en) 2021-09-30 2023-04-06 西门子股份公司 Data query method, data service and electronic device
WO2023050570A1 (en) 2021-09-30 2023-04-06 明度智云(浙江)科技有限公司 User-defined examination and approval method and apparatus for medical electronic experiment records and storage medium

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL., J. MOL. BIOL, vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
ASAADI ET AL., BIOMARKER RESEARCH, vol. 9, no. 87, 2021
BEDFORD ET AL., BIOPHYSICAL REVIEWS, vol. 9, 2017, pages 299 - 308
E. MEYERSW. MILLER, CABIOS, vol. 4, 1989, pages 11 - 17
GONZALEZ-PEREZ ET AL., LANGMUIR, vol. 25, 2009, pages 10447 - 10450
IVANOV AP ET AL., NANO LETT, vol. 11, no. 1, 12 January 2011 (2011-01-12), pages 279 - 85
LIDE, D. R.: "Handb. Chem. Phys", vol. 53, 2003, CRC HANDBOOK OF CHEMISTRY AND PHYSICS, pages: 2616
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
ZHANG ET AL., NAT CHEM., vol. 13, no. 12, 2021, pages 1192 - 1199

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114981450A (en) * 2019-11-19 2022-08-30 格罗宁根大学 Artificial nanopores and related uses and methods

Also Published As

Publication number Publication date
GB202313202D0 (en) 2023-10-11

Similar Documents

Publication Publication Date Title
WO2025046236A1 (en) Methods for nanopore-based analyte detection
Bürmann et al. A folded conformation of MukBEF and cohesin
US20240159768A1 (en) Nanopore proteomics
Pao et al. Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity
Maini et al. Protein synthesis with ribosomes selected for the incorporation of β-amino acids
Gentle et al. Direct production of proteins with N-terminal cysteine for site-specific conjugation
Kaur et al. Evolution of structural biology through the lens of mass spectrometry
Klammt et al. High level cell‐free expression and specific labeling of integral membrane proteins
Rösner et al. Click chemistry for targeted protein ubiquitylation and ubiquitin chain formation
Calabrese et al. Amphipols outperform dodecylmaltoside micelles in stabilizing membrane protein structure in the gas phase
Rey et al. Advanced in vivo cross-linking mass spectrometry platform to characterize proteome-wide protein interactions
JP2022500074A (en) Biological nanopores with adjustable pore diameter and their use as analytical tools
Xuan et al. Site-specific incorporation of a thioester containing amino acid into proteins
Pavlenok et al. Control of subunit stoichiometry in single-chain MspA nanopores
Thakur et al. Aberrantly large single-channel conductance of polyhistidine arm-containing protein nanopores
US20250369044A1 (en) Nanopore-based analysis of analytes
Tsirigotaki et al. Analysis of translocation-competent secretory proteins by HDX-MS
Pelletier et al. A general system for studying protein− Protein interactions in Gram-negative bacteria
Akashi Investigation of molecular interaction within biological macromolecular complexes by mass spectrometry
Shimada et al. Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell-free synthesis systems
Popova et al. Recent Contributions of Proteomics to Our Understanding of Reversible Nε-Lysine Acylation in Bacteria
Apostolidou et al. Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations
Kane et al. Discovery of periplasmic solute binding proteins with specificity for ketone bodies: β‐hydroxybutyrate binding proteins
WO2024091123A1 (en) Nanopore systems and methods for single-molecule polymer profiling
WO2025230413A1 (en) Ion-selective nanopore systems and methods for single-molecule polymer profiling.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24769430

Country of ref document: EP

Kind code of ref document: A1