WO2025229321A1

WO2025229321A1 - Nanopores and uses thereof for analysis of analytes

Info

Publication number: WO2025229321A1
Application number: PCT/GB2025/050915
Authority: WO
Inventors: Giovanni Maglia; Andrew Heron; Vasiliki TSOUSI
Original assignee: Portal Biotech Ltd
Current assignee: Portal Biotech Ltd
Priority date: 2024-04-30
Filing date: 2025-04-29
Publication date: 2025-11-06
Anticipated expiration: 2026-10-30

Abstract

The present disclosure provides methods, systems, and compositions for characterizing an analyte using a nanopore. The system may comprise a a fluidic chamber. The system may also comprise a membrane comprising an engineered biological nanopore. The membrane may separate the fluidic chamber into a first side and a second side. The engineered biological nanopore may comprise a channel. The channel may comprise a first region and a second region. The second region may comprise a constriction region. The first region may be modified to be more net negative than a respective region of a wild-type biological nanopore. The first region may also be modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore. The first region may be adjacent to the second region. The engineered biological nanopore may be contacted with a biopolymer.

Description

NANOPORES AND USES THEREOF FOR ANALYSIS OF ANALYTES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of Greek Patent Application No. 20240100311, filed April 30, 2024, which is herein incorporated by reference in its entirety.

BACKGROUND

[0002] Determination of analytes is an important part of scientific studies. Improvements in the characterization of analytes can be important for further scientific studies or clinical aspects.

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, proteins, 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 comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein the second region of the channel has a neutral charge; and (b) contacting the engineered biological nanopore with a biopolymer.

[0005] In some embodiments, the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of a wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the neutral charge of the second region of the channel generates the EOF.

[0006] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0007] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0008] In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the constriction region of the wild-type biological nanopore.

[0009] In some embodiments, the second region is more neutral as compared to the constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the constriction region of a wild-type biological nanopore.

[0010] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0011] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0012] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0013] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0014] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0015] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0016] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0017] In some embodiments, the engineered biological nanopore comprises the straight geometry. [0018] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0019] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0020] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0021] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0022] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0023] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0024] In some embodiments, the method further comprises measuring a signal generated by translocating the biopolymer through the engineered biological nanopore. In some embodiments, the measuring the signal comprises measuring the signal for a state of (a) an open channel of the engineered biological nanopore; (b) capture of the biopolymer by a first opening of the engineered biological nanopore; or (c) exit of the biopolymer through a second opening of the engineered biological nanopore. In some embodiments, the measuring comprises detecting differences in the signal between states (a), (b), and (c). In some embodiments, the signal comprises an ionic current, a change in ionic current, or derivations thereof. In some embodiments, the measuring comprises detecting a presence of the biopolymer, a concentration of the biopolymer, or any combination thereof. In some embodiments, the measuring comprises detecting one or more characteristics of the biopolymer. In some embodiments, the one or more characteristics of the biopolymer comprise a shape of the biopolymer, a structure of the biopolymer, one or more mutations of the biopolymer, a surface charge of the biopolymer, one or more post-translation modifications of the biopolymer, one or more ligands coupled to the biopolymer, or any combination thereof.

[0025] In some embodiments, (b) comprises contacting the biopolymer with the first side of the fluidic chamber.

[0026] In some embodiments, (b) comprises contacting the biopolymer with the second side of the fluidic chamber.

[0027] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0028] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0029] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0030] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0031] In another aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore; and (b) contacting the engineered biological nanopore with a biopolymer.

[0032] In some embodiments, the net charge of the second region of the channel is at least about 70% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 85% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 90% more neutral as compared to the respective region of the wild-type biological nanopore.

[0033] In some embodiments, the net charge of the second region of the channel is less cationic as compared to the respective region of the wild-type biological nanopore.

[0034] In some embodiments, the net charge of the second region of the channel is less anionic as compared to the respective region of the wild-type biological nanopore.

[0035] The method of any one of claims 13-18, wherein the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, a negative charge of the first region of the channel and the net charge of the second region of the channel generates the EOF.

[0036] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0037] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0038] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0039] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0040] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm. [0041] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0042] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0043] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0044] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0045] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0046] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0047] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0048] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0049] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0050] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore. [0051] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0052] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0053] In some embodiments, the method further comprises measuring a signal generated by translocating the biopolymer through the engineered biological nanopore. In some embodiments, the measuring the signal comprises measuring the signal for a state of (a) an open channel of the engineered biological nanopore; (b) capture of the biopolymer by a first opening of the engineered biological nanopore; or (c) exit of the biopolymer through a second opening of the engineered biological nanopore. In some embodiments, the measuring comprises detecting differences in the signal between states (a), (b), and (c). In some embodiments, the signal comprises an ionic current, a change in ionic current, or derivations thereof. In some embodiments, the measuring comprises detecting a presence of the biopolymer, a concentration of the biopolymer, or any combination thereof. In some embodiments, the measuring comprises detecting one or more characteristics of the biopolymer. In some embodiments, the one or more characteristics of the biopolymer comprise a shape of the biopolymer, a structure of the biopolymer, one or more mutations of the biopolymer, a surface charge of the biopolymer, one or more post-translation modifications of the biopolymer, one or more ligands coupled to the biopolymer, or any combination thereof.

[0054] In some embodiments, (b) comprises contacting the biopolymer with the first side of the fluidic chamber.

[0055] In some embodiments, (b) comprises contacting the biopolymer with the second side of the fluidic chamber.

[0056] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0057] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N 108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0058] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0059] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0060] In another aspect, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein the second region of the channel has a neutral charge, wherein the engineered biological nanopore is configured to contact a biopolymer.

[0061] In some embodiments, the engineered biological nanopore is configured to generate an electro -osmotic force (EOF) greater than an EOF of a wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the neutral charge of the second region of the channel is configured to generate the EOF. In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0062] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel. [0063] In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the constriction region of the wild-type biological nanopore.

[0064] In some embodiments, the second region is more neutral as compared to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the constriction region of a wild-type biological nanopore.

[0065] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0066] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0067] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0068] In some embodiments, wherein the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0069] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0070] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0071] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0072] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0073] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0074] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0075] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0076] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0077] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0078] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0079] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0080] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0081] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0082] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0083] In another aspect, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore, wherein the engineered biological nanopore is configured to a biopolymer.

[0084] In some embodiments, the net charge of the second region of the channel is at least about 70% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 85% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 90% more neutral as compared to the respective region of the wild-type biological nanopore.

[0085] In some embodiments, the net charge of the second region of the channel is less cationic as compared to the respective region of the wild-type biological nanopore.

[0086] In some embodiments, the net charge of the second region of the channel is less anionic as compared to the respective region of the wild-type biological nanopore.

[0087] In some embodiments, the engineered biological nanopore is configured to generate an electro -osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the net charge of the second region of the channel is configured to generate the EOF.

[0088] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

-l i [0089] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0090] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0091] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0092] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0093] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0094] In some embodiments, wherein the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue.

[0095] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0096] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0097] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0098] In some embodiments, the engineered biological nanopore comprises the straight geometry. [0099] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0100] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0101] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0102] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0103] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0104] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0105] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0106] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. [0107] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0108] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position N 15, N 17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0109] In another aspect, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein the second region of the channel has a neutral charge.

[0110] In some embodiments, the engineered biological nanopore is configured to generate an electro -osmotic force (EOF) greater than an EOF of a wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the neutral charge of the second region of the channel is configured to generate the EOF.

[0111] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0112] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0113] In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the constriction region of the wild-type biological nanopore [0114] In some embodiments, the second region is more neutral as compared to the constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to a constriction region of a wild-type biological nanopore.

[0115] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0116] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0117] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0118] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue

[0119] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0120] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi -conical geometry.

[0121] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0122] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0123] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0124] In some embodiments, the engineered biological nanopore comprises the vestibule geometry. [0125] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0126] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0127] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0128] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0129] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position N 15, N 17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0130] In another aspect, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore.

[0131] In some embodiments, the net charge of the second region of the channel is at least about 70% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 85% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, the net charge of the second region of the channel is at least about 90% more neutral as compared to the respective region of the wild-type biological nanopore.

[0132] In some embodiments, the net charge of the second region of the channel is less cationic as compared to the respective region of the wild-type biological nanopore.

[0133] In some embodiments, the net charge of the second region of the channel is less anionic as compared to the respective region of the wild-type biological nanopore.

[0134] In some embodiments, the engineered biological nanopore is configured to generate an electro -osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the negative charge of the first region of the channel and the net charge of the second region of the channel is configured to generate the EOF.

[0135] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0136] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0137] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0138] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0139] In some embodiments, a shortest distance between a first ring of charge in the first region and a second ring of charge in the second region is at most about 5 nm.

[0140] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue.

[0141] In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue. [0142] In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, the mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0143] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry.

[0144] In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi- conical geometry.

[0145] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0146] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0147] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0148] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0149] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0150] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0151] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. [0152] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0153] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position N 15, N 17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0154] In some embodiments, the CsgG nanopore further comprises an additional monomer comprising a mutation corresponding to position N 15, N17, A20, L23, N24, Q27, or any combination thereof, of a wildtype amino acid sequence as set forth in SEQ ID NO: 5.

[0155] In another aspect, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm; and contacting the engineered biological nanopore with a biopolymer.

[0156] In some embodiments, the first region may be more net negative than the second region. In some embodiments, one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids. In some embodiments, one or more amino acids in the first region is mutated to one or more negative amino acids. In some embodiments, when one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the first region is mutated to one or more negative amino acids. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective region of a wild-type biological nanopore. In some embodiments, the first region comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective region of the wild-type biological nanopore. In some embodiments, the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region. In some embodiments, the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the mutated at least one amino acid in the second region. In some embodiments, the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the second ring of charge comprising the mutated at least one amino acid in the second region. In some embodiments, the second region comprise a C(alpha)-C(alpha) diameter of at most 10 nm.

[0157] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, the at least one mutated amino acid in the first portion of each of one or more monomers (or less than all monomers) forms the first ring of charge, wherein the at least one mutated amino acid in the second portion of each of one or more monomer (or less than all monomers) forms the second ring of charge, wherein each monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, each monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion.

[0158] In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or the increased net negative charge, respectively, as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the at least one mutated amino acid in the first portion of each monomer forms the first ring of charge. In some embodiments, the at least one mutated amino acid in the second portion of each monomer forms the second ring of charge. In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, the engineered biological nanopore generates an electro -osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region of the channel and the second region of the channel generates the EOF. In some embodiments, the EOF acts in an opposite direction to an electrophoretic force in the nanopore system. In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0159] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, an increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel. In some embodiments, the first region is more negative as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to the respective region of the wild-type biological nanopore. In some embodiments, the second region is more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net neutral charge of the constriction region is increased. In some embodiments, a net negative charge of the constriction region is increased. In some embodiments, the channel comprises a length of at least about 2 nanometers. In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges. In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue. In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0160] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0161] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0162] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0163] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0164] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0165] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0166] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0167] In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0168] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0169] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0170] In some embodiments, the method further comprises measuring a signal generated by translocating the biopolymer through the engineered biological nanopore. In some embodiments, the measuring the signal comprises measuring a signal for a state of (a) an open channel of the engineered biological nanopore; (b) capture of the biopolymer by a first opening of the engineered biological nanopore; or (c) exit of the biopolymer through a second opening of the engineered biological nanopore. In some embodiments, the measuring comprises detecting differences in the signal between states (a), (b), and (c). In some embodiments, the signal comprises an ionic current, a change in ionic current, or derivations thereof. In some embodiments, the measuring comprises detecting a presence of the biopolymer, a concentration of the biopolymer, or any combination thereof. In some embodiments, the measuring comprises detecting one or more characteristics of the biopolymer. In some embodiments, the one or more characteristics of the biopolymer comprise a shape of the biopolymer, a structure of the biopolymer, one or more mutations of the biopolymer, a surface charge of the biopolymer, one or more post-translation modifications of the biopolymer, one or more ligands coupled to the biopolymer, or any combination thereof.

[0171] In some embodiments, (b) comprises contacting the biopolymer with the first side of the fluidic chamber.

[0172] In some embodiments, (b) comprises contacting the biopolymer with the second side of the fluidic chamber. [0173] In some embodiments, the nanopore system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0174] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N 108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0175] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0176] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0177] In another aspect, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm, wherein the engineered biological nanopore is configured to contact a biopolymer. [0178] In some embodiments, the engineered biological nanopore is configured to generate an electro -osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region of the channel and the second region of the channel is configured to generate the EOF. In some embodiments, EOF acts in an opposite direction to an electrophoretic force in the system.

[0179] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0180] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, an increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the increased net negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel. [0181] In some embodiments, the first region is more net negative as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to the respective region of the wild-type biological nanopore.

[0182] In some embodiments, the second region is more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of a wild-type biological nanopore.

[0183] In some embodiments, a net neutral charge of the constriction region is increased.

[0184] In some embodiments, a net negative charge of the constriction region is increased.

[0185] In some embodiments, the channel comprises a length of at least about 2 nanometers.

[0186] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0187] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue. In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm. [0188] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0189] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0190] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0191] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0192] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0193] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0194] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0195] In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0196] In some embodiments, the biopolymer comprises a non-nucleic acid based polymer analyte. In some embodiments, the non-nucleic acid based polymer analyte comprises a protein, a polypeptide, a peptide, a saccharide, a lipid, a polymer, an inorganic material, or any combination thereof. In some embodiments, the non-nucleic acid based polymer analyte is the peptide, the protein, or the polypeptide.

[0197] In some embodiments, the first side of the fluidic chamber comprises a first solution and the second side of the fluidic chamber comprises a second solution. In some embodiments, the first solution comprises a first concentration of a solute and the second solution comprises a second concentration of the solute. In some embodiments, the solute comprises an ion or an osmolyte. In some embodiments, a difference between the first concentration of the solute and the second concentration of the solute is configured to generate an electroosmotic force.

[0198] In some embodiments, the system further comprises a pair of electrodes. In some embodiments, the pair of electrodes is configured to provide an applied voltage to generate an electrophoretic force. In some embodiments, the applied voltage is a negative voltage on the first side of the fluidic chamber. In some embodiments, the applied voltage is a negative voltage on the second side of the fluidic chamber.

[0199] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0200] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0201] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0202] In another aspect, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm. [0203] In some embodiments, the engineered biological nanopore is configured to generate an electro -osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region of the channel and the second region of the channel are configured to generate the EOF. In some embodiments, the EOF acts in an opposite direction to an electrophoretic force in the engineered biological nanopore.

[0204] In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5.

[0205] In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0206] In some embodiments, the first region is more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

[0207] In some embodiments, the second region is more neutral as compared to the respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

[0208] In some embodiments, a net neutral charge of the constriction region is increased.

[0209] In some embodiments, a net negative charge of the constriction region is increased.

[0210] In some embodiments, the channel comprises a length of at least about 2 nanometers.

[0211] In some embodiments, the first region of the channel comprises at least about 5 unitary negative charges.

[0212] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the one or more monomers comprises a first portion and at least a second portion. In some embodiments, the first portion comprises one or more mutated amino acid residues. In some embodiments, the one or more mutated amino acid residues comprises one or more negative charged amino acid residue. In some embodiments, the second portion comprises another one or more mutated amino acid residues. In some embodiments, the another one or more mutated amino acid residues comprises one or more neutrally charged amino acid residue. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 5 nm. In some embodiments, a mutated amino acid residue of the one or more mutated amino acid residues and another mutated amino acid residue of the another one or more mutated amino acid residues comprises a distance of at most about 3 nm.

[0213] In some embodiments, the engineered biological nanopore comprises a conical geometry, a semi- conical geometry, a straight geometry, or a vestibule geometry. In some embodiments, the engineered biological nanopore comprises the conical geometry or the semi-conical geometry.

[0214] In some embodiments, the engineered biological nanopore comprises an engineered T7 nanopore, an engineered SPP 1 nanopore, an engineered Phi29 nanopore, an engineered Mycobacterium smegmatis porin A (MspA) nanopore, an engineered fragaceatoxin C (FraC) nanopore, an engineered cytolysin A (ClyA) nanopore, an engineered TMH4C4 nanopore, or any combination thereof.

[0215] In some embodiments, the engineered biological nanopore comprises the straight geometry.

[0216] In some embodiments, the engineered biological nanopore comprises an engineered stable protein 1 (SP1) nanopore, an engineered pleurotolysin toxin (Ply AB) nanopore, an engineered outer membrane protein G (OmpG) nanopore, an engineered aerolysin nanopore, an engineered ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof.

[0217] In some embodiments, the engineered biological nanopore comprises the vestibule geometry.

[0218] In some embodiments, the engineered biological nanopore comprises an engineered alpha-hemolysin nanopore, an engineered curli specific gene G (CsgG) nanopore, or any combination thereof.

[0219] In some embodiments, the engineered biological nanopore has a first opening and a second opening. In some embodiments, the first region of the channel comprises the first opening. In some embodiments, the second region of the channel comprises the second opening. In some embodiments, the first region of the channel comprises the second opening. In some embodiments, the second region of the channel comprises the first opening. In some embodiments, the second region of the channel is located between the first opening of the biological nanopore and the second opening of the engineered biological nanopore.

[0220] In some embodiments, the engineered biological nanopore is an engineered MspA nanopore. In some embodiments, 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: 1. In some embodiments, 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: 1. In some embodiments, the monomer comprises a mutation corresponding to position T83, L88, 1105, N108, or any combination thereof of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0221] In some embodiments, the engineered biological nanopore is an engineered CsgG nanopore. In some embodiments, the engineered CsgG 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: 4. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4.

[0222] In some embodiments, the engineered biological nanopore is an engineered CsgG/F nanopore. In some embodiments, the engineered CsgG/F 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: 5. In some embodiments, the monomer comprises a mutation corresponding to position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the monomer comprises a mutation corresponding to position F48, T58, N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0223] 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.

[0224] 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.

[0225] In some aspects, the present disclosure provides a method comprising: (a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which the second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and/or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel; and (b) contacting the engineered biological nanopore with a biopolymer.

[0226] In some aspects, the present disclosure provides an engineered biological nanopore comprising a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and/or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, and wherein the first region of the channel is adjacent to the second region of the channel.

[0227] In some embodiments, the first region is more net negative than the second region. In some embodiments, one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids. In some embodiments, one or more amino acids in the first region is mutated to one or more negative amino acids. In some embodiments, when one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the adjacent region is mutated to one or more negative amino acids. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first region comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective region of a wild-type biological nanopore. In some embodiments, the first region comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective region of the wild-type biological nanopore. In some embodiments, the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region. In some embodiments, a first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the mutated at least one amino acid in the second region. In some embodiments, the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from a second ring of charge comprising the mutated at least one amino acid in the second region. In some embodiments, the second region comprise a C(alpha)-C(alpha) diameter of at most 5 nm.

[0228] In some embodiments, the engineered biological nanopore comprises one or more monomers. In some embodiments, a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore.

[0229] In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids.

[0230] In some embodiments, the at least one mutated amino acid in a first portion of each of one or more monomers (or less than all monomers) forms a first ring of charge, wherein the at least one mutated amino acid in a second portion of each of one or more monomer (or less than all monomers) forms a second ring of charge, wherein each monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region. In some embodiments, each monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge. In some embodiments, the first portion comprises at least one amino acid that is mutated to exhibit the increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore. In some embodiments, the first portion comprises at least one amino acid that is mutated to a negative amino acid to exhibit the increased net negative charge as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge. In some embodiments, the second portion comprises at least one amino acid that is mutated to exhibit the increased net neutral charge or increased net negative charge as compared to a respective portion of a monomer of a wild-type biological nanopore.

[0231] In some embodiments, the second portion comprises at least one amino acid that is mutated to a neutral amino acid or a negative amino acid to exhibit the increased net neutral charge or the increased net negative charge, respectively, as compared to the respective portion of the monomer of the wild-type biological nanopore. In some embodiments, the at least one mutated amino acid in the first portion of each monomer forms the first ring of charge. In some embodiments, the at least one mutated amino acid in the second portion of each monomer forms the second ring of charge. In some embodiments, the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the first portion is mutated to one or more negative amino acids. In some embodiments, one or more amino acids in the second portion is modified to one or more neutral amino acids or one or more negative amino acids. In some embodiments, when one or more amino acids in the second portion is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the first portion is mutated to one or more negative amino acids.

[0232] In some embodiments, the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region modified to be more net negative and the second region modified to be more net neutral or more net negative generate the EOF. In some embodiments, the engineered biological nanopore has a cation-selectivity P(+)/P(-) of at least about 1.5. In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel.

[0233] In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel. In some embodiments, the first region is more negative as compared to another region adjacent to a constriction region of the wild-type biological nanopore. In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore. In some embodiments, the second region is more net neutral as compared to a respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

[0234] In some aspects, the present disclosure provides a system comprising: (a) a fluidic chamber; and (b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, which second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and/or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel, wherein the engineered biological nanopore is configured to contact a biopolymer.

[0235] In some embodiments, the engineered biological nanopore is configured to generate an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore. In some embodiments, the first region modified to be more net negative and the second region modified to be more net neutral or more net negative is configured to generate the EOF. In some embodiments, the engineered biological nanopore has a cationselectivity P(+)/P(-) of at least about 1.5. In some embodiments, the second region of the channel comprises a first entrance and a second entrance. In some embodiments, a negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the second entrance of the second region of the channel. In some embodiments, the negative charge of the first region of the channel is adjacent to the first entrance of the second region of the channel and the second entrance of the second region of the channel.

[0236] In some embodiments, a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore. In some embodiments, the second region is more neutral as compared to a respective region of the wild-type biological nanopore. In some embodiments, a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of a wild-type biological nanopore.

[0237] In some aspects, the present disclosure provides a method comprising (a) providing a mixture containing or suspected of containing an analyte comprising polypeptide or protein, and (b) using an engineered biological nanopore to (1) determine a sequence or (2) generate a measure of a concentration or relative amount of said analyte in said mixture at an accuracy of at least 80%.

[0238] In some embodiments, said mixture contains or is suspected of containing an additional analyte comprising an additional polypeptide or protein. In some embodiments, the method further comprises using said engineered biological nanopore to generate a measure of a concentration or relative amount of said additional analyte in said mixture at an accuracy of greater than 80%. In some embodiments, (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 90%. In some embodiments, (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 95%.

[0239] 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 departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0240] 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 [0241] 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:

[0242] FIGs. 1A-1B shows schematic representations of nanopore geometries. FIG. 1A depicts a nanopore with a first opening (101) and a second opening (102). The nanopore has a channel and constriction (103) with a channel region on each side of the constriction (103). FIG. IB depicts a nanopore with example areas of charge. The figure also shows a dimension of a first entrance (107), a dimension of a second entrance (108), and a dimension of a constriction region (109).

[0243] FIGs. 2A-2B show a schematic representation of a nanopore with a constriction region and channel. FIG. 2A shows the structure of the MspA nanopore with an octameric configuration comprising 8 monomers. FIG. 2B shows the locations of amino acid residues T83, L88, N108, 1105, D90, and D91. Residues D90 and D91 are in the constriction region. Residues T83, L88, N108, and 1105 are in the channel regions.

[0244] FIGs. 3A-3G show electrical current (picoAmps) versus time (seconds) recordings of protein translocations through Msp nanopores. FIG. 3A shows a current recording from a wild-type MspA pore. FIG. 3B shows a current recording from a MspA pore with D90N mutation. FIG. 3C shows a current recording from a MspA pore with D90N, D91N, and I105E mutations. FIG. 3D shows a current recording from a MspA pore with D90N, D91N, and N108E mutations. FIG. 3E shows a current recording from a MspA pore with D90N, D91N, I105E, and N108E mutations. FIG. 3F shows a current recording from a MspA pore with D90N, D91N, and L88E mutations. FIG. 3G shows a current recording from a MspA pore with D90N, D91N, L88E, and T83E. FIG. 3H shows a current reading from a MspA pore with D90N.

[0245] FIG. 4 shows a schematic representation of a CsgG nanopore. The nanopore has a wide channel region and a narrower constriction. The constriction is near the center of the channel.

[0246] FIG. 5 shows a schematic representation of a CsgG-CsgF (CsgG/F) nanopore. The nanopore has a wide channel region (501) with a narrower constriction region (502). The nanopore has an internal CsgF peptide adapter (503) in the trans entrance of the CsgG channel that creates another channel (504).

[0247] FIG. 6 shows a depiction of a computer system that is programmed or otherwise configured to implement the methods provided herein.

[0248] FIG. 7 shows a schematic representation of a nanopore comprising one or more regions and diameter.

DETAILED DESCRIPTION

[0249] While various cases of the invention have been shown and described herein, it will be obvious to those skilled in the art that such cases 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 cases of the invention described herein can be employed.

[0250] 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.

[0251] 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.”

[0252] It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.

[0253] The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is 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.

[0254] 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” is 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” is 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. [0255] The term “variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence. In some cases, the variant is a functional variant.

[0256] The term “functional variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence, and is capable of having one or more activities of the reference amino acid sequence.

[0257] 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 cases, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 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 is 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” is 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, functional homolog, functional ortholog, or functional paralog thereof. “Homologs” can refer to proteins, peptides, oligopeptides, polypeptides having amino acid substitutions, deletions, 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 is a product of gene duplication of a common ancestral gene.

[0258] 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 cases, the percent identity between two amino acid sequences is 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 yet another preferred embodiment, the percent identity between two nucleotide sequences is 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 particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0259] 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. 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.

[0260] 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.

[0261] A “conservative amino acid substitution” can be one in which the amino acid residue is 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), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), or any combination thereof.

[0262] 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 cases, 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 cases, 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. The term “negative mutation” and “negatively-charged mutation” may be used interchangeably herein. The terms “positive mutation” and “positively -charged mutation” may be used interchangeably herein. The terms “neutral mutation” and “neutrally charged mutation” may be used interchangeably herein.

[0263] Nanopores selective for ions (e.g., cation-selective nanopores for positively-charged ions) may generate strong EOFs that can facilitate the capture and/or translocation of heterogeneously charged biological molecules. The constriction region can also represent a sensing region of a nanopore described herein, wherein the sensing region may be where signal is generated (e.g., signal generated from capture and/or translocation of an analyte). A constriction region engineered with charged amino acid residues (e.g., negatively-charged amino acid residues) may generate a strong EOF and be selective for cation and/or anion species. However, without wishing to be bound by theory, an engineered biological nanopore with a charged constriction may also result in a small pool of available modifications (e.g., modifications to the nanopore) to tune a current signal.

[0264] It can be advantageous to engineer a nanopore (e.g., a biological nanopore) and/or a monomer of a nanopore to generate strong EOF with modifications outside of the constriction region. For example, the present disclosure provides engineered nanopores (e.g., biological nanopores) with modifications to the constriction region to contain one or more neutral charges and/or negative charges. These engineered biological nanopores may then be modified to introduce one or more negatively-charged amino acid residues to a region other than the constriction region (e.g., a region adjacent to the constriction region). This combination of charges can result in a strong EOF, and methods of generating a strong EOF that may not involve engineering a charged constriction region.

[0265] In some aspects, the present disclosure provides nanopores, systems, methods, or any combination thereof for analysis of an analyte (e.g., a biopolymer or a non-nucleic acid-based polymer analyte). The nanopores, systems, methods, or any combination thereof described herein may be used to determine one or more characteristics of an analyte. A characteristic of an analyte can comprise a length of the analyte (e.g., a contour length, in the case of polymeric analyte), a volume of the analyte, a mass of the analyte, a shape of the analyte, a secondary structure of the analyte, a tertiary structure of the analyte, a charge distribution of the analyte, an identity of the analyte, a sequence of the analyte, any chemical modifications of the analyte, or any combination thereof. The nanopores, systems, methods, or any combination thereof described herein may be used for single molecule analysis. The single molecule analysis may be analysis of a nucleic acid analyte or a non-nucleic acid analyte (e.g., a peptide, polypeptide, protein, or any combination thereof). The methods, systems, nanopores, or any combination thereof described herein may be used for characterizing at least one feature of a target analyte (e.g., protein, polypeptide, nucleic acid conjugated to a protein/polypeptide/peptide, or combination thereof). The detection and/or analysis may be of one or more analytes. As an example, the detection and/or analysis of the one or more analytes may be at a single molecule level.

Nanopores

[0266] 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 cases, the pore (e.g., nanopore) comprises a transmembrane region. In some cases, the pore comprises a hydrophilic portion. In some cases, the pore comprises a hydrophobic portion. In some cases, the pore comprises a hydrophilic and a hydrophobic portion. In some cases, a pore comprises an opening (e.g., an entrance). In some cases, a pore comprises at least one opening. In some cases, 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, a circumference, or any combination thereof.

[0267] 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 cases, an analyte may be trapped in the channel of the nanopore. In some cases, an analyte may translocate through the channel of the nanopore. In some cases, 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. [0268] 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 cases, 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, 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 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 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. A first region of the channel may be adjacent to a first end of a second region (e.g., constriction region) and a third region may be adjacent to a second end of a second region (e.g., constriction region). As an example, a first region of the channel may be adjacent to a first entrance of a second region (e.g., constriction region) and a third region may be adjacent to a second entrance of a second region (e.g., constriction region). In other cases, the first region and/or third region may be separated from the second region of the channel by a distance of at most about 4.0 run, at most about 3.0 nm, at most about 2.0 rim, at most about 1.5 run, at most about 1.0 run, 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 cases, a first region and/or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) 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 cases, a first region and/or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) 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 cases, a first region and/or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) by a distance from about 0.001 nm to about 15 nm. In some cases, a first region and/or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) by a distance from at least about 0.001 nm. In some cases, a first region and/or third region of the channel may be separated from a second region of the channel (e.g., comprising the constriction region) 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, 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 run, about 4 nm to about 15 run, about 5 run to about 10 run, about 5 run to about 15 run, or about 10 nm to about 15 nm.

[0269] Unless specifically indicated otherwise, when referring to “diameter” herein, a diameter may be determined by measuring center-to-center distances or atomic surface-to-surface distances. A diameter may be measured along a plane from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid (e.g., between a first alpha carbon to a second alpha carbon that is opposite to the first alpha carbon). The second region (e.g., comprising a constriction region) of the nanopore may be a narrower region of the channel than another region of the channel (e.g., a first region and/or third region). In some cases, 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. Without wishing to be bound by theory, modifying a constriction region of a nanopore to shift an electrical resistance may modulate the electro-osmotic force (EOF) and/or may improve the ability of the nanopore to characterize an analyte. Characterization of an analyte may occur at the second region (e.g., constriction region). In the second region (e.g., constriction region), a current flow may be modulated by a composition of the analyte within. For example, a current flow may be modulated by local composition of an analyte and/or an amino acid composition of the analyte. The electro-osmotic flow (EOF) may be maximally created at a second region (e.g., a constriction region or narrowest region). For example, the EOF may be maximally created at a second region (e.g., a constriction region or narrowest region) due to a maximal electrostatic effect on cation or anion flux in the constrained dimensions of the second region.

[0270] The constriction region of the nanopore may be a narrower region of the channel than another region of the channel. In some cases, the constriction region of the nanopore can contribute to the electrical resistance of the nanopore. For example, the presence of charged amino acid residues may contribute to an electrical resistance. A constriction region comprising a net negative charge from one or more negatively- charged amino acid residues may affect a flow of electric current of the nanopore system and/or translocation of an analyte (e.g., a charged or uncharged analyte). Alternatively, a constriction region comprising a net negative charge from one or more negatively-charged amino acid residues may affect a flow of electric current of the nanopore system and/or translocation of an analyte (e.g., a charged or uncharged analyte). 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. [0271] In some cases, the nanopore comprises a shape (e.g., a geometry). For example, a nanopore may be cylindrical. In some cases, the nanopore can be conical shape. In some cases, the nanopore can be globular shape. In some cases, the nanopore can be hourglass shape. In some cases, the nanopore can be a toroidal shape, comprising a ring and a channel. In some cases, 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 toroidal shape can comprise a ring or a donut shape. 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, 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).

[0272] In some cases in which the nanopore comprises an hourglass shape, the nanopore may have two or more flanking regions. The regions may be the first region and the third region described herein. For example, the first region and third region may flank a second region of the nanopore. The second region may be a constriction region of the nanopore. The first and third regions (e.g., funnel regions) can have the same diameter or different (e.g., distinct) diameters. The first and third regions can each have a diameter of at least about 2.2, at least about 2.4, at least about 2.6, at least about 2.8, at least about 3.0, at least about 3.2, at least about 3.4, at least about 3.6, at least about 3.8, at least about 4.0, at least about 4.2, at least about 4.4, at least about 4.6, at least about 4.8, at least about 5.0, at least about 5.2, at least about 5.4, at least about 5.6, at least about 5.8, at least about 6.0, at least about 6.2, at least about 6.4, at least about 6.6, at least about 6.8, at least about 7.0, at least about 7.5, at least about 8.0, at least about 8.5, at least about 9.0, at least about 9.5, at least about 10, or greater than about 10 times the diameter of the second region (e.g., constriction region).

[0273] 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 (e.g., run substantially 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., 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 cases a channel length is less than a linear length or a contour length of an analyte. In some cases a channel length is greater than a linear length or a contour length of an analyte.

[0274] In some cases, a channel comprises a channel length from about 0.5 nm to about 40 nm. In some cases, the channel can comprise a channel length of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 5 nm, at least about 10, 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, or more than 40 nm. In some cases, the channel can comprise a channel length of 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 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5nm, or less than 0.5 nm. In some cases, the channel can comprise a channel length of about 0.5 nm, about 1 nm, about 1.5 nm, about 2 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, or about 40 nm.

[0275] In some cases, a first region and/or third region of the channel of the engineered biological nanopore described herein may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) measured along a plane from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid (e.g., between a first alpha carbon to a second alpha carbon that is opposite to the first alpha carbon). The dimension may be a diameter of the nanopore (e.g., the engineered biological nanopore). The diameter may be expressed as a Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). The diameter can be of a widest region of the first region and/or third region. In some cases, the diameter may be of a narrowest region of the first region and/or third region. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) may be at least about 1 nanometer (nm), at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm, wherein the dimension (e.g., diameter) is expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). In some cases, the first region and/or third region of the channel of the engineered biological nanopore described herein may comprise a diameter of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1 nm, or less than about 1 nm, wherein the diameter may be expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). In some cases, the first region and/or third region of the channel of the engineered biological nanopore described herein may comprise a diameter of about 1 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm. A first region and/or third region of the channel (e.g., a region adjacent to a nanopore) may be a wider than a second region of the nanopore (e.g., comprising the constriction region). In some cases, the first region and/or third region may have one or more mutations (e.g., a plurality of mutations). The one or more mutations may be at an area of the first region and/or third region with a diameter (e.g., narrowest diameter) wider than a second region (e.g., a narrowest region). A dimension (e.g., width, length, diameter, circumference, or widest dimension) may be determined by measuring from a first alpha carbon of a first amino acid (e.g., a first mutated amino acid) to a second alpha carbon of a second amino acid (e.g., a second mutated amino acid). As an example, one or more mutations of a first region and/or third region may be positioned in an area of the first region and/or third region comprising a diameter (e.g., narrowest diameter) of at least about 1 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm, wherein the diameter is expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). As another example, one or more mutations of a first region and/or third region may be positioned in an area of the first region and/or third region comprising a diameter (e.g., narrowest diameter) of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1 nm, or less than about 1 nm, wherein the diameter may be expressed as the Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). In some cases, the one or more mutations of a first region and/or third region may be positioned in an area of the first region and/or third region comprising a diameter (e.g., narrowest diameter) of about 1 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm. For example, as shown in FIG. 7, a nanopore 700 may comprise a first region 710 and a second region (e.g., constriction region) 720. The first region can be adjacent to the second region. The first region can have a diameter 730. This diameter 730 can be expressed as a dimension from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid (e.g., between a first alpha carbon to a second alpha carbon that is opposite to the first alpha carbon). A mutation 740 can be introduced at the area of the first region 710 where there is a diameter (e.g., narrowest diameter) 730.

[0276] A nanopore can comprise at least one opening (e.g., entrance). The opening can be a first opening. The nanopore may comprise two or more opening (e.g., entrances). For example, a nanopore described herein may comprise a first opening and a second opening. An opening of a nanopore can face a side (e.g., compartment) of a nanopore system described herein. For example, a first opening of a nanopore may face a first side (e.g., cis side) of a nanopore system. A second opening of a nanopore may face a second side (e.g., trans side) of a nanopore system. As shown in FIG. 1A, a nanopore described herein may comprise a first opening (101). The first opening (101) can face a first side (e.g., a cis side) of the nanopore system. The engineered biological nanopore may comprise a second opening (102). The second opening (102) can face a second side (e.g., a trans side) of the nanopore system. [0277] In some cases, a first region and/or third region of a nanopore may comprise a larger dimension (e.g., width, length, diameter, circumference, or widest dimension) compared to a second region (e.g., comprising a constriction region) of the channel of the nanopore. For example, the first region and/or third region of the channel of the nanopore may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) that may be at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than about 90% larger than that of a second region (e.g., comprising a constriction region) of the channel of the nanopore. For example, the first region and/or third region of the channel of the nanopore may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) that may be at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or less than about 5% larger than that of a second region (e.g., comprising a constriction region) of the channel of the nanopore. In some cases, an engineered biological nanopore described herein may comprise a first region and/or third region of a channel (e.g., adjacent to a constriction region) that can be at least about 1.5x, 2x, 3x, 4x, 5x, lOx, 50x, or greater than about 50x wider than a second region of a channel (e.g., comprising a constriction region). In some cases, an engineered biological nanopore described herein may comprise a first region and/or third region of a channel (e.g., adjacent to a constriction region) that can be at most about 50x, lOx, 5x, 4x, 3x, 2x, 1.5x or less than about 1.5x wider than a second region of a channel (e.g., comprising a constriction region). In some cases, an engineered biological nanopore described herein may comprise a first region and/or third region of a channel (e.g., adjacent to a constriction region) that can be from about 2x to about 50x wider than a second region of a channel (e.g., comprising a constriction region). In some cases, an engineered biological nanopore described herein may comprise a first region and/or third region of a channel (e.g., adjacent to a constriction region) that can be from about 2x to about 3x, about 2x to about 4x, about 2x to about 5x, about 2x to about 6x, about 2x to about 7x, about 2x to about 8x, about 2x to about 9x, about 2x to about lOx, about 2x to about 20x, about 2x to about 25x, about 2x to about 5 Ox, about 3x to about 4x, about 3x to about 5x, about 3x to about 6x, about 3x to about 7x, about 3x to about 8x, about 3x to about 9x, about 3x to about lOx, about 3x to about 20x, about 3x to about 25x, about 3x to about 5 Ox, about 4x to about 5x, about 4x to about 6x, about 4x to about 7x, about 4x to about 8x, about 4x to about 9x, about 4x to about lOx, about 4x to about 20x, about 4x to about 25x, about 4x to about 50x, about 5x to about 6x, about 5x to about 7x, about 5x to about 8x, about 5x to about 9x, about 5x to about lOx, about 5x to about 20x, about 5x to about 25x, about 5x to about 5 Ox, about 6x to about 7x, about 6x to about 8x, about 6x to about 9x, about 6x to about lOx, about 6x to about 20x, about 6x to about 25x, about 6x to about 5 Ox, about 7x to about 8x, about 7x to about 9x, about 7x to about lOx, about 7x to about 20x, about 7x to about 25x, about 7x to about 50x, about 8x to about 9x, about 8x to about lOx, about 8x to about 20x, about 8x to about 25x, about 8x to about 5 Ox, about 9x to about lOx, about 9x to about 20x, about 9x to about 25x, about 9x to about 50x, about lOx to about 20x, about lOx to about 25x, about lOx to about 50x, about 20x to about 25x, about 20x to about 50x, or about 25x to about 50x wider than a second region of a channel (e.g., comprising a constriction region). [0278] In some cases, a first opening of a nanopore may be the same dimension (e.g., diameter, circumference, and/or widest dimension) as a second opening. In some cases, a first opening of a nanopore may be a different dimension (e.g., diameter, circumference, and/or widest dimension) as a second opening. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a first opening of 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 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, or greater than about 30 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a first opening of at most about 30 nm, at most about 25 nm, at most about 20 nm, at most about 19 nm, at most about 18 nm, at most about 17 nm, at most about 16 nm, at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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, or less than about 0. 1 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a first opening (e.g., cis opening) from about 1 nm to about 8 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a first opening (e.g., cis opening) from 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 4.5 nm, about 1 nm to about 5 nm, about 1 nm to about 6 nm, about 1 nm to about 7 nm, about 1 nm to about 8 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 4.5 nm, about 1.5 nm to about 5 nm, about 1.5 nm to about 6 nm, about 1.5 nm to about 7 nm, about 1.5 nm to about 8 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 4.5 nm, about 2 nm to about 5 nm, about 2 nm to about 6 nm, about 2 nm to about 7 nm, about 2 nm to about 8 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 4.5 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about 6 nm, about 2.5 nm to about 7 nm, about 2.5 nm to about 8 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about 4.5 nm, about 3 nm to about 5 nm, about 3 nm to about 6 nm, about 3 nm to about 7 nm, about 3 nm to about 8 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 4.5 nm, about 3.5 nm to about 5 nm, about 3.5 nm to about 6 nm, about

3.5 nm to about 7 nm, about 3.5 nm to about 8 nm, about 4 nm to about 4.5 nm, about 4 nm to about 5 nm, about 4 nm to about 6 nm, about 4 nm to about 7 nm, about 4 nm to about 8 nm, about 4.5 nm to about 5 nm, about 4.5 nm to about 6 nm, about 4.5 nm to about 7 nm, about 4.5 nm to about 8 nm, about 5 nm to about 6 run, about 5 nm to about 7 run, about 5 run to about 8 run, about 6 run to about 7 run, about 6 run to about 8 run, or about 7 nm to about 8 nm. In some cases, a nanopore provided herein may comprise a dimension of a first opening (e.g., cis opening) (e.g., diameter, circumference, and/or widest dimension) from about 8 nm to about 30 nm. In some cases, a nanopore provided herein may comprise a dimension of a first opening (e.g., cis opening) (e.g., diameter, circumference, and/or widest dimension) from at most about 30 nm. In some cases, a nanopore provided herein may comprise a dimension of a first opening (e.g., cis opening) (e.g., diameter, circumference, and/or widest dimension) from about 8 nm to about 9 nm, about 8 nm to about 10 nm, about 8 nm to about 11 nm, about 8 nm to about 12 nm, about 8 nm to about 13 nm, about 8 nm to about 14 nm, about 8 nm to about 15 nm, about 8 nm to about 20 nm, about 8 nm to about 25 nm, about 8 nm to about 30 nm, about 9 nm to about 10 nm, about 9 nm to about 11 nm, about 9 nm to about 12 nm, about 9 nm to about 13 nm, about 9 nm to about 14 nm, about 9 nm to about 15 nm, about 9 nm to about 20 nm, about 9 nm to about 25 nm, about 9 nm to about 30 nm, about 10 nm to about 11 nm, about 10 nm to about 12 nm, about 10 nm to about 13 nm, about 10 nm to about 14 nm, about 10 nm to about 15 nm, about 10 nm to about 20 nm, about 10 nm to about 25 nm, about 10 nm to about 30 nm, about 11 nm to about 12 nm, about 11 nm to about 13 nm, about 11 nm to about 14 nm, about 11 nm to about 15 nm, about 11 nm to about 20 nm, about 11 nm to about 25 nm, about 11 nm to about 30 nm, about 12 nm to about 13 nm, about 12 nm to about 14 nm, about 12 nm to about 15 nm, about 12 nm to about 20 nm, about 12 nm to about 25 nm, about 12 nm to about 30 nm, about 13 nm to about 14 nm, about 13 nm to about 15 nm, about 13 nm to about 20 nm, about 13 nm to about 25 nm, about 13 nm to about 30 nm, about 14 nm to about 15 nm, about 14 nm to about 20 nm, about 14 nm to about 25 nm, about 14 nm to about 30 nm, about 15 nm to about 20 nm, about 15 nm to about 25 nm, about 15 nm to about 30 nm, about 20 nm to about 25 nm, about 20 nm to about 30 nm, or about 25 nm to about 30 nm.

[0279] In some cases, a nanopore provided herein may comprise a dimension of a first opening of about 1 nanometer (nm), about 2 nm, about 3 nm, about 4 nm, or about 5 nm. In some cases, a nanopore provided herein may comprise a dimension of a second opening of about 1 nm, about 2 nm, about 3 nm, about 4 nm, or about 5 nm.

[0280] In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a second opening (e.g., trans opening) of at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, or greater than about 15 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a second opening (e.g., trans opening) of at most about 15 nm, at most about 14 nm, at most about 13 nm, at most about 12 nm, at most about 11 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 run, at most about 6 run, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or less than about 1 nm.

[0281] In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a second opening (e.g., trans opening) from about 0.5 nm to about 6 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a second opening (e.g., trans opening) from 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 4.5 nm, about 0.5 nm to about 5 nm, about 0.5 nm to about 5.5 nm, about 0.5 nm to about 6 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

I nm to about 4 nm, about 1 nm to about 4.5 nm, about 1 nm to about 5 nm, about 1 nm to about 5.5 nm, about 1 nm to about 6 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 4.5 nm, about 1.5 nm to about 5 nm, about 1.5 nm to about 5.5 nm, about 1.5 nm to about 6 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 4.5 nm, about 2 nm to about 5 nm, about 2 nm to about 5.5 nm, about 2 nm to about 6 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 4.5 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about 5.5 nm, about 2.5 nm to about 6 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about 4.5 nm, about 3 nm to about 5 nm, about 3 nm to about 5.5 nm, about 3 nm to about 6 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 4.5 nm, about 3.5 nm to about 5 nm, about 3.5 nm to about 5.5 nm, about 3.5 nm to about 6 nm, about 4 nm to about 4.5 nm, about 4 nm to about 5 nm, about 4 nm to about 5.5 nm, about 4 nm to about 6 nm, about 4.5 nm to about 5 nm, about 4.5 nm to about 5.5 nm, about 4.5 nm to about 6 nm, about 5 nm to about 5.5 nm, about 5 nm to about 6 nm, or about 5.5 nm to about 6 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a second opening (e.g., trans opening) from about 6 nm to about 15 nm. In some cases, a nanopore provided herein may comprise a dimension (e.g., diameter, circumference, and/or widest dimension) of a second opening (e.g., trans opening) from about 6 nm to about

7 nm, about 6 nm to about 8 nm, about 6 nm to about 9 nm, about 6 nm to about 10 nm, about 6 nm to about

I I nm, about 6 nm to about 12 nm, about 6 nm to about 13 nm, about 6 nm to about 14 nm, about 6 nm to about 15 nm, about 7 nm to about 8 nm, about 7 nm to about 9 nm, about 7 nm to about 10 nm, about 7 nm to about 11 nm, about 7 nm to about 12 nm, about 7 nm to about 13 nm, about 7 nm to about 14 nm, about 7 nm to about 15 nm, about 8 nm to about 9 nm, about 8 nm to about 10 nm, about 8 nm to about 11 nm, about

8 nm to about 12 nm, about 8 nm to about 13 nm, about 8 nm to about 14 nm, about 8 nm to about 15 nm, about 9 nm to about 10 nm, about 9 nm to about 11 nm, about 9 nm to about 12 nm, about 9 nm to about 13 nm, about 9 nm to about 14 run, about 9 run to about 15 run, about 10 nm to about 11 nm, about 10 run to about 12 nm, about 10 nm to about 13 nm, about 10 nm to about 14 nm, about 10 nm to about 15 nm, about 11 nm to about 12 nm, about 11 nm to about 13 nm, about 11 nm to about 14 nm, about 11 nm to about 15 nm, about 12 nm to about 13 nm, about 12 nm to about 14 nm, about 12 nm to about 15 nm, about 13 nm to about 14 nm, about 13 nm to about 15 nm, or about 14 nm to about 15 nm.

[0282] The constriction region of the nanopore may also have a dimension (e.g., diameter, circumference, and/or widest dimension). The constriction region may comprise a length, where the length can be a length along a longitudinal axis of a channel region of a nanopore. In some cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be 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 2.0 nm, at least about 3.0 nm, at least about 4.0 nm, at least about 5.0 nm, or greater than about 5.0 nm. In some cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be at most about 5.0 nm, at most about 4.0 nm, at most about 3.0 nm, at most about 2.0 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 cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be from about 0. 1 nm to about 1.2 nm. In some cases, the length of the constriction region of the nanopore (e.g., biological nanopore) can be from 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 0.6 nm, about 0. 1 nm to about 0.7 nm, about 0. 1 nm to about 0.8 nm, about 0. 1 nm to about 0.9 nm, about 0. 1 nm to about 1 nm, about 0. 1 nm to about 1.1 nm, about 0. 1 nm to about 1.2 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 0.6 nm, about 0.2 nm to about 0.7 nm, about 0.2 nm to about 0.8 nm, about 0.2 nm to about 0.9 nm, about 0.2 nm to about 1 nm, about 0.2 nm to about 1. 1 nm, about 0.2 nm to about 1.2 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 0.6 nm, about 0.3 nm to about 0.7 nm, about 0.3 nm to about 0.8 nm, about 0.3 nm to about 0.9 nm, about 0.3 nm to about 1 nm, about 0.3 nm to about 1.1 nm, about 0.3 nm to about 1.2 nm, about 0.4 nm to about 0.5 nm, about 0.4 nm to about 0.6 nm, about 0.4 nm to about 0.7 nm, about 0.4 nm to about 0.8 nm, about 0.4 nm to about 0.9 nm, about 0.4 nm to about 1 nm, about 0.4 nm to about 1.1 nm, about 0.4 nm to about 1.2 nm, about 0.5 nm to about 0.6 nm, about 0.5 nm to about 0.7 nm, about 0.5 nm to about 0.8 nm, about 0.5 nm to about 0.9 nm, about 0.5 nm to about 1 nm, about 0.5 nm to about 1.1 nm, about 0.5 nm to about 1.2 nm, about 0.6 nm to about 0.7 nm, about 0.6 nm to about 0.8 nm, about 0.6 nm to about 0.9 nm, about 0.6 nm to about 1 nm, about 0.6 nm to about 1. 1 nm, about 0.6 nm to about 1.2 nm, about 0.7 nm to about 0.8 nm, about 0.7 nm to about 0.9 nm, about 0.7 nm to about 1 nm, about 0.7 nm to about 1. 1 nm, about 0.7 nm to about 1.2 nm, about 0.8 nm to about 0.9 nm, about 0.8 nm to about 1 nm, about 0.8 nm to about 1.1 nm, about 0.8 nm to about 1.2 nm, about 0.9 nm to about 1 nm, about 0.9 nm to about 1. 1 nm, about 0.9 nm to about 1.2 nm, about 1 nm to about 1.1 nm, about 1 nm to about 1.2 nm, or about 1.1 nm to about 1.2 nm. [0283] In some cases, a second region of the channel of the engineered biological nanopore described herein may comprise a dimension (e.g., width, length, diameter, circumference, or widest dimension) measured along a plane from a first alpha carbon of a first amino acid to a second alpha carbon of a second amino acid. The dimension may be a diameter of the nanopore (e.g., the engineered biological nanopore) expressed as a Ca-Ca distance (e.g., the alpha-carbon to alpha-carbon distance). The diameter can be of any region of the second region, for example a narrowest region. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be at least about 0. 1 nm, at least about 0.5 nm, at least about 1.0 nm, at least about 1.5 nm, at least about 2.0 nm, at least about 2.5 nm, at least about 3.0 nm, at least about 3.5 nm, at least about 4.0 nm, at least about 4.5 nm, at least about 5.0 nm, or greater than about 5.0 nm. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be 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. 1 nm, or less than about 0. 1 nm. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be about 0.1 nm, 0.5 nm, 1.0 nm, 2.0 nm, 2.5 nm, 3.0 nm, 3.5 nm, 4.0 nm, 4.5 nm, or 5.0 nm. In some cases, the dimension (e.g., width, length, diameter, circumference, or widest dimension) of the second region may be from about 0.5 nm to 2.0 nm.

[0284] In some cases, a first region and/or third region can comprise a length. The length of the first region and/or third region can be a length as measured along a longitudinal axis of the channel. This longitudinal axis may run perpendicular to a membrane (e.g., run substantially perpendicular to a membrane). The length of the first region and/or third region can be measured as the distance along the longitudinal axis of the first region and/or third region between the most distant points of the first region and/or third region along the longitudinal axis of the channel. In some cases, a length of a first region and/or third region of the nanopore may be 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, at least about 20 nm, or greater than about 20 nm. In some cases, a length of a first region and/or third region of the nanopore may be at most about 20 nm, 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, or less than about 0.5 nm. In some cases, a length of a first region and/or third region of the nanopore may be about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 10 nm, about 15 nm, or about 20 nm. In some cases, a length of the first region and/or third region may be from about 3 nm to 10 nm.

[0285] In some cases, a second region (e.g., constriction region) can comprise a length. The length of the second region (e.g., constriction region) can be a length as measured along a longitudinal axis of the channel. This longitudinal axis may run perpendicular to a membrane (e.g., run substantially perpendicular to a membrane). The length of the second region (e.g., constriction region) can be measured as the distance along the longitudinal axis of the second region (e.g., constriction region) between the most distant points of the second region (e.g., constriction region) along the longitudinal axis of the channel. In some cases, a length of a second region (e.g., constriction region) of the nanopore may be at least about 0. 1 run, at least about 0.5 run, at least about 1 run, at least about 1.5 nm, at least about 2 nm, at least about 2.5 run, at least about 3 run, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 10 nm, or greater than about 10 nm. In some cases, a length of a second region (e.g., constriction region) of the nanopore may be at most about 10 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, at most about 0. 1 nm, or less than about 0. 1 nm. In some cases, a length of a second region (e.g., constriction region) of the nanopore may be about 0.5 nm, about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 10 nm, about 15 nm, or about 20 nm. In some cases, a length of the second region (e.g., constriction region) may be from about 0. 1 nm to 5 nm.

[0286] In some cases, one or more amino acid mutations may be present in a region of the nanopore comprising a constriction region (e.g., a narrowest region). The one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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. For example, one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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 1.0 nm, at least about 1.5 nm, at least about 2.0 nm, at least about 2.5 nm, at least about 3.0 nm, at least about 3.5 nm, at least about 4.0 nm, at least about 4.5 nm, at least about 5.0 nm, or greater than about 5.0 nm. As another example, one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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 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. 1 nm, or less than about 0.1 nm.

[0287] As another example, the one or more amino acid mutations may be present in a region (e.g., constriction region) comprising 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 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.

[0288] In some cases, 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 cases, 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 nanopore 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 cases, 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 nanopore 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, atmost 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 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.

[0289] In some cases, 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 nanopore described herein may be from about 0.0001 nm to about 2 nm. In some cases, 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 nanopore 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.

[0290] A constriction region may be located at any region of a nanopore (e.g., a region of the channel of the biological nanopore). In some cases, the constriction region can be located at a distance from a first entrance of the nanopore (e.g., the biological nanopore). For example, the constriction region can be adjacent to a first entrance of the nanopore. A constriction region may be located at least about 0.0001 nm (nanometers), 0.001 nm, 0.01 nm, 0.05 nm, 0. 1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, or greater than about 20 nm from a first entrance of a nanopore. A constriction region may be located at most about 20 nm, 15 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0. 1 nm, 0.05 nm, 0.01 nm, 0.001 nm, 0.0001 nm, or less than about 0.0001 nm from a first entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 10 nm from a first entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 0.0005 nm, 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 1 nm, about 0.0001 nm to about 2 nm, about 0.0001 nm to about 3 nm, about 0.0001 nm to about 4 nm, about 0.0001 nm to about 5 nm, about 0.0001 nm to about 10 nm, about 0.0005 nm to about 0.001 nm, about 0.0005 run to about 0.005 run, about 0.0005 nm to about 0.01 run, about 0.0005 nm to about 0.05 nm, about 0.0005 nm to about 1 nm, about 0.0005 nm to about 2 nm, about 0.0005 nm to about 3 nm, about 0.0005 nm to about 4 nm, about 0.0005 nm to about 5 nm, about 0.0005 nm to about 10 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 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 0.005 nm to about 0.01 nm, about 0.005 nm to about 0.05 nm, about 0.005 nm to about 1 nm, about 0.005 nm to about 2 nm, about 0.005 nm to about 3 nm, about 0.005 nm to about 4 nm, about 0.005 nm to about 5 nm, about 0.005 nm to about 10 nm, about 0.01 nm to about 0.05 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 0.05 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 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 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 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3 nm to about 10 nm, about 4 nm to about 5 nm, about 4 nm to about 10 nm, or about 5 nm to about 10 nm from a first entrance of a nanopore.

[0291] In some cases, the constriction region can be located at a distance from a second entrance of the nanopore (e.g., the biological nanopore). For example, the constriction region can be located adjacent to the second entrance of the nanopore. A constriction region may be located at least about 0.0001 nm, 0.001 nm, 0.01 nm, 0.05 nm, 0. 1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, 15 nm, 20 nm, or greater than about 20 nm from a second entrance of a nanopore. A constriction region may be located at most about 20 nm, 15 nm, 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0. 1 nm, 0.05 nm, 0.01 nm, 0.001 nm, 0.0001 nm, or less than about 0.0001 nm from a second entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 10 nm from a second entrance of a nanopore. In some cases, a constriction region may be located from about 0.0001 nm to about 0.0005 nm, 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 1 nm, about 0.0001 nm to about 2 nm, about 0.0001 nm to about 3 nm, about 0.0001 nm to about 4 nm, about 0.0001 nm to about 5 nm, about 0.0001 nm to about 10 nm, about 0.0005 nm to about 0.001 nm, about 0.0005 nm to about 0.005 nm, about 0.0005 nm to about 0.01 nm, about 0.0005 nm to about 0.05 nm, about 0.0005 nm to about 1 nm, about 0.0005 nm to about 2 nm, about 0.0005 nm to about 3 nm, about 0.0005 nm to about 4 nm, about 0.0005 nm to about 5 nm, about 0.0005 nm to about 10 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 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 0.005 nm to about 0.01 nm, about 0.005 run to about 0.05 run, about 0.005 run to about 1 run, about 0.005 run to about 2 run, about 0.005 nm to about 3 nm, about 0.005 nm to about 4 nm, about 0.005 nm to about 5 nm, about 0.005 nm to about 10 nm, about 0.01 nm to about 0.05 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 0.05 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 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 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 3 nm to about 4 nm, about 3 nm to about 5 nm, about 3 nm to about 10 nm, about 4 nm to about 5 nm, about 4 nm to about 10 nm, or about 5 nm to about 10 nm from a second entrance of a nanopore.

[0292] A constriction region can be located between the first and second entrance of a nanopore (e.g., the biological nanopore). For example, a constriction region may reside in a channel of a nanopore described herein at any distance between a first entrance and a second entrance of the nanopore. A nanopore may comprise a constriction region with an adjacent channel region. An adjacent channel region can comprise a region of the nanopore channel that may be at least about 0.0001 nm, 0.001 nm, 0.01 nm, 0.05 nm, 0. 1 nm, 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 10 nm, or greater than about 10 nm from a constriction region. An adjacent channel region can comprise a region of the nanopore channel that may be at most about 10 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0. 1 nm, 0.05 nm, 0.01 nm, 0.001 nm, 0.0001 nm, or less than about 0.0001 nm from a constriction region.

[0293] The methods, systems, nanopores, or any combination thereof described herein can comprise increasing the net negative charge at a constriction area and/or in a funnel region just outside of the at least one constriction area. The distance can be a length between a first region (e.g., a funnel region) and a second region (e.g., a constriction region). The distance (e.g., length) of a funnel region to the constriction area (measured along the central pore axis) can be from about 0.2 nm, about 0.3 nm, about 0.4 nm, about 0.5 nm, about 0.6 nm, about 0.7 nm, about 0.8 nm, about 0.9 nm, about 1.0 nm, about 1.2 nm, about 1.5 nm, about 2.0 and an upper limit independently selected from about 3 nm, about 2.9 nm, about 2.8 nm, about 2.7 nm, about 2.6 nm, about 2.5 nm, about 2.4 nm, about 2.3 nm, about 2.2 nm, or about 2. 1 nm.

[0294] In some cases, the nanopore can be a transmembrane protein pore derived from beta-barrel pores or alpha-helix bundle pores, beta-barrel pores comprising a barrel or channel that is formed from beta-strands. Examples of beta-barrel pores can include, but are not limited to, beta-toxins, such as alpha-hemolysin, anthrax toxin and leukocidins, and outer membrane proteins/porins of bacteria, such as Mycobacterium smegmatis porin (Msp), for example MspA, MspB, MspC or MspD, CsgG from the E. colt curb secretion system, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, outer membrane protein FhuA, outer membrane protein A (OmpA) and Neisseria autofransporter lipoprotein (NalP) and other pores, such as lysenin, bacterial nucleoside transporter Tsx. The nanopore (e.g., engineered biological nanopore) can comprise one or more monomeric units. The one or more monomeric units may be from a betabarrel pore or alpha-helix pore. The terms “monomer” and “monomeric unit” may be used interchangeably herein. The monomeric unit can be the individual subunit protein of a pore described herein. One or more monomeric units can assemble to form a functional porin channel. As an example, a nanopore (e.g., engineered biological nanopore) described herein may comprise one or more monomeric units of a MspA pore, CsgG pore, CsgF pore, OmpF pore, OmpG pore, outer membrane phospholipase A pore, outer membrane protein FhuA pore, OmpA pore, NalP pore, aHL pore, FraC pore, lysenin pore, bacterial nucleoside transporter Tsx pore, or any combinations thereof, or any functional homologs thereof, or any functional paralogs thereof, or any functional orthologs thereof. In some cases, an engineered biological nanopore described herein may comprise one or more monomers (e.g., mutant monomers) of MspA/MsmegO965, MspB/Msmeg0520, MspC/Msmeg5483, MspD/Msmeg6057, MppA, PorMl, PorM2, PorMl, Mmcs4296, Mmcs4297, Mmcs3857, Mmcs4382, Mmcs4383, Mjls3843, Mjls3857, Mjls3931 Mjls4674, Mjls4675, Mjls4677, Map3123c, Mav3943, Mvanl836, Mvan4117, Mvan4839, Mvan4840, Mvan5016, Mvan5017, Mvan5768, MUL —2391, Mflvl734, Mflvl735, Mflv2295, Mflvl891, MCH4691c, MCH4689c, MCH4690c, MAB1080, MAB1081, MAB2800, RHA1 ro08561, RHA1 ro04074, RHA1 ro03127, or any combinations thereof.

[0295] In some cases, alpha-helix bundle pores comprise a barrel or channel that is formed from alpha-helices. Examples of alpha-helix bundle pores can include, but are not limited to, inner membrane proteins and outer membrane proteins, such as WZA polysaccharide transporter and FraC. In a specific embodiment, the nanopore is selected from the group consisting of Aerolysin (Aer), Cytolysin K (CytK), Mycobacterium smegmatis porin A (MspA), alpha-hemolysin (aHL), E. colt curli secretion system component CsgG, Fragaceatoxin C (FraC) or an engineered mutant thereof. In one embodiment the nanopore is 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).

[0296] In some cases, 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 may comprise a T7 nanopore, a SPP 1 nanopore, a Phi29 nanopore, a Mycobacterium smegmatis porin A (MspA) nanopore, a fragaceatoxin C (FraC) nanopore, a cytolysin A (ClyA) nanopore, a TMH4C4 nanopore, or any combination thereof. In some cases, 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 may comprise a stable protein 1 (SP1) nanopore, a pleurotolysin toxin (Ply AB) nanopore, an outer membrane protein G (OmpG) nanopore, an aerolysin nanopore, a ferric hydroxamate uptake component A (FhuA) nanopore, or any combination thereof. In some cases, 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 alphahemolysin nanopore, a curli specific gene G (CsgG) nanopore, or any combination thereof.

[0297] In some cases, the nanopore comprises a pore-forming toxin. The nanopore can comprise an a- helical pore-forming toxin, a [3-barrel 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 cases, 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.

[0298] In some cases, the nanopore can be a T7 pore, a PN pore, a SP1 pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (a-HL) pore, a SPP 1 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. In some cases, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, a-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, TMH4C4, or any combination thereof. In some cases, an engineered biological nanopore described herein may comprise one or monomers from a T7 pore, a PN pore, a SP 1 pore, a Phi29 pore, a PlyAB pore, an alpha-hemolysin (a-HL) pore, a SPP 1 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. In some cases, the nanopore described herein may comprise one or more monomers from T7, PN, SP1, Phi29, PlyAB, a-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, TMH4C4, or any combination thereof. In some cases, the nanopore may comprise a one or more monomers from T7, PN, SP1, Phi29, PlyAB, a-HL, SPP1, FraC, MspA, CsgG, OmpG, aerolysin, ClyA, FhuA, PFO, or TMH4C4, or a mutant thereof, or a functional homolog thereof, or a functional ortholog thereof, or a functional paralog thereof. The monomer described herein can be a pore-forming protein. Pore-forming proteins can represent a group of molecules that create channels or pores in membranes. The proteins can assemble into pores and allow the passage of analytes (e.g., molecules, ions, nucleic acids, peptides, polypeptides, proteins, or combinations thereof) through a membrane (e.g., lipid bilayer).

[0299] In some cases, the nanopore may comprise an assembly of monomers. The nanopore may comprise a number of monomers in an arrangement. Monomers may be arranged vertically, horizontally, layered as rings, or any combination thereof, to form a nanopore described herein. In some cases, 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 cases, 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 cases, a nanopore may comprise 1 monomer. For example, the nanopore may be an OmpG nanopore. In some cases, a nanopore (e.g., biological nanopore) comprises from about 3 monomers to about 40 monomers. In some cases, 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 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.

[0300] A monomer can comprise one or more portions. For example, a monomer may comprise a first portion, a second portion, a third portion, or any combination thereof. In some cases, a monomer may comprise more than three portions. A first portion of a monomer can correspond to a first region of a nanopore described herein (e.g., an engineered biological nanopore). A second portion of a monomer can correspond to a second region of a nanopore described herein (e.g., an engineered biological nanopore). The second portion of the monomer may be a constriction region-forming portion. A third portion of a monomer can correspond to a third region of a nanopore described herein (e.g., an engineered biological nanopore). [0301] In some cases, a nanopore described herein may comprise a homogeneous monomeric composition. A homogeneous monomeric composition can comprise, any of the monomers disclosed herein, with the same amino acid composition. For example, a nanopore may be comprised of 8 monomers. Each monomer may comprise at least one portion. For example, each monomer may comprise a first portion, a second portion, a third portion, or any combination thereof. In some cases, the first portions of monomers of the nanopore may be the same amino acid composition. The same amino acid composition can refer to two or more portions of monomers (e.g., first portions of the monomers) with the same amino acid sequence. In some cases, the second portions of monomers of the nanopore may be the same amino acid composition. In some cases, the third portions of monomers of the nanopore may be the same amino acid composition. In some cases, the first, second, and third portions between each monomer of the nanopore may be the same amino acid composition (e.g., comprise the same charge).

In some cases, a nanopore described herein may comprise a heterogeneous monomeric composition. A heterogeneous monomeric composition can comprise monomers with different amino acid compositions. A heterogeneous monomeric composition can comprise one or more of any of the monomers disclosed herein. A heterogeneous monomeric composition can comprise, any of the monomers disclosed here, wherein a monomer the nanopore has different amino acid composition than another monomer of the nanopore. Monomers comprising different amino acid compositions may have different charges. As an example, a different amino acid composition between two monomers (e.g., one or more portions of two monomers) may be monomers (e.g., one or more portions of two monomers) with different amino acid sequences. As another example, a different amino acid composition between two monomers (e.g., one or more portions of two monomers) may be monomers (e.g., one or more portions of two monomers) with different net charges. In some cases, a first portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion of another monomer of the nanopore. In some cases, a second portion of at least one monomer of the nanopore may have a different amino acid composition than a second portion of another monomer of the nanopore. In some cases, a third portion of at least one monomer of the nanopore may have a different amino acid composition than a third portion of another monomer of the nanopore. In some cases, a first portion and a second portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion and a second portion of another monomer of the nanopore. In some cases, a first portion and a third portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion and a third portion of another monomer of the nanopore. In some cases, a second portion and a third portion of at least one monomer of the nanopore may have a different amino acid composition than a second portion and a third portion of another monomer of the nanopore. In some cases, a first portion, a second portion, and a third portion of at least one monomer of the nanopore may have a different amino acid composition than a first portion, a second portion, and a third portion of another monomer of the nanopore. [0302] In some cases, if the nanopore comprises a single monomer (e.g., from one proteinaceous chain), there may be a ring of charge if there are multiple mutations around the single monomer (e.g., proteinaceous chain). For example, a monomer described herein (e.g., a single monomer) may comprise two or more mutations forming a ring of charge around the single monomer (e.g., proteinaceous chain). The monomer may comprise a plurality of mutations forming the ring of charge. In some cases, the monomer may comprise at least about 2 mutations, at least about 3 mutations, at least about 4 mutations, at least about 5 mutations, at least about 10 mutations, or greater than about 10 mutations. In some cases, the monomer may comprise at most about 10 mutations, at most about 5 mutations, at most about 4 mutations, at most about 3 mutations, at most about 2 mutations, or less than about 2 mutations.

[0303] One or more of the monomers of the nanopore may be arranged vertically, horizontally, layered, or any combination thereof, the amino acid residues (e.g., positively-charged amino acid residues, negatively- charged amino acid residues, neutral amino acid residues, or any combination thereof) to form one or more rings of charge. In some cases, a pore may be engineered to contain regions of separate rings of charge 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 charge 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 charge along the longitudinal length of the channel. A nanopore may be engineered to contain regions of charges (e.g., rings of charge). For example, a nanopore may have about 2 to about 20 separate rings of charge in regions 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 20, about 10 to about 15, about 10 to about 20, or about 15 to about 20 separate rings of charge along the longitudinal length of the channel.

[0304] A region of a channel of a nanopore described herein may comprise one or more rings of charge. A first region of the nanopore may have a number of rings of charge. A third region of a nanopore may have a number of rings of charge. In some cases, a total number of rings of charge of a nanopore may be rings of charge in both a first region and a third region of the nanopore. In some cases, a first region and/or third region of the channel may comprise at least about 1, 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 15, at least about 20, or greater than about 20 rings of charge. In some cases, a first region and/or third region of the channel may comprise at most about 20, at most about 15, 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, at most about 1, or less than about 1 ring of charge. In some cases, a first region and/or third region of the channel may comprise from about 1 ring of charge to about 20 rings of charge. In some cases, a first region and/or third region of the channel may comprise from about 1 ring of charge to about 2 rings of charge, about 1 ring of charge to about 3 rings of charge, about 1 ring of charge to about 4 rings of charge, about 1 ring of charge to about 5 rings of charge, about 1 ring of charge to about 6 rings of charge, about 1 ring of charge to about 7 rings of charge, about 1 ring of charge to about 8 rings of charge, about 1 ring of charge to about 9 rings of charge, about 1 ring of charge to about 10 rings of charge, about 1 ring of charge to about 15 rings of charge, about 1 ring of charge to about 20 rings of charge, about 2 rings of charge to about 3 rings of charge, about 2 rings of charge to about 4 rings of charge, about 2 rings of charge to about 5 rings of charge, about 2 rings of charge to about 6 rings of charge, about 2 rings of charge to about 7 rings of charge, about 2 rings of charge to about 8 rings of charge, about 2 rings of charge to about 9 rings of charge, about 2 rings of charge to about 10 rings of charge, about 2 rings of charge to about 15 rings of charge, about 2 rings of charge to about 20 rings of charge, about 3 rings of charge to about 4 rings of charge, about 3 rings of charge to about 5 rings of charge, about 3 rings of charge to about 6 rings of charge, about 3 rings of charge to about 7 rings of charge, about 3 rings of charge to about 8 rings of charge, about 3 rings of charge to about 9 rings of charge, about 3 rings of charge to about 10 rings of charge, about 3 rings of charge to about 15 rings of charge, about 3 rings of charge to about 20 rings of charge, about 4 rings of charge to about 5 rings of charge, about 4 rings of charge to about 6 rings of charge, about 4 rings of charge to about 7 rings of charge, about 4 rings of charge to about 8 rings of charge, about 4 rings of charge to about 9 rings of charge, about 4 rings of charge to about 10 rings of charge, about 4 rings of charge to about 15 rings of charge, about 4 rings of charge to about 20 rings of charge, about 5 rings of charge to about 6 rings of charge, about 5 rings of charge to about 7 rings of charge, about 5 rings of charge to about 8 rings of charge, about 5 rings of charge to about 9 rings of charge, about 5 rings of charge to about 10 rings of charge, about 5 rings of charge to about 15 rings of charge, about 5 rings of charge to about 20 rings of charge, about 6 rings of charge to about 7 rings of charge, about 6 rings of charge to about 8 rings of charge, about 6 rings of charge to about 9 rings of charge, about 6 rings of charge to about 10 rings of charge, about 6 rings of charge to about 15 rings of charge, about 6 rings of charge to about 20 rings of charge, about 7 rings of charge to about 8 rings of charge, about 7 rings of charge to about 9 rings of charge, about 7 rings of charge to about 10 rings of charge, about 7 rings of charge to about 15 rings of charge, about 7 rings of charge to about 20 rings of charge, about 8 rings of charge to about 9 rings of charge, about 8 rings of charge to about 10 rings of charge, about 8 rings of charge to about 15 rings of charge, about 8 rings of charge to about 20 rings of charge, about 9 rings of charge to about 10 rings of charge, about 9 rings of charge to about 15 rings of charge, about 9 rings of charge to about 20 rings of charge, about 10 rings of charge to about 15 rings of charge, about 10 rings of charge to about 20 rings of charge, or about 15 rings of charge to about 20 rings of charge.

[0305] In some cases, a second region of the channel may comprise at least about 1, 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 15, at least about 20, or greater than about 20 rings of charge. In some cases, a second region of the channel may comprise at most about 20, at most about 15, 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, at most about 1, or less than about 1 ring of charge. In some cases, a second region of the channel may comprise from about 1 ring of charge to about 20 rings of charge. In some cases, a second region of the channel may comprise from about 1 ring of charge to about 2 rings of charge, about 1 ring of charge to about 3 rings of charge, about 1 ring of charge to about 4 rings of charge, about 1 ring of charge to about 5 rings of charge, about 1 ring of charge to about 6 rings of charge, about 1 ring of charge to about 7 rings of charge, about 1 ring of charge to about 8 rings of charge, about 1 ring of charge to about 9 rings of charge, about 1 ring of charge to about 10 rings of charge, about 1 ring of charge to about 15 rings of charge, about 1 ring of charge to about 20 rings of charge, about 2 rings of charge to about 3 rings of charge, about 2 rings of charge to about 4 rings of charge, about 2 rings of charge to about 5 rings of charge, about 2 rings of charge to about 6 rings of charge, about 2 rings of charge to about 7 rings of charge, about 2 rings of charge to about 8 rings of charge, about 2 rings of charge to about 9 rings of charge, about 2 rings of charge to about 10 rings of charge, about 2 rings of charge to about 15 rings of charge, about 2 rings of charge to about 20 rings of charge, about 3 rings of charge to about 4 rings of charge, about 3 rings of charge to about 5 rings of charge, about 3 rings of charge to about 6 rings of charge, about 3 rings of charge to about 7 rings of charge, about 3 rings of charge to about 8 rings of charge, about 3 rings of charge to about 9 rings of charge, about 3 rings of charge to about 10 rings of charge, about 3 rings of charge to about 15 rings of charge, about 3 rings of charge to about 20 rings of charge, about 4 rings of charge to about 5 rings of charge, about 4 rings of charge to about 6 rings of charge, about 4 rings of charge to about 7 rings of charge, about 4 rings of charge to about 8 rings of charge, about 4 rings of charge to about 9 rings of charge, about 4 rings of charge to about 10 rings of charge, about 4 rings of charge to about 15 rings of charge, about 4 rings of charge to about 20 rings of charge, about 5 rings of charge to about 6 rings of charge, about 5 rings of charge to about 7 rings of charge, about 5 rings of charge to about 8 rings of charge, about 5 rings of charge to about 9 rings of charge, about 5 rings of charge to about 10 rings of charge, about 5 rings of charge to about 15 rings of charge, about 5 rings of charge to about 20 rings of charge, about 6 rings of charge to about 7 rings of charge, about 6 rings of charge to about 8 rings of charge, about 6 rings of charge to about 9 rings of charge, about 6 rings of charge to about 10 rings of charge, about 6 rings of charge to about 15 rings of charge, about 6 rings of charge to about 20 rings of charge, about 7 rings of charge to about 8 rings of charge, about 7 rings of charge to about 9 rings of charge, about 7 rings of charge to about 10 rings of charge, about 7 rings of charge to about 15 rings of charge, about 7 rings of charge to about 20 rings of charge, about 8 rings of charge to about 9 rings of charge, about 8 rings of charge to about 10 rings of charge, about 8 rings of charge to about 15 rings of charge, about 8 rings of charge to about 20 rings of charge, about 9 rings of charge to about 10 rings of charge, about 9 rings of charge to about 15 rings of charge, about 9 rings of charge to about 20 rings of charge, about 10 rings of charge to about 15 rings of charge, about 10 rings of charge to about 20 rings of charge, or about 15 rings of charge to about 20 rings of charge.

[0306] In some cases, the rings of charge of the first region and/or third region may all be of the same charge (e.g., all the rings may be negatively -charged or neutral charged). One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise a negative charge. For example, one or more rings of charge may comprise one or more negatively-charged amino acids. To achieve a negative charge, one or more amino acids may be modified to be one or more negatively - charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and/or neutral amino acids) may be substituted for one or more negatively-charged amino acids. One or more negatively-charged amino acids may be added. One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may be more net negative as compared to one or more rings of charge of a respective region of a wild-type nanopore. One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise more negatively- charged amino acids as compared to one or more rings of charge of a respective region of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively -charged amino acids and/or neutral amino acids) may be substituted for one or more negatively-charged amino acids. As another example, one or more negatively-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and/or neutral amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more positively-charged amino acids may be substituted for one or more neutral amino acids in the region (e.g., a first region, second region, third region, or any combination thereof). One or more rings of charge of a first region and/or third region may be net negative. A net negative charge may be achieved when there may be a greater number of negatively -charged amino acids compared to a number of positively -charged amino acids and/or a number of neutral charged amino acids. A net negative charge of one or more rings of charge of the region (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively -charged amino acids and/or neutral amino acids) for one or more negatively -charged amino acids; (ii) deleting one or more positively -charged amino acids and/or neutral amino acids; (iii) adding one or more negatively -charged amino acids; or (iv) any combinations thereof.

[0307] One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise a neutral charge. For example, one or more rings of charge may comprise one or more neutral-charged amino acids. To achieve a neutral charge, one or more amino acids may be modified to be one or more neutral-charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and/or negatively -charged amino acids) may be substituted for one or more neutral-charged amino acids. One or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may be more net neutral as compared to one or more rings of charge of a respective region of a wild-type nanopore. One or more rings of charge of a region (e.g., a first region, second region, third region, or any combination thereof) may comprise more neutral-charged amino acids as compared to one or more rings of charge of a respective region of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively- charged amino acids and/or negatively-charged amino acids) may be substituted for one or more neutral- charged amino acids. As another example, one or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively-charged amino acids and/or negatively -charged amino acids may be added to achieve a same number of positively -charged amino acids and negatively-charged amino acids (e.g., and achieve a neutral charge). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and/or negatively-charged amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively -charged amino acids and/or negatively-charged amino acids may be deleted to achieve a same number of positively-charged amino acids and negatively -charged amino acids (e.g., and achieve a neutral charge). One or more rings of charge of a first region and/or third region may be net neutral. A net neutral charge may be achieved when there may be a same number of positively-charged amino acids and negatively-charged amino acids. A net neutral charge may be achieved when there may be a same number of positively-charged amino acids and negatively- charged amino acids when the ring of charge comprises all neutral charged amino acids. A net neutral charge of one or more rings of charge of the region (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and/or negatively -charged amino acids) for one or more neutral-charged amino acids; (ii) deleting one or more positively-charged amino acids and/or negatively-charged amino acids; (iii) adding one or more neutral-charged amino acids; or (iv) any combinations thereof. A net neutral charge of one or more rings of charge of the region (e.g., a first region, second region, third region, or any combination thereof) may also be achieved by (i) adding one or more positively-charged amino acids and/or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively- charged amino acids (e.g., and thus comprise an overall neutral charge) and/or (ii) deleting one or more positively-charged amino acids and/or negatively -charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge).

[0308] In some cases, the rings of charge of the second region may all be of the same charge (e.g., all the rings may be negatively-charged, neutral charged, or any combination thereof). In some cases, a ring of charge of the one or more rings of charge of the first region may comprise at least one negative charge, at least one positive charge, at least one neutral charge, or any combination thereof. In some cases, a ring of charge of the one or more rings of charge of the third region may comprise at least one negative charge, at least one positive charge, at least one neutral charge, or any combination thereof. In some cases, a ring of charge of the one or more rings of charge of the second region may comprise at least one negative charge, at least one positive charge, at least one neutral charge, or any combination thereof. In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be above a second region of the channel (e.g., comprising the constriction region). In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be below a second region of the channel (e.g., comprising the constriction region). In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be immediately above (e.g., adjacent to) a second region of the channel (e.g., comprising the constriction region). For example, a first ring of charge of the first region and/or third region may comprise a net negative charge (e.g., greater amount of negatively-charged amino acid residues than an amount of positively -charged amino acid residues and/or neutrally -charged amino acid residues). A first ring of charge of the first region and/or third region may comprise a greater negative charge distribution as compared to a charge distribution of a second ring of charge of the first region and/or third region. In some cases, one or more rings of charge of the rings of the charges in the channel of the engineered biological nanopore may be immediately below (e.g., adjacent to) a second region of the channel (e.g., comprising the constriction region). For example, a first ring of charge of the second region may comprise a net neutral charge (e.g., equal (e.g., balanced) amount of negatively-charged amino acid residues and positively-charged amino acid residues or 100% neutral amino acid residue composition). A first ring of charge of the second region may comprise a more neutral charge distribution as compared to a charge distribution of a second ring of charge of the first region and/or third region. A neutral charge distribution of the rings of charge of the second region of the channel may be 50% more than a respective region of a wild-type nanopore. For example, a first ring of charge may comprise a neutral charge distribution and a second ring of charge may comprise a positive charge distribution or a negative charge distribution. A neutral charge distribution of the rings of charge of the second region of the channel may be 100% more than a respective region of a wild-type nanopore. For example, a first ring of charge may comprise a neutral charge distribution and a second ring of charge may comprise a neutral charge distribution.

[0309] A separation of the rings may modulate an EOF of the nanopore. The number of rings, spacing of rings, or any combination thereof may affect a net charge of a channel of a nanopore. In some cases, the rings of charge (e.g., between a ring of charge in the first region and/or third region and a second ring of charge in the second region) may each be spaced 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 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at last about 9 nm, or greater than about 10 nm apart from each other along the longitudinal length of the channel. In some cases, the rings of charge (e.g., between a ring of charge in the first region and/or third region and a second ring of charge in the second region) may each be spaced at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from each other along the longitudinal length of the channel.

[0310] In some cases, the rings of charge (e.g., between a ring of charge in the first region and/or third region and a second ring of charge in the second region) may each be spaced from about 0. 1 nm to about 5 nm apart from each other along the longitudinal length of the channel. In some cases, the rings of charge (e.g., between a ring of charge in the first region and/or third region and a second ring of charge in the second region) may each be spaced from 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 1.5 nm, about 0. 1 nm to about 2 nm, about 0. 1 nm to about 2.5 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 0.2 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 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 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 run, about 0.4 nm to about 3 run, about 0.4 run to about 4 run, about 0.4 run to about 5 run, about 0.5 run 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 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 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 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 4 nm, about 2 nm to about 5 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 5 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 4 nm to about 5 nm, about 5 nm to about 6 nm, about 6 nm to about 7 nm, about 7 nm to about 8 nm, about 8 nm to about 9 nm, or about 9 nm to about 10 nm apart from each other along the longitudinal length of the channel.

[0311] In some cases, a ring of charge may be spaced 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 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at last about 9 nm, or greater than about 10 nm apart from another ring of charge along the longitudinal length of the channel. In some cases, a ring of charge may be spaced at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from another ring of charge along the longitudinal length of the channel.

[0312] In some cases, a ring of charge may be spaced from about 0. 1 nm to about 5 nm apart from another ring of charge along the longitudinal length of the channel. In some cases, a ring of charge may be spaced from 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 1.5 nm, about 0. 1 nm to about 2 nm, about 0. 1 nm to about 2.5 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 0.2 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 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 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 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 run to about 2 run, about 0.5 run to about 2.5 run, about 0.5 run to about 3 run, 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 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 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 4 nm, about 2 nm to about 5 nm, about 2.5 nm to about 3 nm, about 2.5 nm to about 4 nm, about 2.5 nm to about 5 nm, about 3 nm to about 4 nm, about 3 nm to about 5 nm, about 4 nm to about 5 nm, about 5 nm to about 6 nm, about 6 nm to about 7 nm, about 7 nm to about 8 nm, about 8 nm to about 9 nm, or about 9 nm to about 10 nm apart from another ring of charge along the longitudinal length of the channel.

[0313] In some cases, a first ring of charge may comprise one or more mutations described herein. The first ring of charge may be separated from a second ring of charge comprising one or more mutations described herein. The first ring of charge and the second ring of charge may be in the same region (e.g., a first region, a second region, or a third region described herein). The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by 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 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm apart from each other along the longitudinal length of the channel. The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by at most about 15 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from each other along the longitudinal length of the channel.

[0314] As another example, the first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be in different regions. For example, a first ring of charge may be in a first region and/or third region, and a second ring of charge may be in a second region. The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by 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 nm, at least about 2 nm, at least about 3 run, at least about 4 run, at least about 5 run, at least about 6 run, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm apart from each other along the longitudinal length of the channel. The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by at most about 15 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 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 0.9 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 apart from each other along the longitudinal length of the channel.

[0315] An engineered biological nanopore described herein may comprise two or more monomers (e.g., 8 monomers or an octameric pore). Each monomer of the nanopore may comprise at least a first portion, a second portion, and a third portion. A portion of a monomer may comprise one or more mutations of one or more residues to one or more negative charges. The first portion of a monomer can contribute to a first region of a channel (e.g., a region of the channel adjacent to the constriction region). The second portion of a monomer (e.g., the constriction-forming portion) can contribute to a second region of the channel (e.g., the region comprising the constriction region). The third portion of a monomer can contribute to a third region of the channel (e.g., another region of the channel adjacent to the constriction region). In the engineered biological nanopore, the second portion of the monomer may comprise one or more neutral charges. In the engineered biological nanopore, the second portion of the monomer may comprise one or more negative charges. In the engineered biological nanopore, the second portion of the monomer may comprise one or more neutral charges and negative charges. In some cases, a first portion of a first monomer of the engineered biological nanopore may comprise a mutation of one or more residues to negative charges to increase a negative charge distribution in the first portion (e.g., a portion forming a first region of the channel). A third portion of a first monomer of the engineered biological nanopore may comprise a mutation of one or more residues to a negative charge to increase a negative charge distribution in the third portion (e.g., a portion forming a third region of the channel). A first portion of a second monomer of the engineered biological nanopore may comprise the same mutation(s) as compared to the first portion of the first monomer. In some cases, a first portion of a second monomer of the engineered biological nanopore may comprise different mutations as compared to the first portion of the first monomer. For example, a first portion of a second monomer of the engineered biological nanopore may comprise one or more mutations of one or more residues to a negative charge to increase a negative charge distribution. The second monomer may comprise one or more mutations of one or more residues in a third portion to increase a negative charge distribution in the third portion (e.g., a portion forming a third region of the channel). As an example, the one or more monomers comprising an engineered biological nanopore (e.g., oligomeric nanopore) described herein may comprise one or more mutations of one or more second portions (e.g., constriction region-forming portions) to contribute to a neutral constriction region and/or negative constriction region of the nanopore, one or more mutations of first portions to contribute to a greater negative charge of a first region of the channel (e.g., a region of the channel adjacent to the constriction region), one or more mutations of third portions to contribute to a greater negative charge of a third region of the channel (e.g., another region of the channel adjacent to the constriction region), or any combination thereof. The neutral second region (e.g., constriction region) may comprise a neutral charge. The neutral second region (e.g., constriction region) may be modified to be more neutral as compared to a respective region of a wild-type nanopore. The neutral second region (e.g., constriction region) may have a net neutral charge. As another example, the monomers comprising an engineered biological nanopore (e.g., oligomeric nanopore) described herein may comprise one or more mutations of one or more second portions (e.g., constriction region-forming portions) to contribute to a negative second region (e.g., constriction region) of the nanopore, one or more mutations of first portions to contribute to a greater negative charge of a first region of the channel (e.g., a region of the channel adjacent to the constriction region), one or more mutations of third portions to contribute to a greater negative charge of a third region of the channel (e.g., another region of the channel adjacent to the constriction region), or any combination thereof. The second region (e.g., negatively-charged second region, e.g., constriction region) may comprise a negative charge. The second region (e.g., negatively-charged second region, e.g., constriction region) may be modified to be more negatively -charged as compared to a respective region of a wild-type nanopore. The second region (e.g., negatively -charged second region, e.g., constriction region) may have a net negative charge.

[0316] In some cases, the second region of the channel (e.g., comprising the constriction region) may comprise a charge (e.g., a neutral charge). The second region of the channel may have a net neutral charge. The net neutral charge may result from an equal number of positively charged amino acid residues and negatively charged amino acid residues. A balance of positively charged amino acid residues and negatively charged amino acid residues may provide for a net neutral charge of a constriction region of a nanopore described herein. A net neutral charge may result from an increase in neutral amino acid residues in the second region of the channel. The charges may be distributed in rings (e.g., rings of charge). The rings can be co-planar with a membrane. An engineered biological nanopore and/or monomers described herein may comprise a number of unitary charges. The unitary charges may be the amino acid residues (e.g., the charged amino acid residues) that comprise the rings of charge of the nanopore.

[0317] A unit of charge carried by a single proton and/or a single electron may be referred to as an “elementary charge”. An elementary charge may be a smallest charge that can exist freely. Elementary charge may be a value representing a charge of an electron or proton. Unitary charge may be a quantization of charge (e.g., a quantization of charge into one or more rings of charge).

[0318] In some cases, the engineered biological nanopore comprises a number of unitary charges. In some cases, the channel of the engineered biological nanopore described herein comprises a number of unitary charges. In some cases, the first region of the channel and/or the third region of the channel of the engineered biological nanopore described herein comprise a number of unitary charges. The channel of the engineered biological nanopore may comprise at least about 2 unitary charges, at least about 3 unitary charges, at least about 4 unitary charges, at least about 5 unitary charges, at least about 6 unitary charges, at least about 7 unitary charges, at least about 8 unitary charges, at least about 9 unitary charges, at least about 10 unitary charges, at least about 12 unitary charges, at least about 15 unitary charges, at least about 18 unitary charges, at least about 20 unitary charges, at least about 25 unitary charges, at least about 30 unitary charges, at least about 40 unitary charges, at least about 50 unitary charges, at least about 75 unitary charges, at least about 100 unitary charges, at least about 200 unitary charges, or greater than about 200 unitary charges. The channel of the engineered biological nanopore may comprise at most about 200 unitary charges, at most about 100 unitary charges, at most about 75 unitary charges, at most about 50 unitary charges, at most about 40 unitary charges, at most about 30 unitary charges, at most about 25 unitary charges, at most about 20 unitary charges, at most about 18 unitary charges, at most about 15 unitary charges, at most about 12 unitary charges, at most about 10 unitary charges, at most about 9 unitary charges, at most about 8 unitary charges, at most about 7 unitary charges, at most about 6 unitary charges, at most about 5 unitary charges, at most about 4 unitary charges, at most about 3 unitary charges, at most about 2 unitary charges, or less than about 2 unitary charges.

[0319] The channel of the engineered biological nanopore may comprise from about 5 unitary charges to about 250 unitary charges, he channel of the engineered biological nanopore may comprise from about 5 unitary charges to about 10 unitary charges, about 5 unitary charges to about 20 unitary charges, about 5 unitary charges to about 30 unitary charges, about 5 unitary charges to about 40 unitary charges, about 5 unitary charges to about 50 unitary charges, about 5 unitary charges to about 75 unitary charges, about 5 unitary charges to about 100 unitary charges, about 5 unitary charges to about 125 unitary charges, about 5 unitary charges to about 150 unitary charges, about 5 unitary charges to about 200 unitary charges, about 5 unitary charges to about 250 unitary charges, about 10 unitary charges to about 20 unitary charges, about 10 unitary charges to about 30 unitary charges, about 10 unitary charges to about 40 unitary charges, about 10 unitary charges to about 50 unitary charges, about 10 unitary charges to about 75 unitary charges, about 10 unitary charges to about 100 unitary charges, about 10 unitary charges to about 125 unitary charges, about 10 unitary charges to about 150 unitary charges, about 10 unitary charges to about 200 unitary charges, about 10 unitary charges to about 250 unitary charges, about 20 unitary charges to about 30 unitary charges, about 20 unitary charges to about 40 unitary charges, about 20 unitary charges to about 50 unitary charges, about 20 unitary charges to about 75 unitary charges, about 20 unitary charges to about 100 unitary charges, about 20 unitary charges to about 125 unitary charges, about 20 unitary charges to about 150 unitary charges, about 20 unitary charges to about 200 unitary charges, about 20 unitary charges to about 250 unitary charges, about 30 unitary charges to about 40 unitary charges, about 30 unitary charges to about 50 unitary charges, about 30 unitary charges to about 75 unitary charges, about 30 unitary charges to about 100 unitary charges, about 30 unitary charges to about 125 unitary charges, about 30 unitary charges to about 150 unitary charges, about 30 unitary charges to about 200 unitary charges, about 30 unitary charges to about 250 unitary charges, about 40 unitary charges to about 50 unitary charges, about 40 unitary charges to about 75 unitary charges, about 40 unitary charges to about 100 unitary charges, about 40 unitary charges to about 125 unitary charges, about 40 unitary charges to about 150 unitary charges, about 40 unitary charges to about 200 unitary charges, about 40 unitary charges to about 250 unitary charges, about 50 unitary charges to about 75 unitary charges, about 50 unitary charges to about 100 unitary charges, about 50 unitary charges to about 125 unitary charges, about 50 unitary charges to about 150 unitary charges, about 50 unitary charges to about 200 unitary charges, about 50 unitary charges to about 250 unitary charges, about 75 unitary charges to about 100 unitary charges, about 75 unitary charges to about 125 unitary charges, about 75 unitary charges to about 150 unitary charges, about 75 unitary charges to about 200 unitary charges, about 75 unitary charges to about 250 unitary charges, about 100 unitary charges to about 125 unitary charges, about 100 unitary charges to about 150 unitary charges, about 100 unitary charges to about 200 unitary charges, about 100 unitary charges to about 250 unitary charges, about 125 unitary charges to about 150 unitary charges, about 125 unitary charges to about 200 unitary charges, about 125 unitary charges to about 250 unitary charges, about 150 unitary charges to about 200 unitary charges, about 150 unitary charges to about 250 unitary charges, or about 200 unitary charges to about 250 unitary charges.

[0320] In some cases, the first region, second region, and/or third region of the channel of the wild-type biological nanopore or engineered biological nanopore may comprise a charge (e.g., a negative charge). In some cases, a first region and/or a third region may be more net negative than a second region of a nanopore (e.g., an engineered biological nanopore). The net negative charge of the first region, second region, and/or third region of the channel may result from a greater number of negatively charged amino acid residues compared to a number of positively charged amino acid residues, neutral amino acid residues, or combination thereof. A net negative charge or increasing a net negative charge may result from substitution of positively charged amino acid residues to neutral amino acid residues. A net negative charge or increasing a net negative charge may result from substitution of positively charged amino acid residues to negatively-charged amino acid residues. For example, a net negative charge or increasing a net negative charge may result from a region (e.g., a first region, second region, and/or third region) comprising five negatively -charged amino acid residues and five positively -charged amino acid residues and substituting one or more positively -charged residues for one or more neutral amino acid residues. For example, a net negative charge or increasing a net negative charge may result from a region comprising five negatively-charged amino acid residues and five positively -charged amino acid residues and substituting one or more positively -charged residues for one or more negatively- charged amino acid residues and/or one or more neutral charged amino acids. The first region and/or third region of the channel of the engineered biological nanopore (e.g., the region adjacent to the constriction region) may be modified to be more net negative as compared to another region adjacent to a constriction region of a wild-type biological nanopore. In some cases, the first region may be more net negative as compared to a respective region of a wild-type biological nanopore. In some cases, a net charge of a first region may be (e.g., or may be modified to be) at least about 50% more negative as compared to a respective region of a wild-type biological nanopore. The second region of the channel of the engineered biological nanopore (e.g., the region having a constriction region) may be modified to be more net negative as compared to another region having of a nanopore described herein may comprise a constriction region of a wild-type biological nanopore. [0321] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise a negative charge. For example, one or more regions (e.g., a first region, second region, third region, or any combination thereof) may comprise one or more negatively -charged amino acids. To achieve a negative charge, one or more amino acids may be modified to be one or more negatively-charged amino acids. One or more amino acids (e.g., one or more positively-charged amino acids and/or neutral amino acids) may be substituted for one or more negatively- charged amino acids. One or more negatively -charged amino acids may be added. In some cases, a region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality of amino acids modified to a negative charge. A region (e.g., a first region, second region, third region, or any combination thereof) may comprise two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids modified to a negative charge. For example, the two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues may be added to the region (e.g., a first region, second region, third region, or any combination thereof). The negatively-charged amino acids may be any negatively -charged amino acid described herein (e.g., negatively-charged natural amino acids and/or negatively-charged non-natural amino acids).

[0322] In some cases, two or more mutations in two or more monomers may make up a ring of charge in one or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein. One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise two or more mutations that can form a ring of charge relative to the two or more mutations. As an example, the two or more mutations may comprise one or more mutations to negatively-charged amino acids (e.g., substitution to one or more negatively -charged amino acids and/or addition of one or more negatively -charged amino acids). Introduction of a negatively-charged amino acid may thus provide at least one ring of charge comprising a negative charge or a net negative charge (e.g., a negative charge or a net negative charge associated with a mutated amino acid).

[0323] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) may be more net negative as compared to one or more respective regions (e.g., a first region, second region, third region, or any combination thereof) of a wild-type nanopore. One or more regions (e.g., a first region, second region, third region, or any combination thereof) may comprise more negatively- charged amino acids as compared to one or more respective regions (e.g., a first region, second region, third region, or any combination thereof) of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively -charged amino acids and/or neutral amino acids) may be substituted for one or more negatively-charged amino acids. As another example, one or more negatively-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and/or neutral amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). As another example, one or more positively-charged amino acids may be substituted for one or more neutral amino acids in the region (e.g., a first region, second region, third region, or any combination thereof). In some cases, a region (e.g., a first region, second region, third region, or any combination thereof) may be more net negative as compared to a respective region of a wild-type nanopore. The region may comprise a plurality of amino acids modified to a negative charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively -charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues may be added to the region (e.g., a first region, second region, third region, or any combination thereof) to make the region more net negative as compared to a respective region of a wild-type nanopore. As a further example, the region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively-charged amino acids and/or neutral-charged amino acids to achieve a more net negative charge as compared to a respective region of a wild-type nanopore. In some cases, the region of the monomer may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of positively -charged amino acids substituted for a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of neutral-charged amino acids (e.g., to thus make the region more net negative as compared to a respective region of a wild-type nanopore). A region of a nanopore disclosed herein may comprise a ring of charge comprising a plurality of mutations. The ring of charge may be net positive, and then mutated with (1) one or more negative charges and/or (2) one or more neutral charges, so that the ring of charge is more net negative than the ring of charge in the wild type nanopore. The ring of charge may be net neutral, and then mutated with (1) one or more negative charges, so that the ring of charge is more net negative than the ring of charge in the wild type nanopore.

[0324] In some cases, if there may be a total number of rings of charge in a region, and the rings of charge are each net positive, then (i) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively -charged amino acids, (ii) one or more positively -charged amino acids in one or more rings can be substituted with one or more neutral -charged amino acids, (iii) one or more positively-charged amino acids in one or more rings can be deleted, (iv) one or more neutral-charged amino acids in one or more rings can be added, (v) one or more negatively-charged amino acids in one or more rings can be added, or (vi) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a total number rings of charge in a region, and the rings of charge are each net neutral, then (i) one or more neutral -charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more negatively-charged amino acids in one or more rings can be added, or (iii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of positively-charged rings of charge in one or more regions as compared to a number of neutral-charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively -charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (iv) one or more positively-charged amino acids can be deleted, (v) one or more negatively -charged amino acids in one or more rings can be added, (vi) one or more neutral- charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore.

[0325] In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of positively -charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively -charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively -charged amino acids, (iv) one or more positively -charged amino acids in one or more rings can be deleted, (v) one or more negatively -charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of negatively -charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more negatively-charged amino acids in one or more rings can be added, (iii) one or more neutral-charged amino acids in one or more rings can be deleted, or (vii) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore.

[0326] In some cases, if there may be a greater number or equal number of negatively-charged rings of charge in a region as compared to a number of positively-charged rings of charge, then (i) one or more negatively-charged amino acids in one or more rings can be added, (ii) one or more positively-charged amino acids in one or more rings can be deleted, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iv) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, or (v) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of negatively -charged rings of charge in a region as compared to a number of neutral-charged rings of charge, then (i) one or more negatively -charged amino acids in one or more rings can be added, (ii) one or more neutral -charged amino acids in one or more rings can be deleted, (iii) one or more neutral -charged amino acids in one or more rings can be substituted with one or more negatively- charged amino acids, or (v) any combination thereof, to arrive at the region that is more net negative (e.g., comprising more net negative rings of charge) as compared to a respective region of a wild-type nanopore.

[0327] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) may be net negative. A net negative charge may be achieved when there may be a greater number of negatively -charged amino acids compared to a number of positively-charged amino acids and/or a number of neutral charged amino acids. A net negative charge of one or more regions (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively -charged amino acids and/or neutral amino acids) for one or more negatively- charged amino acids; (ii) deleting one or more positively-charged amino acids and/or neutral amino acids; (iii) adding one or more negatively-charged amino acids; or (iv) any combinations thereof. In some cases, a region (e.g., a first region, second region, third region, or any combination thereof) may be net negative. The region (e.g., a first region, second region, third region, or any combination thereof) may comprise negatively -charged amino acids. The portion may comprise a plurality of amino acids modified to a negative charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively -charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) negatively-charged amino acid residues may be added to the region (e.g., a first region, second region, third region, or any combination thereof). In some cases, the region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality of substituted and/or added negatively-charged amino acids such that the total amino acids of the region are negatively charged (e.g., and the region is net negative). The negatively-charged amino acids may be any negatively -charged amino acid described herein. As a further example, the region (e.g., a first region, second region, third region, or any combination thereof) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively- charged amino acids and/or neutral -charged amino acids to achieve a negative charge (e.g., a greater number of negatively -charged amino acids in the region as compared to a number of positively-charged amino acids and/or neutral charged amino acids). In some cases, two or more monomers making up a ring of charge in a region may be net negative. For example, for an octameric nanopore (e.g., a nanopore comprising eight monomeric units) two more monomers may be net negative in a region (e.g., a first region, second region, third region, or any combination thereof). As another example, each monomer making up a ring of charge in a region may be net negative. The ring of charge may be net neutral or net positive, and then mutated so that the ring of charge is net negative. For example, the ring of charge may be net positive, then mutated with one or more negative or one or more neutral charges so that the ring of charge is net negative. As another example, the ring of charge may be net neutral, then mutated with one or more negative charges so that the ring of charge is net negative.

[0328] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise a neutral charge. For example, one or more regions may comprise one or more neutral-charged amino acids. To achieve a neutral charge, one or more amino acids may be modified to be one or more neutral -charged amino acids. One or more amino acids (e.g., one or more positively -charged amino acids and/or negatively-charged amino acids) may be substituted for one or more neutral -charged amino acids. One or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). In some cases, a region (e.g., a second region, e.g., a constriction region) may comprise a plurality of amino acids modified to a neutral charge. A region (e.g., a second region, e.g., a constriction region) may comprise two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids modified to a neutral charge. For example, the two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues may be added to the region (e.g., a second region, e.g., a constriction region). The neutral- charged amino acids may be any neutral-charged amino acid described herein. In some cases, two or more mutations in two or more monomers may make up a ring of charge in one or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein. One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may comprise two or more mutations that can form a ring of charge relative to the two or more mutations. As an example, the two or more mutations may comprise one or more mutations to neutral-charged amino acids (e.g., substitution to one or more neutral-charged amino acids and/or addition of one or more neutral -charged amino acids). Introduction of a neutral-charged amino acid may thus provide at least one ring of charge comprising a neutral charge or a net negative charge (e.g., a neutral charge or a net negative charge associated with a mutated amino acid).

[0329] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may be more net neutral as compared to one or more respective regions of a wild-type nanopore. One or more regions (e.g., a first region, second region, third region, or any combination thereof) may comprise more neutral-charged amino acids as compared to one or more respective regions of a wild-type nanopore. As an example, one or more amino acids (e.g., one or more positively-charged amino acids and/or negatively -charged amino acids) may be substituted for one or more neutral-charged amino acids. As another example, one or more neutral-charged amino acids may be added to the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively-charged amino acids and/or negatively-charged amino acids may be added to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and achieve a neutral charge). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and/or negatively-charged amino acids) may be deleted from the region (e.g., a first region, second region, third region, or any combination thereof). A number of one or more positively-charged amino acids and/or negatively-charged amino acids may be deleted to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and achieve a neutral charge). In some cases, a region (e.g., a second region, e.g., a constriction region) may be more net neutral as compared to a respective region (e.g., a constriction region) of a wild-type nanopore. The region (e.g., a second region, e.g., a constriction region) may comprise more neutral-charged amino acids as compared to a respective region (e.g., a constriction region) of a wild-type nanopore. The region may comprise a plurality of amino acids modified to a neutral charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues may be added to the region (e.g., a second region, e.g., a constriction region). The neutral -charged amino acids may be any neutral-charged amino acid described herein. As a further example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively-charged amino acids and/or negatively -charged amino acids to achieve a same number of positively -charged amino acids and negatively-charged amino acids in the region (e.g., and thus be more net neutral as compared to a respective region (e.g., a constriction region) of a wild-type nanopore). As another example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of added positively-charged amino acids and/or negatively-charged amino acids to achieve a same number of positively- charged amino acids and negatively-charged amino acids in the region (e.g., and thus be more net neutral as compared to a respective region (e.g., a constriction region) of a wild-type nanopore). A region of a nanopore disclosed herein may comprise a ring of charge comprising a plurality of mutations. The ring of charge may be net negative, and then mutated with (1) one or more positive charges and/or (2) one or more neutral charges, so that the ring of charge is more net neutral than the ring of charge in the wild type nanopore. The ring of charge may be net positive, and then mutated with (1) one or more negative charges and/or (2) one or more neutral charges, so that the ring of charge is more net neutral than the ring of charge in the wild type nanopore. [0330] In some cases, if there may be a total number of rings of charge in a region, and the rings of charge are each net positive, then (i) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively -charged amino acids, (ii) one or more positively -charged amino acids in one or more rings can be substituted with one or more neutral -charged amino acids, (iii) one or more positively-charged amino acids in one or more rings can be deleted, (iv) one or more neutral-charged amino acids in one or more rings can be added, (v) one or more negatively-charged amino acids in one or more rings can be added, or (vi) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a total number of rings of charge in a region, and the rings of charge are each net negative, then (i) one or more negatively-charged amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (ii) one or more negatively -charged amino acids in one or more rings can be substituted with one or more neutral-charged amino acids, (iii) one or more negatively -charged amino acids in one or more rings can be deleted, (iv) one or more neutral-charged amino acids in one or more rings can be added, (v) one or more positively-charged amino acids in one or more rings can be added, or (vi) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore.

[0331] In some cases, if there may be a greater number or equal number of positively -charged rings of charge in a region as compared to a number of neutral -charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively -charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively -charged amino acids, (iv) one or more positively -charged amino acids in one or more rings can be deleted, (v) one or more negatively -charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of positively -charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more negatively-charged amino acids, (ii) one or more positively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more positively-charged amino acids in one or more rings can be substituted with one or more negatively -charged amino acids, (iv) one or more positively- charged amino acids in one or more rings can be deleted, (v) one or more negatively -charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore.

[0332] In some cases, if there may be a greater number or equal number of neutral-charged rings of charge in a region as compared to a number of negatively-charged rings of charge, then (i) one or more neutral amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (ii) one or more negatively -charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iii) one or more negatively-charged amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (iv) one or more negatively -charged amino acids in one or more rings can be deleted, (v) one or more positively -charged amino acids in one or more rings can be added, (vi) one or more neutral-charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of negatively -charged rings of charge in a region as compared to a number of positively-charged rings of charge, then (i) one or more positively-charged amino acids in one or more rings can be added, (ii) one or more neutral-charged amino acids in one or more rings can be added, (iii) one or more negatively- charged amino acids in one or more rings can be deleted, (iii) one or more negatively-charged amino acids in one or more rings can be substituted with one or more neutral amino acids, (iv) one or more negatively- charged amino acids in one or more rings can be substituted with one or more positively -charged amino acids, or (v) any combination thereof, to arrive at the region that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. In some cases, if there may be a greater number or equal number of negatively-charged rings of charge in a region as compared to a number of neutral-charged rings of charge, then (i) one or more positively -charged amino acids in one or more rings can be added, (ii) one or more neutral -charged amino acids in one or more rings can be added, (iii) one or more negatively -charged amino acids in one or more rings can be substituted with one or more positively-charged amino acids, (iv) one or more negatively -charged amino acids in one or more rings can be substituted with one or more neutral-charged amino acids, (v) one or more negatively charged amino acids in one or more rings can be deleted, or (v) any combination thereof, to arrive at the regions that is more net neutral (e.g., comprising more net neutral rings of charge) as compared to a respective region of a wild-type nanopore. [0333] One or more regions (e.g., a first region, second region, third region, second protein unit, or any combination thereof) of a nanopore described herein may be net neutral. A net neutral charge may be achieved when there may be a same number of positively-charged amino acids and negatively-charged amino acids. A net neutral charge may be achieved when the one or more regions (e.g., a first region, second region, third region, or any combination thereof) comprises all neutral charged amino acids. A net neutral charge of one or more regions (e.g., a first region, second region, third region, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and/or negatively- charged amino acids) for one or more neutral-charged amino acids; (ii) deleting one or more positively -charged amino acids and/or negatively-charged amino acids; (iii) adding one or more neutral -charged amino acids; or (iv) any combinations thereof. A net neutral charge of one or more regions (e.g., a first region, second region, third region, or any combination thereof) may also be achieved by (i) adding one or more positively -charged amino acids and/or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge) and/or (ii) deleting one or more positively-charged amino acids and/or negatively -charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge). In some cases, a region (e.g., a second region, e.g., a constriction region) may be net neutral. The region (e.g., a second region, e.g., a constriction region) may comprise neutral-charged amino acids. The region may comprise a plurality of amino acids modified to a neutral charge. For example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) amino acids may be substituted with two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues. As another example, two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) neutral-charged amino acid residues may be added to the region (e.g., a second region, e.g., a constriction region). In some cases, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality of substituted and/or added neutral -charged amino acids such that the total amino acids of the region are neutral charged (e.g., and the region is net neutral). The neutral-charged amino acids may be any neutral-charged amino acid described herein. As a further example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of deleted positively -charged amino acids and/or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids in the region (e.g., and thus be net neutral). As another example, the region (e.g., a second region, e.g., a constriction region) may comprise a plurality (e.g., 2 or more, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, or greater than about 30) of added positively-charged amino acids and/or negatively -charged amino acids to achieve a same number of positively -charged amino acids and negatively-charged amino acids in the region (e.g., and thus be net neutral). In some cases, two or more monomers making up a ring of charge in a region may be net neutral. For example, for an octameric nanopore (e.g., a nanopore comprising eight monomeric units) two more monomers (e.g., about 2, 3, 4, 5, 6, 7, or 8 monomers) may be net neutral in a region (e.g., a first region, second region, third region, or any combination thereof). As another example, each monomer making up a ring of charge in a region may be net neutral. The ring of charge may be net negative or net positive, and then mutated so that the ring of charge is net neutral. For example, the ring of charge may be net negative, then mutated with one or more positive or one or more neutral charges so that the ring of charge is net neutral. As another example, the ring of charge may be net positive, then mutated with one or more negative or one or more neutral charges so that the ring of charge is net neutral.

[0334] There may be a distance between an amino acid (e.g., a modified amino acid) in one region of the nanopore described herein (e.g., an engineered biological nanopore) and another amino acid (e.g., another modified amino acid) in another region of the nanopore. In some cases, a mutated amino acid in a first region may be separated by a distance from a mutated amino acid in a second region (e.g., a different region). As an example, a mutated amino acid in a region (e.g., a first region and/or a third region) may be separated by a distance from another mutated amino acid in another region (e.g., the second region). A mutated amino acid in a region (e.g., a first region and/or a third region) may be separated from another mutated amino acid in another region (e.g., the second region) by a distance of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. A mutated amino acid in a region (e.g., a first region and/or a third region) may be separated from another mutated amino acid in a region (e.g., the second region) by a distance of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0335] Two amino acids (e.g., two mutated amino acids) may be separated from one another, wherein the two amino acids may be in the same region of a nanopore. In some cases, an amino acid (e.g., a modified amino acid) in one region of a nanopore described herein (e.g., an engineered biological nanopore) may be separated by a distance from another amino acid (e.g., another modified amino acid) in the same region. A mutated amino acid in a region (e.g., a first region, a second region, a third region, a second protein unit, or any combination thereof) may be separated from another mutated amino acid in the same region (e.g., a first region, a second region, a third region, a second protein unit, or any combination thereof) by a distance of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. A mutated amino acid in a region (e.g., a first region, a second region, a third region, a second protein unit, or any combination thereof) may be separated from another mutated amino acid in the same region (e.g., a first region, a second region, a third region, a second protein unit, or any combination thereof) by a distance of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about

6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0336] A mutated amino acid in a first region or third region may be at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm away from a mutated amino acid in a second region. A mutated amino acid in a first region or third region may be at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about

7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm away from a mutated amino acid in a second region.

[0337] In some cases, a first portion, second portion, and/or third portion of a monomer described herein may comprise a charge (e.g., a negative, a neutral charge, or any combination thereof). A first portion, second portion, and/or third portion of a monomer may comprise a negative charge. A first portion, second portion, and/or third portion of a monomer may be modified to be more net negative as compared to a respective portion of a wild-type monomer. A first portion, second portion, and/or third portion of a monomer may comprise a net negative charge. The net negative charge may result from a greater number of negatively charged amino acid residues compared to a number of positively charged amino acid residues, neutral amino acid residues, or combination thereof. A net negative charge or increasing a net negative charge of a first portion of a monomer described herein may result from substitution of positively charged amino acid residues to neutral amino acid residues. A net negative charge or increasing a net negative charge of a first portion of a monomer described herein may result from substitution of positively charged amino acid residues to negatively-charged amino acid residues. For example, a net negative charge or increasing a net negative charge of a first portion, second portion, and/or third portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively-charged residues for one or more neutral amino acid residues. For example, a net negative charge or increasing a net negative charge of a first portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively-charged residues for one or more negatively-charged amino acid residues. A third portion of a monomer may comprise a net negative charge. The net negative charge may result from a greater number of negatively charged amino acid residues compared to a number of positively charged amino acid residues, neutral amino acid residues, or combination thereof. A net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from substitution of positively charged amino acid residues to neutral amino acid residues. A net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from substitution of positively charged amino acid residues to negatively -charged amino acid residues. For example, a net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively -charged residues for one or more neutral amino acid residues. For example, a net negative charge or increasing a net negative charge of a third portion of a monomer described herein may result from a portion comprising five negatively-charged amino acid residues and five positively-charged amino acid residues, and substituting one or more positively-charged residues for one or more negatively-charged amino acid residues. The first portion and/or third portion of an engineered monomer (e.g., the portion(s) adjacent to the constriction-forming portion of the monomer) may be more negative as compared to another portion adjacent to a constriction-forming portion of a wild-type monomer. The second portion of the engineered monomer (e.g., the constriction-forming portion) may be more negative as compared to another constriction-forming portion of a wild-type biological nanopore.

[0338] In some cases, a first portion and/or a third portion may comprise a negative charge (e.g., net negative charge). The negative charge may be from one or more negatively -charged amino acids (e.g., one or more negatively-charged natural amino acids and/or one or more negatively-charged non-natural amino acids). In some cases, a first portion or a third portion may comprise at least one amino acid that is mutated to exhibit an increased net negative charge. For example, one or more positively -charged amino acids and/or one or more neutral amino acids may be modified (e.g., mutated) to one or more negatively-charged amino acids. In some cases, a first portion and/or a third portion may comprise a plurality of amino acids that are mutated to exhibit an increased net negative charge. For example, a first portion may comprise two amino acids that are mutated to exhibit an increased net negative charge and a third portion may comprise two amino acids that are mutated to exhibit an increased net negative charge. In some cases, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, or greater than about 30 amino acids in a first portion and/or third portion may be modified to one or more amino acids that increase a net negative charge (e.g., one or more negatively -charged amino acids). In some cases, at most about 30, at most about 20, at most about 10, 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 amino acid in a first portion and/or third portion may be modified to one or more amino acids that increase a net negative charge (e.g., one or more negatively-charged amino acids). Modifications of one or more amino acids of a portion of a monomer described herein may comprise (i) substitution of one or more amino acids (e.g., natural and/or non-natural amino acids); (ii) addition of one or more amino acids (e.g., natural and/or non-natural amino acids); (iii) deletion of one or more amino acids (e.g., natural and/or non-natural amino acids); (iv) or any combinations thereof. Modifications of amino acids may also comprise post-translational modifications. For example, modifications of one or more amino acids of a portion of a monomer described herein may comprise phosphorylation, methylation, acetylation, glycosylation, addition of disulfide bonds, ubiquitination, hydroxylation, carboxylation, lipidation, amidation, or any combinations thereof. Any post-translational modification may be combined with one or more of addition of at least one amino acid, deletion of at least one amino acid, or substitution of at least one amino acid.

[0339] A first portion and/or a third portion may comprise at least one amino acid that is mutated to exhibit an increased net negative charge as compared to a respective portion of a wild-type biological nanopore. For example, a first portion of an engineered biological nanopore described herein may have an amino acid in a second portion (e.g., constriction portion). A wild-type biological nanopore may have an amino acid at an identical position in a second portion. The amino acid in the engineered biological nanopore may be mutated to increase a net negative charge. Thus, the first portion of the engineered biological nanopore comprises at least one amino acid that is mutated to exhibit an increased net negative charge as compared to a respective portion of a wild-type biological nanopore. As another example, a first portion and/or a third portion may comprise at least one amino acid that is mutated to a negatively-charged amino acid to exhibit an increased net negative charge as compared to a respective portion of a wild-type biological nanopore.

[0340] In some cases, at least one mutated amino acid in a first portion of each of one or more monomers may form a first ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a first portion and/or third portion of each of the 8 monomers may form a first ring of charge. In other cases, at least one mutated amino acid in a first portion and/or third portion of less than all monomers may form a first ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a first portion of 6 monomers may form a first ring of charge. In some cases, at least one mutated amino acid in a second portion of each of one or more monomers may form a second ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a second portion of each of the 8 monomers may form a second ring of charge. In other cases, at least one mutated amino acid in a second portion of less than all monomers may form a second ring of charge. For example, in an octameric nanopore, at least one mutated amino acid in a second portion of 6 monomers may form a second ring of charge.

[0341] One or more portions (e.g., a first portion, second portion, third portion, second protein unit, or any combination thereof) of a monomer described herein may comprise a neutral charge. For example, one or more portions may comprise one or more neutral-charged amino acids. To achieve a neutral charge, one or more amino acids may be modified to be one or more neutral -charged amino acids. One or more amino acids (e.g., one or more positively -charged amino acids and/or negatively-charged amino acids) may be substituted for one or more neutral-charged amino acids. One or more neutral-charged amino acids may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof). In some cases, a portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality of amino acids modified to a neutral charge. A portion (e.g., a second portion, e.g., a constriction forming portion) may comprise two or more (e.g., three or more, e.g., five or more) amino acids modified to a neutral charge. For example, the two or more (e.g., three or more, e.g., five or more) amino acids may be substituted with two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues. For example, the eight or less (e.g., five or less, e.g., three or less) amino acids may be substituted with eight or less (e.g., five or less, e.g., three or less) neutral -charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). As another example, eight or less (e.g., five or less, e.g., three or less) neutral-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). The neutral -charged amino acids may be any neutral -charged amino acid described herein.

[0342] One or more portions (e.g., a first portion, second portion, third portion, second protein unit, or any combination thereof) of a monomer described herein may be more net neutral as compared to one or more respective portions of a wild-type monomer. One or more portions (e.g., a first portion, second portion, third portion, or any combination thereof) may comprise more neutral-charged amino acids as compared to one or more respective portions of a wild-type monomer. As an example, one or more amino acids (e.g., one or more positively-charged amino acids and/or negatively -charged amino acids) may be substituted for one or more neutral-charged amino acids. As another example, one or more neutral-charged amino acids may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof). A number of one or more positively -charged amino acids and/or negatively-charged amino acids may be added to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and achieve a more net neutral charge as compared to a respective portion of a wild-type monomer). As another example, one or more amino acids (e.g., one or more positively -charged amino acids and/or negatively-charged amino acids) may be deleted from the portion (e.g., a first portion, second portion, third portion, or any combination thereof). A number of one or more positively-charged amino acids and/or negatively-charged amino acids may be deleted to achieve a same number of positively-charged amino acids and negatively -charged amino acids (e.g., and achieve a more net neutral charge as compared to a respective portion of a wild-type monomer). In some cases, a portion (e.g., a second portion, e.g., a constriction forming portion) may be more net neutral as compared to a respective portion (e.g., a constriction forming portion) of a wild-type monomer. The portion (e.g., a second portion, e.g., a constriction forming portion) may comprise more neutral-charged amino acids as compared to a respective portion (e.g., a constriction forming portion) of a wild-type monomer. The portion may comprise a plurality of amino acids modified to a neutral charge. For example, two or more (e.g., three or more, e.g., five or more) amino acids may be substituted with two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues. For example, eight or less (e.g., five or less, e.g., three or less) amino acids may be substituted with eight or less (e.g., five or less, e.g., three or less) neutral -charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). As another example, eight or less (e.g., five or less, e.g., three or less) neutral-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). The neutral-charged amino acids may be any neutral-charged amino acid described herein. As a further example, the portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of deleted positively-charged amino acids and/or negatively -charged amino acids to achieve a same number of positively -charged amino acids and negatively-charged amino acids in the portion (e.g., and thus be more net neutral as compared to a respective portion (e.g., a constriction forming portion) of a wild-type monomer). As another example, the portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of added positively -charged amino acids and/or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids in the portion (e.g., and thus be more net neutral as compared to a respective portion (e.g., a constriction forming portion) of a wild-type monomer).

[0343] In some cases, if there may be a total number of positively-charged amino acids in a portion of a monomer, and the portion of the monomer is net positive, then (i) one or more positively -charged amino acids can be substituted with one or more negatively -charged amino acids, (ii) one or more positively-charged amino acids can be substituted with one or more neutral-charged amino acids, (iii) one or more positively -charged amino acids can be deleted, (iv) one or more neutral-charged amino acids can be added, (v) one or more negatively-charged amino acids can be added, or (vi) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a total number of negatively-charged amino acids in a portion of a monomer, and the portion of the monomer is net negative, then (i) one or more negatively-charged amino acids can be substituted with one or more positively-charged amino acids, (ii) one or more negatively -charged amino acids can be substituted with one or more neutral -charged amino acids, (iii) one or more negatively-charged amino acids can be deleted, (iv) one or more neutral-charged amino acids can be added, (v) one or more positively -charged amino acids can be added, or (vi) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer.

[0344] In some cases, if there may be a greater number or an equal number of positively -charged amino acids as compared to neutral amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more negatively -charged amino acids, (ii) one or more positively -charged amino acids can be substituted with one or more neutral amino acids, (iii) one or more positively -charged amino acids can be substituted with one or more negatively -charged amino acids, (iv) one or more positively-charged amino acids can be deleted, (v) one or more negatively-charged amino acids can be added, (vi) one or more neutral- charged amino acids can be added, or (vii) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number or an equal number of neutral -charged amino acids as compared to positively -charged amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more negatively -charged amino acids, (ii) one or more positively-charged amino acids can be substituted with one or more neutral amino acids, (iii) one or more positively -charged amino acids can be substituted with one or more negatively -charged amino acids, (iv) one or more positively -charged amino acids can be deleted, (v) one or more negatively- charged amino acids can be added, (vi) one or more neutral -charged amino acids in one or more rings can be added, or (vii) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number or an equal number of neutral-charged amino acids as compared to negatively-charged amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more positively-charged amino acids, (ii) one or more negatively -charged amino acids can be substituted with one or more neutral amino acids, (iii) one or more negatively -charged amino acids can be substituted with one or more positively-charged amino acids, (iv) one or more negatively-charged amino acids can be deleted, (v) one or more positively-charged amino acids can be added, (vi) one or more neutral-charged amino acids can be added, or (vii) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer. [0345] In some cases, if there may be a greater number or an equal number of negatively -charged amino acids as compared to positively-charged amino acids in a portion of a monomer, then (i) one or more positively- charged amino acids can be added, (ii) one or more neutral -charged amino acids can be added, (iii) one or more negatively-charged amino acids can be deleted, (iii) one or more negatively-charged amino acids can be substituted with one or more neutral amino acids, (iv) one or more negatively -charged amino acids can be substituted with one or more positively-charged amino acids, or (v) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number or an equal number of negatively -charged amino acids as compared to neutral amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more positively-charged amino acids, (ii) one or more negatively -charged amino acids can be substituted with one or more neutral amino acids, (iii) one or more negatively-charged amino acids can be substituted with one or more positively-charged amino acids, (iv) one or more negatively-charged amino acids can be deleted, (v) one or more positively -charged amino acids can be added, (vi) one or more neutral-charged amino acids can be added, or (vii) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number positively-charged amino acids as compared to negatively-charged amino acids, then (i) one or more negatively-charged amino acids and/or one or more positively -charged amino acids can be substituted with one or more neutral-charged amino acids, (ii) one or more positively -charged amino acids can be deleted, (iii) one or more negatively -charged amino acids can be added, or (iv) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number negatively- charged amino acids as compared to positively-charged amino acids, then (i) one or more negatively -charged amino acids and/or one or more positively-charged amino acids can be substituted with one or more neutral- charged amino acids, (ii) one or more negatively-charged amino acids can be deleted, (iii) one or more positively-charged amino acids can be added, or (iv) any combination thereof, to arrive at more net neutral portion of the monomer as compared to a portion of a wild-type monomer.

[0346] One or more portions (e.g., a first portion, second portion, third portion, second protein unit, or any combination thereof) of a monomer described herein may be net neutral. A net neutral charge may be achieved when there may be a same number of positively-charged amino acids and negatively-charged amino acids. A net neutral charge may be achieved when the one or more portions (e.g., a first portion, second portion, third portion, or any combination thereof) comprises all neutral charged amino acids. A net neutral charge of one or more portions (e.g., a first portion, second portion, third portion, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and/or negatively-charged amino acids) for one or more neutral-charged amino acids; (ii) deleting one or more positively-charged amino acids and/or negatively-charged amino acids; (iii) adding one or more neutral- charged amino acids; or (iv) any combinations thereof. A net neutral charge of one or more portions (e.g., a first portion, second portion, third portion, or any combination thereof) may also be achieved by (i) adding one or more positively-charged amino acids and/or negatively -charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge) and/or (ii) deleting one or more positively -charged amino acids and/or negatively -charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids (e.g., and thus comprise an overall neutral charge). In some cases, a portion (e.g., a second portion, e.g., a constriction forming portion) may be net neutral. The portion (e.g., a second portion, e.g., a constriction forming portion) may comprise neutral-charged amino acids. The portion may comprise a plurality of amino acids modified to a neutral charge. For example, two or more (e.g., three or more, e.g., five or more) amino acids may be substituted with two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues. For example, eight or less (e.g., five or less, e.g., three or less) amino acids may be substituted with eight or less (e.g., five or less, e.g., three or less) neutral-charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) neutral-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). As another example, eight or less (e.g., five or less, e.g., three or less) neutral-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). In some cases, the portion (e.g., the second portion, e.g., the constriction forming portion) may comprise a plurality of substituted and/or added neutral-charged amino acids such that the total amino acids of the portion are neutral charged (e.g., and the portion is net neutral). The neutral-charged amino acids may be any neutral-charged amino acid described herein. As a further example, the portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of deleted positively-charged amino acids and/or negatively -charged amino acids to achieve a same number of positively -charged amino acids and negatively-charged amino acids in the portion (e.g., and thus be net neutral). As a further example, the portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of deleted positively- charged amino acids and/or negatively-charged amino acids to achieve a same number of positively -charged amino acids and negatively-charged amino acids in the portion (e.g., and thus be net neutral). As another example, the portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of added positively -charged amino acids and/or negatively-charged amino acids to achieve a same number of positively-charged amino acids and negatively-charged amino acids in the portion (e.g., and thus be net neutral).

[0347] One or more portions (e.g., a first portion, second portion, third portion, second protein unit, or any combination thereof) of a monomer described herein may comprise a negative charge. For example, one or more portions may comprise one or more negatively-charged amino acids. To achieve a negative charge, one or more amino acids may be modified to be one or more negatively-charged amino acids (e.g., aspartic acid (D) and/or glutamic acid (E)). One or more amino acids (e.g., one or more positively-charged amino acids and/or neutral-charged amino acids) may be substituted for one or more negatively -charged amino acids. One or more negatively -charged amino acids may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof). In some cases, a portion (e.g., a second portion, e.g., a constriction forming portion) may comprise a plurality of amino acids modified to a negative charge. A portion (e.g., a first portion, second portion, third portion, or any combination thereof) may comprise two or more (e.g., three or more, e.g., five or more) amino acids modified to a negative charge.

[0348] For example, the two or more (e.g., three or more, e.g., five or more) amino acids may be substituted with two or more (e.g., three or more, e.g., five or more) negatively -charged amino acid residues. For example, the eight or less (e.g., five or less, e.g., three or less) amino acids may be substituted with eight or less (e.g., five or less, e.g., three or less) negatively-charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) negatively-charged amino acid residues may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof). As another example, eight or less (e.g., five or less, e.g., three or less) negatively-charged amino acid residues may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof). The negatively-charged amino acids may be any negatively-charged amino acid described herein (e.g., negatively-charged natural amino acids and/or negatively-charged non-natural amino acids). [0349] One or more portions (e.g., a first portion, second portion, third portion, second protein unit, or any combination thereof) of a monomer described herein may be more net negative as compared to one or more respective portions of a wild-type monomer. One or more portions (e.g., a first portion, second portion, third portion, or any combination thereof) may comprise more negatively -charged amino acids as compared to one or more respective portions of a wild-type monomer. As an example, one or more amino acids (e.g., one or more positively-charged amino acids and/or neutral -charged amino acids) may be substituted for one or more negatively-charged amino acids. As an example, one or more positively-charged amino acids may be substituted for one or more neutral-charged amino acids. As another example, one or more negatively -charged amino acids may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof). As another example, one or more amino acids (e.g., one or more positively-charged amino acids and/or neutral-charged amino acids) may be deleted from the portion (e.g., a first portion, second portion, third portion, or any combination thereof). In some cases, a portion (e.g., a first portion, second portion, third portion, or any combination thereof) may be more net negative as compared to a respective portion of a wild-type monomer. The portion may comprise a plurality of amino acids modified to a negative charge. For example, two or more (e.g., three or more, e.g., five or more) amino acids may be substituted with two or more (e.g., three or more, e.g., five or more) negatively -charged amino acid residues. For example, eight or less (e.g., five or less, e.g., three or less) amino acids may be substituted with eight or less (e.g., five or less, e.g., three or less) negatively-charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) negatively-charged amino acid residues may be added to the portion (e.g., a first portion, second portion, third portion, or any combination thereof) to make the portion more net negative as compared to a respective portion of a wild-type monomer. As a further example, the portion (e.g., a first portion, second portion, third portion, or any combination thereof) may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of deleted positively-charged amino acids and/or neutral-charged amino acids to achieve a more net negative charge as compared to a respective portion of a wild-type monomer. In some cases, the portion of the monomer may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of positively -charged amino acids substituted for a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of neutral-charged amino acids (e.g., to thus make the portion more net negative as compared to a respective portion of a wild-type monomer).

[0350] In some cases, if there may be a number of positively-charged amino acids and a portion of a monomer is net positive, then (i) one or more positively-charged amino acids can be substituted with one or more negatively-charged amino acids, (ii) one or more positively -charged amino acids can be substituted with one or more neutral -charged amino acids, (iii) one or more positively-charged amino acids can be deleted, (iv) one or more neutral-charged amino acids can be added, (v) one or more negatively -charged amino acids can be added, or (vi) any combination thereof, to arrive at more net negative portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a number of neutral-charged amino acids and a portion of a monomer is net neutral, then i) one or more neutral-charged amino acids can be substituted with one or more negatively -charged amino acids, (ii) one or more negatively -charged amino acids can be added, or (iii) any combination thereof, to arrive at more net negative portion of the monomer as compared to a portion of a wild-type monomer.

[0351] In some cases, if there may be a greater number or an equal number of positively -charged amino acids as compared to neutral amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more negatively -charged amino acids, (ii) one or more positively -charged amino acids can be substituted with one or more neutral amino acids, (iii) one or more positively -charged amino acids can be substituted with one or more negatively -charged amino acids, (iv) one or more positively-charged amino acids can be deleted, (v) one or more negatively-charged amino acids can be added, or (vi) any combination thereof, to arrive at more net negative portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number or an equal number of neutral -charged amino acids as compared to positively-charged amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more negatively-charged amino acids, (ii) one or more positively -charged amino acids can be substituted with one or more neutral amino acids, (iii) one or more positively -charged amino acids can be substituted with one or more negatively-charged amino acids, (iv) one or more positively- charged amino acids can be deleted, (v) one or more negatively-charged amino acids can be added, (vi) one or more neutral -charged amino acids can be added, or (vii) any combination thereof, to arrive at more net negative portion of the monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number or an equal number of negatively-charged amino acids as compared to positively -charged amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more negatively -charged amino acids, (ii) one or more negatively-charged amino acids can be added, (iii) one or more neutral -charged amino acids in one or more rings can be deleted, or (vii) any combination thereof, to arrive at more net negative portion of the monomer as compared to a portion of a wild-type monomer.

[0352] In some cases, if there may be a greater number or an equal number of negatively -charged amino acids as compared to positively-charged amino acids in a portion of a monomer, then (i) one or more negatively- charged amino acids can be added, (ii) one or more positively-charged amino acids can be deleted, (iii) one or more positively-charged amino acids can be substituted with one or more neutral amino acids, (iv) one or more positively-charged amino acids can be substituted with one or more negatively-charged amino acids, or (v) any combination thereof, to arrive at more net negative portion of the monomer as compared to a portion of a wildtype monomer. In some cases, if there may be a greater number or an equal number of negatively -charged amino acids as compared to neutral amino acids in a portion of a monomer, then (i) one or more neutral amino acids can be substituted with one or more negatively -charged amino acids, (ii) one or more negatively -charged amino acids can be added, or (iii) any combination thereof, to arrive at more net negative one or more portions of a monomer as compared to a portion of a wild-type monomer. In some cases, if there may be a greater number positively-charged amino acids as compared to negatively-charged amino acids, then (i) one or more positively-charged amino acids can be substituted with one or more neutral-charged amino acids, (ii) one or more positively-charged amino acids can be substituted with one or more negatively-charged amino acids, (iii) one or more positively -charged amino acids can be deleted, (iv) one or more negatively-charged amino acids can be added, or (v) any combination thereof, to arrive at more net negative one or more portions of a monomer as compared to one or more respective portions of a wild-type monomer. In some cases, if there may be a greater number negatively-charged amino acids as compared to positively-charged amino acids, then (i) one or more positively-charged amino acids can be substituted with one or more neutral-charged amino acids, (ii) one or more positively -charged amino acids can be substituted with one or more negatively-charged amino acids, (iii) one or more positively-charged amino acids can be deleted, (iv) one or more negatively -charged amino acids can be added, or (v) any combination thereof, to arrive at more net negative portion of the monomer as compared to a portion of a wild-type monomer.

[0353] One or more portions (e.g., a first portion, second portion, third portion, second protein unit, or any combination thereof) of a monomer described herein may be net negative. A net negative charge may be achieved when there may be greater number of negatively -charged amino acids in the one or more portions. A net negative charge may be achieved when the one or more portions (e.g., a first portion, second portion, third portion, or any combination thereof) comprises all negatively -charged amino acids. A net negative charge of one or more portions (e.g., a first portion, second portion, third portion, or any combination thereof) may be achieved by (i) substituting one or more amino acids (e.g., one or more positively-charged amino acids and/or neutral-charged amino acids) for one or more negatively-charged amino acids; (ii) deleting one or more positively-charged amino acids and/or neutral-charged amino acids; (iii) adding one or more negatively- charged amino acids; or (iv) any combinations thereof. In some cases, a portion (e.g., a second portion, e.g., a constriction forming portion) may be net negative. The portion (e.g., a first portion, second portion, third portion, or any combination thereof) may comprise negatively-charged amino acids. The portion may comprise a plurality of amino acids modified to a negative charge. For example, two or more (e.g., three or more, e.g., five or more) amino acids may be substituted with two or more (e.g., three or more, e.g., five or more) negatively-charged amino acid residues. For example, eight or less (e.g., five or less, e.g., three or less) amino acids may be substituted with eight or less (e.g., five or less, e.g., three or less) negatively-charged amino acid residues. As another example, two or more (e.g., three or more, e.g., five or more) negatively-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). As another example, eight or less (e.g., five or less, e.g., three or less) negatively-charged amino acid residues may be added to the portion (e.g., a second portion, e.g., a constriction forming portion). In some cases, the portion (e.g., a first portion, second portion, third portion, or any combination thereof) may comprise a plurality of substituted and/or added negatively-charged amino acids such that the total amino acids of the portion are negatively charged (e.g., and the portion is net negative). The negatively-charged amino acids may be any negatively-charged amino acid described herein. As a further example, the portion (e.g., a first portion, second portion, third portion, or any combination thereof) may comprise a plurality (e.g., two or more (e.g., three or more, e.g., five or more), or eight or less (e.g., five or less, e.g., three or less)) of deleted positively-charged amino acids and/or neutral-charged amino acids to achieve a negative charge (e.g., a greater number of negatively-charged amino acids in the portion as compared to a number of positively-charged amino acids and/or neutral charged amino acids).

[0354] In some cases, each monomer of a nanopore described herein (e.g., an engineered biological nanopore) may comprise a net charge in the first portion and/or third portion that may be more negative as compared to a net charge in the second portion. Each monomer of the nanopore may be modified in a manner described herein (e.g., the monomer may comprise a mutation described herein at a first portion, second portion, third portion, or any combination thereof). For example, a first portion and/or third portion may comprise at least one amino acid that is mutated. The amino acid may be mutated to exhibit an increased net negative charge. In some cases, at least about 1, 2, 3, 4, 5, or greater than about 5 amino acids may be mutated in first portion and/or third portion to exhibit an increased net negative charge.

[0355] In some cases, a mutated amino acid residue in a first portion or third portion of the monomer may be at most about 100 amino acids, 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 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, or 1 amino acid apart from a mutated amino acid in the second portion. In some cases, a mutated amino acid residue in a first portion or third portion of the monomer may be 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, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 100 amino acids, or greater than about 100 amino acids apart from a mutated amino acid in the second portion.

[0356] A monomer may comprise one or more amino acid mutations described herein. The amino acid mutations may modify a charge of one or more amino acid residues of the monomer. In some cases, a monomer may comprise one or more amino acid mutations. The amino acid mutations may modify a charge (e.g., increase a charge, e.g., increase negative charge) in the residues that contribute to a region of the engineered biological nanopore. The region may comprise a diameter (e.g., narrowest diameter) of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. The region may comprise a diameter (e.g., narrowest diameter) of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 run, at least about 5 run, at least about 6 run, at least about 7 run, at least about 8 run, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0357] The amino acid mutations may modify a charge (e.g., increase a charge, e.g., increase neutral charge) in the residues that contribute to a region of the engineered biological nanopore. The region may comprise a diameter (e.g., narrowest diameter) of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about

3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. The region may comprise a diameter (e.g., narrowest diameter) of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about

2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0358] A monomer may comprise one or more amino acid mutations described herein, such that the one or more amino acid mutations may be in a ring of charge. The ring of charge may be formed from at least one charge of one or more monomers. For example, a plurality of monomers may assemble in a nanopore described herein to form one or more rings of charge. The one or more rings of charge may be in a region (e.g., a first region, a second region, a third region, or any combination thereof). In some cases, one or more amino acid mutations may modify a charge (e.g., increase a charge, e.g., increase negative charge) in the residues that contribute to a ring of charge (e.g., a ring of charge in a region) of the engineered biological nanopore. For example, a ring of charge in a region (e.g., a first region, a second region, a third region, or any combination thereof) may comprise one or more modifications (e.g., one or more mutations to negatively-charged amino acids) in residues of a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer). The ring of charge (e.g., the ring of charge in a region comprising one or more amino acid mutations to introduce a negative charge) may comprise a diameter of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. The ring of charge (e.g., the ring of charge in a region comprising one or more amino acid mutations to introduce a negative charge) may comprise a diameter of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0359] As another example, a ring of charge in a region (e.g., a first region, a second region, a third region, or any combination thereof) may comprise one or more modifications (e.g., one or more mutations to neutral- charged amino acids) in residues of a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer). The ring of charge (e.g., the ring of charge in a region comprising one or more amino acid mutations to introduce a neutral charge) may comprise a diameter of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm. The ring of charge (e.g., the ring of charge in a region comprising one or more amino acid mutations to introduce a neutral charge) may comprise a diameter of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0360] A second portion of a monomer (e.g., constriction-forming portion) may comprise a negative charge. A second portion of a monomer (e.g., constriction-forming portion) may be modified to be more net negative as compared to a respective portion of a wild-type monomer. A second portion of a monomer (e.g., constriction-forming portion) may comprise a net negative charge. The net negative charge may result from a greater number of negatively charged amino acid residues compared to a number of positively charged amino acid residues, neutral amino acid residues, or combination thereof. A negative charge, a net negative charge or increasing a net negative charge of a second portion of a monomer described herein may result from mutation (e.g., insertion, deletion, and/or substitution) of positively-charged amino acid residues, negatively -charged amino acid residues, neutral amino acid residues, or any combination thereof. A negative charge, a net negative charge or increasing a net negative charge of a second portion of a monomer (e.g., constriction-forming portion) described herein may result from substitution of positively charged amino acid residues to neutral amino acid residues. A negative charge, a net negative charge or increasing a net negative charge of a second portion of a monomer (e.g., constriction-forming portion) described herein may result from substitution of positively charged amino acid residues to negatively -charged amino acid residues. For example, a negative charge, a net negative charge or increasing a net negative charge of a second portion of a monomer (e.g., constriction-forming portion) described herein may result from a portion comprising two negatively -charged amino acid residues and two positively-charged amino acid residues, and substituting one or more positively- charged residues for one or more neutral amino acid residues. For example, a negative charge, a net negative charge or increasing a net negative charge of a second portion of a monomer (e.g., constriction-forming portion) described herein may result from a portion comprising two negatively -charged amino acid residues and two positively-charged amino acid residues, and substituting one or more positively -charged residues for one or more negatively -charged amino acid residues.

[0361] A second portion of a monomer (e.g., constriction-forming portion) may comprise a neutral charge. A second portion of a monomer (e.g., constriction-forming portion) may be modified to be more net neutral as compared to a respective portion of a wild-type monomer. A second portion of a monomer may comprise a neutral charge. The neutral charge may result from a number of neutrally-charged amino acid residues in the second portion of the monomer. The neutral charge may result from an equal number of positively -charged amino acid residues and negatively-charged amino acid residues in the second portion of the monomer. A neutral charge, a net neutral charge, or increasing a net neutral charge of a second portion of a monomer described herein may result from substitution of positively-charged amino acid residues, negatively -charged amino acid residues, or any combination thereof, to neutral amino acid residues. A neutral charge, a net neutral charge or increasing a net neutral charge of a second portion of a monomer described herein may result from mutation (e.g., insertion, deletion, and/or substitution) of positively-charged amino acid residues, negatively- charged amino acid residues, neutral amino acid residues, or any combination thereof. For example, a neutral charge, a net neutral charge or increasing a net neutral charge of a second portion of a monomer described herein may result from a second portion comprising 3 positively-charged amino acid residues and 2 negatively- charged amino acid residues, and deleting one of the positively -charged amino acid residues. The deletion may create an equal number of positive and negative charges, and provide a net neutral charge in the second portion of the monomer.

[0362] In some cases, a second portion may comprise at least one amino acid that is mutated to exhibit a negative charge and/or net negative charge. In some cases, a second portion may comprise at least one amino acid that is mutated such that the second portion may be more net negative as compared to a respective portion of a wild-type nanopore. In some cases, a second portion may comprise a plurality of amino acids that are mutated to exhibit a negative charge and/or net negative charge. In some cases, a second portion may comprise at least one amino acid that is mutated to exhibit a neutral charge and/or net neutral charge. In some cases, a second portion may comprise at least one amino acid that is mutated such that the second portion may be more net neutral as compared to a respective portion of a wild-type nanopore. In some cases, a second portion may comprise a plurality of amino acids that are mutated to exhibit a neutral charge and/or net neutral charge. In some cases, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, or greater than about 30 amino acids in a second portion may be modified to one or more amino acids that increase a net neutral charge or net negative charge. In some cases, at most about 30, at most about 20, at most about 10, 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 amino acid in a second portion may be modified to one or more amino acids that increase a net neutral charge or net negative charge.

[0363] For example, a second portion of an engineered monomer described herein may have an amino acid at a distance from another amino acid of a third portion. A wild-type biological monomer may have an amino acid at an identical position (e.g., at a same distance from another amino acid of a third portion). Thus, the mutated amino acid in the second portion of the engineered monomer may be respective to the amino acid in the second portion of the wild-type monomer. The amino acid in the second portion may be mutated to increase a net neutral charge. Thus, the second portion of the engineered biological monomer comprises at least one amino acid that is mutated to exhibit an increased net neutral charge as compared to a respective portion of a wild-type biological monomer. As another example, a second portion may comprise at least one amino acid that is mutated to a negatively-charged amino acid to exhibit an increased net negative charge as compared to a respective portion of a wild-type biological monomer.

[0364] There may be a distance between an amino acid (e.g., a modified amino acid) in one portion of a monomer described herein (e.g., an engineered monomer) and another amino acid (e.g., another modified amino acid) in another portion of the monomer. In some cases, a mutated amino acid in a first portion may be separated by a distance from a mutated amino acid in a second portion. As an example, a mutated amino acid in a portion (e.g., a first portion and/or a third portion) may be separated by a distance from another mutated amino acid in another portion (e.g., the second portion). A mutated amino acid in a portion (e.g., a first portion and/or a third portion) may be separated from another mutated amino acid in a portion (e.g., the second portion) by a distance of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about

3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.2 nm, or less than about 0.2 nm. A mutated amino acid in a portion (e.g., a first portion and/or a third portion) may be separated from another mutated amino acid in a portion (e.g., the second portion) by a distance of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm. In some cases, two amino acids (e.g., two mutated amino acids) may be separated from one another in two portions of a monomer (e.g., two different portions).

[0365] Two amino acids (e.g., two mutated amino acids) may be separated from one another in a same portion of a monomer. As an example, an amino acid (e.g., a modified amino acid) in one portion of a monomer described herein (e.g., an engineered monomer) may be separated by a distance from another amino acid (another modified amino acid) in the same portion. A mutated amino acid in a portion (e.g., a first portion, a second portion, a third portion, a second protein unit, or any combination thereof) may be separated from another mutated amino acid in the same portion (e.g., a first portion, a second portion, a third portion, a second protein unit, or any combination thereof) by a distance of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about

4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, at most about 0.2 nm, or less than about 0.2 nm. A mutated amino acid in a portion (e.g., a first portion, a second portion, a third portion, a second protein unit, or any combination thereof) may be separated from another mutated amino acid in the same portion (e.g., a first portion, a second portion, a third portion, a second protein unit, or any combination thereof) by a distance of at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 run, at least about 5 run, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm.

[0366] A mutated amino acid in a first portion or third portion may be at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, or less than about 0.5 nm away from a mutated amino acid in a second portion. A mutated amino acid in a first portion or third portion may be at least about 0.5 nm, at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm away from a mutated amino acid in a second portion.

[0367] In some cases, a nanopore described herein may be an engineered biological nanopore. The nanopore may be a synthetic nanopore. The nanopore may comprise a proteinaceous nanopore. In some cases, the nanopore described herein may be derived or originate from a wild-type biological nanopore. In other cases, the nanopore described herein may not be derived or may not originate from a wild-type biological nanopore. In some cases, the nanopore described herein may be derived or originate from a wild-type biological nanopore. In some cases, the nanopore described herein may be a de novo nanopore or assembled from scratch using nanopore technology and without relying on a reference genome. The proteinaceous nanopore may be designed de novo with predictive protein engineering software. In some cases, the engineered biological nanopore described herein may be a de novo nanopore based on de novo alpha-helical transmembrane regions, beta-barrel transmembrane regions, or any combination thereof. The engineered biological nanopore may be synthesized to have a charge at a first region of the channel, a second region of the channel, a third region of the channel, or any combination thereof. In some cases, the engineered biological nanopore may comprise a first region and/or third region of the channel having a negative charge, a neutral charge, or any combination thereof. In some cases, the engineered biological nanopore may comprise a second region of the channel having a negative charge, a neutral charge, or any combination thereof. For example, the engineered biological nanopore may comprise a first region and/or third region of the channel having a negative charge and/or a second region of the channel having a neutral charge. For example, the engineered biological nanopore may comprise a first region and/or third region of the channel having a negative charge and/or a second region of the channel having a negative charge. For example, the engineered biological nanopore may comprise a first region and/or third region of the channel having a neutral charge and/or a second region of the channel having a neutral charge. For example, the engineered biological nanopore may comprise a first region and/or third region of the channel having a neutral charge and/or a second region of the channel having a negative charge. [0368] In some cases, a nanopore may comprise multiple parts (e.g., a nanopore may be complexed with two or more proteins). In some cases, a nanopore (e.g., an engineered biological nanopore) may be formed of different proteins. An engineered biological nanopore may be assembled from two or more (e.g., 2, 3, 4, 5, 6, or more) different proteins. In some cases, the two or more proteins can assemble to form the channel (e.g., a primary channel), a constriction region, or any combination thereof, of the engineered biological nanopore. In some cases, a first protein may form the channel region of the nanopore and/or a second protein may form the constriction region of the nanopore. The second protein may comprise an adapter that can bind to the inside of the channel. Binding of the adapter protein to the channel may form one or more constriction region. For example, a nanopore (e.g., an engineered biological nanopore) may be a CsgG/F nanopore comprising a CsgG protein and a CsgF protein (e.g., adapter). In some cases, the adapter comprises a proteinaceous adapter or a chemical adapter. In some cases, the proteinaceous adapter comprises a CsgF subunit, a CsgF subunit truncation, or a CsgF subunit homolog, paralog, ortholog, or any combination thereof. In some cases, the chemical adapter comprises cyclodextrin, cucurbituril, crown ethers, calixarenes, porphyrins, cyclosporines, cyclems, or cyclams. In some cases, the proteinaceous adapter can be a monomeric adapter. In some cases, the proteinaceous adapter can be an oligomeric adapter. In some cases, the adapter can be coupled to the channel of the nanopore. In some cases, the adapter can be coupled to the channel of the nanopore via a covalent bond. In some cases, the adapter is coupled to the channel of the nanopore via a non-covalent bond. In some cases, the adapter is coupled to the channel of the nanopore via a linker.

[0369] In some cases, a monomer may comprise a first protein unit. In some cases, a monomer may comprise a first protein unit and a second protein unit. The first protein unit may be a monomer of CsgG. The second protein unit may be a monomer of CsgF. The first protein unit (e.g., CsgG) and the second protein unit (e.g., CsgF) may be derived from different sequences. In some cases, an engineered biological nanopore described herein may comprise the first protein unit. In some embodiments, an engineered biological nanopore described herein may comprise the second protein unit. In some cases, an engineered biological nanopore described herein may comprise the first protein unit and the second protein unit. For example, an engineered biological nanopore described herein may comprise a CsgG/CsgF nanopore comprising the first protein unit (e.g., CsgG) and the second protein unit (e.g., CsgF). The first protein unit may comprise one or more monomers. The second protein unit may comprise one or more monomers. In some cases, a monomer of the first protein unit may comprise a first portion, second portion, third portion, or any combination thereof described herein. In some cases, a monomer of the second protein unit may comprise a first portion, second portion, third portion, or any combination thereof described herein. For example, a first protein unit and/or a second protein unit may comprise one or more modified monomers (e.g., a plurality of modified monomers). As another example, a first protein unit and/or a second protein unit may comprise one or more unmodified monomers (e.g., a plurality of unmodified monomers). The first protein unit and/or a second protein unit may comprise a heterogenous composition of monomers, for example where the first protein unit and/or a second protein unit comprise a combination of one or more modified monomers and one or more unmodified monomers.

[0370] In some cases, a first portion (e.g., of a first protein unit) may comprise at least one modification, a second portion (e.g., of a first protein unit) may comprise at least one modification, a third portion (e.g., of a first protein unit) may comprise at least one modification, or any combination thereof. In some cases, a first portion (e.g., of a second protein unit) may comprise at least one modification, a second portion (e.g., of a second protein unit) may comprise at least one modification, a third portion (e.g., of a second protein unit) may comprise at least one modification, or any combination thereof. In some cases, a first portion of a second protein unit may couple to a portion (e.g., a first portion, second portion, third portion, or combination thereof) of a first protein unit. In some cases, a first portion, second portion, third portion, or combination thereof, of a second protein unit may couple to a first protein unit.

[0371] A second portion of a monomer (e.g., an engineered monomer) can comprises one or more modifications to reduce the aromaticity in the second portion. For example, a second portion (e.g., a second portion of a first protein unit) may have a modified amino acid residue, wherein the modified amino acid comprises an aromatic amino acid substituted for a non-aromatic amino acid. As another example, a second portion (e.g., a second portion of a first protein unit) may have a modified amino acid residue, wherein the modified amino acid comprises a deleted aromatic amino acid. The modified second portion (e.g., second portion of the first protein unit) may have a reduced number of one or more phenylalanine (F) amino acids, tryptophan (W) amino acids, tyrosine (Y) amino acids, or combination thereof, as compared to an unmodified second portion of the first protein unit. As another example, the modified second portion (e.g., second portion of the first protein unit) may have a reduced number of one or more aromatic non-natural amino acids, as compared to an unmodified second portion of the first protein unit. In some cases, the second portion (e.g., of the first protein unit) may comprise one or more modifications to a neutral charge and/or one or more modifications to a negative charge. The second portion (e.g., of the first protein unit) may comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, or greater than about 15 modifications to a neutral charge and/or negative charge. The second portion (e.g., of the first protein unit) may comprise at most about 15, at most about 10, 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 modification to a neutral charge and/or negative charge.

[0372] The second portion (e.g., of the first protein unit) may comprise one or more amino acids that are mutated to a negative charge or neutral charge. Amino acids may be mutated by any combination of substituting, adding, or deleting one or more natural and/or non-natural amino acids in a second portion (e.g., second portion of the first protein unit). For example, one or more amino acids may be mutated to a negative charge by substituting one or more positively -charged amino acids and/or one or more neutral amino acids with one or more negatively-charged amino acids. The second portion (e.g., of the first protein unit) may comprise two or more amino acids that are mutated to a negative charge and/or neutral charge. For example, the second portion (e.g., of the first protein unit) may comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, or greater than about 15 amino acids that are mutated to a negative charge and/or neutral charge. As another example, the second portion (e.g., of the first protein unit) may comprise at most about 15, at most about 10, 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 amino acid that is mutated to a negative charge and/or neutral charge.

[0373] In some cases, the first portion and/or third portion (e.g., of the first protein unit) may comprise a modification to a negative charge and one or more other modifications. For example, the first portion and/or third portion (e.g., of the first protein unit) may comprise a modification to a negative charge and a neutral charge. As another example, the first portion and/or third portion (e.g., of the first protein unit) may comprise one or more amino acids modified to a negative charge. In some cases, the first portion and/or third portion (e.g., of the first protein unit) may comprise two or more amino acids (e.g., 2, 3, 4, 5, 10, 15, or greater than about 15 amino acids) modified to a negative charge and/or a neutral charge. For example, the first portion and/or third portion (e.g., of the first protein unit) may comprise three amino acids all mutated to lysine. As another example, the first portion and/or third portion (e.g., of the first protein unit) may comprise three amino acids with two amino acids mutated to lysine (K) and one amino acid mutated to arginine (R). As a third example, the first portion and/or third portion (e.g., of the first protein unit) may comprise three amino acids with one amino acid mutated to lysine (K), one amino acid mutated to arginine (R), and one amino acid mutated to asparagine (N).

[0374] In some cases, the second portion (e.g., of the first protein unit) may comprise a modification to a negative charge and one or more other modifications. For example, the second portion (e.g., of the first protein unit) may comprise a modification to a negative charge and a neutral charge. As another example, the second portion (e.g., of the first protein unit) may comprise one or more amino acids modified to a negative charge. In some cases, the second portion (e.g., of the first protein unit) may comprise two or more amino acids (e.g., 2, 3, 4, 5, 10, 15, or greater than about 15 amino acids) modified to a negative charge and/or a neutral charge. For example, the second portion (e.g., of the first protein unit) may comprise three amino acids all mutated to lysine. As another example, the second portion (e.g., of the first protein unit) may comprise three amino acids with two amino acids mutated to lysine (K) and one amino acid mutated to arginine (R). As a third example, the second portion (e.g., of the first protein unit) may comprise three amino acids with one amino acid mutated to lysine (K), one amino acid mutated to arginine (R), and one amino acid mutated to asparagine (N).

[0375] One or more monomers comprises one or more second protein units. The one or more second protein units may comprise one or more modifications. For example, a first portion and/or third portion of the second protein unit may comprise one or more mutated amino acid residues. For example, a first portion and/or third portion of the second protein unit may comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, or greater than about 15 mutated amino acids. As another example, a first portion and/or third portion of the second protein unit may comprise at most about 15, at most about 10, 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 mutated amino acid. The first portion and/or third portion of the second protein unit can comprise one or more amino acid modifications to a negative charge and/or a neutral charge. In some cases, the first portion and/or third portion of the second protein unit can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, or greater than about 15 amino acids that are mutated to a negative charge and/or a neutral charge. In some cases, the first portion and/or third portion of the second protein unit can comprise at most about 15, at most about 10, 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 amino acids that are mutated to a negative charge and/or a neutral charge.

[0376] As another example, a second portion of the second protein unit may comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, or greater than about 15 mutated amino acids. A second portion of the second protein unit may comprise at most about 15, at most about 10, 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 mutated amino acid. The second portion of the second protein unit can comprise one or more amino acid modifications to a negative charge and/or a neutral charge. In some cases, the second portion of the second protein unit can comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, or greater than about 15 amino acids that are mutated to a negative charge and/or a neutral charge. In some cases, the second portion of the second protein unit can comprise at most about 15, at most about 10, 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 amino acids that are mutated to a negative charge and/or a neutral charge.

[0377] The second protein unit may comprise one or more amino acid mutations in a second portion. The second portion may comprise a constriction-forming portion. In some cases, the constriction-forming portion can comprise a narrowest lumen-facing region. The second protein unit can comprise a second portion (e.g., constriction-forming portion) that may be narrower than an adjacent region. A constriction region formed by a plurality of second portions (e.g., constriction-forming portions) may comprise a C(alpha)-C(alpha) diameter of 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, or less than about 0.5 nm. In some cases, the second protein unit may comprise at least about 1, 2, 3, 4, 5, or greater than about 5 mutated amino acids in a narrowest region (e.g., narrowest lumen-facing region). In some cases, the second portion (e.g., constriction-forming portion) of the second protein unit may comprise 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 mutated amino acid. The second portion (e.g., constriction-forming portion) of the second protein unit may comprise one or more amino acid mutations that increase a charge (e.g., increase a negative charge). The amino acid mutations may be in residues that contribute to a narrowest C(alpha)- C(alpha) diameter region of the engineered biological nanopore. For example, the narrowest C(alpha)- C(alpha) diameter region of the engineered biological nanopore may be 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, or less than about 0.5 nm. The amino acid mutations (e.g., substitutions, deletions, or insertions) in the second portion (e.g., constriction-forming portion) of the second protein unit may increase a negative charge in the second region of the second protein unit.

[0378] In some cases, the engineered biological nanopore may comprise one or more monomers with at least one different charge from one or more monomers of a wild-type nanopore. In some cases, the engineered biological nanopore may comprise one or more monomers with at least one different amino acid residue from one or more monomers of a wild-type nanopore. For example, the engineered biological nanopore may comprise at least one different amino acid residue in a first region and/or third region compared to a first region and/or third region of a wild-type nanopore. For example, the engineered biological nanopore may comprise at least one different amino acid residue in a second region compared to a second region of a wild-type nanopore. In some cases, an engineered biological nanopore described herein may comprise two or more different amino acids in a first region, second region, third region, or any combination thereof, compared to that of a first region, second region, third region, or any combination thereof of a wild-type nanopore.

[0379] In some cases, a first ring of charge (e.g., a first ring of charge of a first protein unit or second protein unit) may comprise one or more mutations described herein. The first ring of charge may be separated from a second ring of charge (e.g., a second ring of charge of a first protein unit or second protein unit) comprising one or more mutations described herein. The first ring of charge and the second ring of charge may be in a same protein unit (e.g., a first protein unit or second protein unit). The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by 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 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, or greater than about 10 nm apart from each other along the longitudinal length of the channel. The first ring of charge (e.g., first ring of charge comprising one or more mutations described herein) and the second ring of charge (e.g., second ring of charge comprising one or more mutations described herein) may be separated by at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4 nm, at most about 3 run, at most about 2 run, at most about 1 run, 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 apart from each other along the longitudinal length of the channel.

[0380] A monomer of an engineered biological nanopore described herein may comprise a number of amino acid residues within a second region of the channel (e.g., comprising the constriction region). In some cases, a monomer may comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 7, at least about 10, or greater than about 10 amino acid residues within a second region of the channel (e.g., comprising the constriction region) of the engineered biological nanopore. In some cases, a monomer may comprise at most about 10, at most about 7, 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 amino acid residue(s) within a second region of the channel (e.g., comprising the constriction region) of the engineered biological nanopore. For example, one or more of the amino acid residues of the monomer within the second region of the channel may be negatively- charged, neutrally-charged, or any combination thereof. A monomer of an engineered biological nanopore described herein may comprise a number of amino acid residues within a first region and/or third region of the channel (e.g., a region adjacent to the constriction region). In some cases, a monomer may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or greater than about 15 amino acid residues within a first region and/or third region of the channel (e.g., a region adjacent to the constriction region) of the engineered biological nanopore. In some cases, a monomer may comprise at most about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue(s) within a first region and/or third region of the channel (e.g., a region adjacent to the constriction region) of the engineered biological nanopore. For example, one or more of the amino acid residues of the monomer within the first region and/or third region of the channel may be negatively-charged, neutrally -charged, or any combination thereof.

[0381] The capture of an analyte may be achieved by generation of a force opposing the movement of the analyte outside of the nanopore. This force may be an electro-osmotic flow (EOF). The terms “electroosmotic flow” and “electro-osmotic force” may be used interchangeably herein. The term “electro-osmotic flow” can refer to the net flux of water generated by the movement of ions through the nanopore under an external applied potential. EOF may be modulated by changing the properties of the solution of the nanopore system (e.g., pH, ionic strength, or any combination thereof) and/or by altering a charge of the nanopore by means of mutagenesis. The size of the nanopore, shape of the nanopore, or any combination thereof may also influence EOF directionality and magnitude in the systems and/or methods described herein.

[0382] In some cases, an electro-osmotic flow can act across the membrane in a first side (e.g., cis side) to a second side (e.g., trans side) direction. In some cases, an electro-osmotic flow can act across the membrane in a second side (e.g., trans side) to a first side (e.g., cis side) direction. An electro-osmotic flow can be the flow that results from a net flow of a mobile layer of ions along a surface as induced by an applied potential (e.g., applied voltage potential). For example, a charged surface may form a static layer of oppositely charged mobile ions. Under an applied potential the charged mobile ions may be induced to move in the direction of higher potential if negative, or in the direction of lower potential if positive. The flow of charged ions can create a drag on the surrounding solvent (e.g., water) molecules, which in turn can result in a net flow that exerts a force acting on the surrounding molecules, both charged and neutral. For example, in a negatively charged nanopore channel, an electro-osmotic flow can result from a net flow of positive ions in a cis to trans direction (e.g., due to a lower potential on the trans side) causing the surrounding water to flow cis to trans and exert a force on surrounding molecules. The amount of ion flow and the corresponding magnitude of the electro-osmotic flow can be influenced by parameters such as an ion concentration difference across the membrane, a difference in potential, a net charge of a nanopore channel, a geometry of a nanopore channel, or any combinations thereof.

[0383] An "applied potential" can refer to an electrical potential (e.g., voltage) that is introduced to a system. The terms "applied potential" and "applied voltage" may be used interchangeably herein. The applied potential may be a force driving charge (e.g., ions) to move through the system. For example, the nanopore system described herein may have a solution (e.g., an electrolyte solution) and the membrane may be immersed in the solution. The solution can contain a concentration of one or more ions that conduct electricity. A potential (e.g., voltage difference) can be applied across the membrane, creating an electric field that drives ionic current through a nanopore. In the nanopore system, a positive potential may be applied to a first side (e.g., a cis side) or a second side (e.g., a trans side). In some cases, a negative potential may be applied to a first side (e.g., a cis side) or a second side (e.g., a trans side). An applied voltage may refer to a first applied voltage and/or a second applied voltage. As an example, 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. In some cases, an applied voltage may 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, 1000 mV, or greater than about 1000 mV in magnitude.

[0384] A potential difference (e.g., voltage difference) can be established between a first side and second side of the nanopore system. For example, a positive electrical voltage may be introduced on one side of the system (e.g., a trans side), relative to another side of the system (e.g., a cis side), where the voltage may be negative. The electrical field may then drive charged molecules (e.g., negatively-charged molecules) through a nanopore from one side of the system to the other. In some cases, a potential difference of the nanopore system may be less than about -10 mV or greater than about +10 mV. For example, a potential difference of the system may be less than about -300 mV, about -300 mV, about -200 mV, about -180 mV, about -160 mV, about -140 mV, about -120 mV, about -100 mV, about -80 mV, about -60 mV, about -40 mV, about -20 mV, about -10 mV, about 0 mV, about +10 mV, about +20 mV, about +40 mV, about +60 mV, about +80 mV, about +100 mV, about +120 mV, about +140 mV, about +160 mV, about +180 mV, about +200 mV, about +300 mV, or greater than about +300 mV.

[0385] The net charge of the channel, 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, 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 cases, 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. For example, changing a channel to have a more negative net charge can reduce a flow of a negatively charged molecule (e.g., a chloride ion). In some cases, 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 cases, changing a channel to have a more negative net charge and a 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. As another example, changing a first portion and/or third portion of one or more monomers (e.g., contributing to a first region of a channel and/or third region of a channel of an engineered biological nanopore) to have a more negative net charge can reduce a flow of a negatively charged molecule (e.g., a chloride ion). In some cases, changing a first portion and/or third portion of one or more monomers (e.g., contributing to a first region and/or third region of an engineered biological nanopore) to have a wider geometry can increase a flow of a larger molecule (e.g., a glucose molecule or a peptide analyte). In some cases, changing a first portion and/or third portion of one or more monomers (e.g., contributing to a first region of a channel and/or third region of a channel of an engineered biological nanopore) to have a more negative net charge and a 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. In some cases, a shift in the net charge can make some charged molecules translocate more easily through the pore. In some cases, a shift in the net charge can make some charged molecules translocate with more difficulty through the pore.

[0386] One or more modifications may be introduced to modify a charge of a first region, second region, third region, or any combination thereof. In some cases, a region (e.g., a first region or a third region) may be modified to be more net negative as compared to a respective region of a wild-type nanopore. A respective region can be an identical region of a wild-type nanopore as compared to the region of a nanopore described herein (e.g., an engineered biological nanopore). A region (e.g., a first region or a third region) may have an amino acid composition comprising a plurality of amino acids. The plurality of amino acids may comprise one or more negatively -charged amino acids, one or more neutral amino acids, one or more positively -charged amino acids, or any combination thereof. A region (e.g., a first region or a third region) may be modified by introducing one or more amino acid mutations to the region (e.g., a first region or a third region). In some cases, a region (e.g., a first region or a third region) may be modified by introducing one or more amino acid mutations to a monomer comprising a portion that corresponds that to that region. As an example, a region (e.g., a first region or a third region) may be modified to be more net negative as compared to a respective region of a wild-type nanopore by substituting one or more positively-charged amino acids and/or one or more neutral charged amino acids with one or more negatively-charged amino acids. As another example, a region (e.g., a first region or a third region) may be modified to be more net negative as compared to a respective region of a wild-type nanopore by deleting one or more positively-charged amino acids and/or one or more neutral charged amino acids. As another example, a region (e.g., a first region or a third region) may be modified to be more net negative as compared to a respective region of a wild-type nanopore by adding one or more negatively-charged amino acids to the region. As another example, if a region (e.g., a first region or a third region) has a net negative charge, the region may be modified to be more net negative as compared to a respective region of a wild-type nanopore by substituting one or more positively-charged amino acids with one or more neutral charged amino acids and/or one or more negatively-charged amino acids. As another example, if the region (e.g., a first region or a third region) has a greater number of negatively-charged amino acid residues compared to a number of negatively-charged amino acids and one or more neutral charged amino acids, the region may still be modified to be more net negative as compared to a respective region of a wildtype nanopore by (i) substituting one or more positively-charged amino acids and/or one or more neutral charged amino acids with one or more negatively-charged amino acids; (ii) deleting one or more positively- charged amino acids and/or one or more neutral charged amino acids; (iii) adding one or more negatively- charged amino acids; (iv) or any combination thereof. One or more natural amino acids and/or non-natural amino acids may be introduced to modify the region to be more net negative as compared to a respective region of a wild-type nanopore. One or more natural amino acids and/or non-natural amino acids may be deleted to modify the region to be more net negative as compared to a respective region of a wild-type nanopore. [0387] For example, introducing one or more negatively-charged amino acids (e.g., lumen-facing amino acid residues) outside of a second region (e.g., constriction region) may alter the selectivity of the nanopore. Introducing one or more negatively -charged amino acids (e.g., lumen-facing amino acid residues) outside of a second region (e.g., constriction region) in a first region (e.g., funnel region) may alter the selectivity of the nanopore. Introducing one or more negatively-charged amino acids (e.g., lumen-facing amino acid residues) outside of a second region (e.g., constriction region) in a first region (e.g., funnel region) having a diameter of at most about 3.5 nm (Ca-Ca), may alter the selectivity of the nanopore. The first region may have a diameter as described herein. The first region may have a diameter (e.g., a diameter expressed as the Ca-Ca distance) of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or less than about 1 nm.

[0388] In some cases, the methods, system, nanopores, or any combination thereof, may be used to increase a net negative charge. The methods, system, nanopores, or any combination thereof, may be used to increase a net negative charge at the second region (e.g., constriction region) and/or in a first region. The methods, system, nanopores, or any combination thereof, may be used to increase a net negative charge at the second region (e.g., constriction region), alternatively in combination with a first region and/or a third region. The first region and/or third region may have a diameter described herein. For example, the first region and/or third region may have a diameter (e.g., a diameter expressed as the Ca-Ca distance) of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or less than about 1 nm. In some cases, one or more amino acids (e.g., lumen-facing amino acid residues) in a second region (e.g., constriction region) and/or in a first region and/or third region may have a negative charge. The one or more amino acids may comprise any of the negatively-charged amino acids described herein. The first region and/or third region may have a diameter (e.g., a diameter expressed as the Ca-Ca distance) of at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or less than about 1 nm.

[0389] In some cases, one or more amino acids (e.g., lumen-facing amino acid residues) in a second region of the nanopore (e.g., a constriction region) may have one or more negative charges. The one or more amino acids may be positioned in the second region, having a diameter (e.g., a diameter expressed as the Ca-Ca distance) of at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, at most about 0.5 nm, at most about 0. 1 nm, or less than about 0. 1 nm. [0390] In some cases, an electro-osmotic flow can be the flow that results from one or more constriction regions present in a nanopore channel. For example, constriction regions in a nanopore can affect the flow of some ions (e.g. larger hydrated ions) more than other ions (e.g. smaller hydrated ions). In some cases, an electro-osmotic flow can be the flow that results from a net flow of mobile ions along a surface as induced by an applied potential and one or more constriction regions present in a nanopore channel.

[0391] In some cases, an electro-osmotic flow can be created or modified by a difference between a solution on a first side (e.g., cis side) of a membrane and a solution on a second side (e.g., trans side) of a membrane. In some cases, the solution on the first side (e.g., cis side) of the membrane can be a first solution. In some cases, the solution on the second side (e.g., trans side) of the membrane can be a second solution. The difference can be a difference in concentration of a molecule, including an ion, an electrolyte, an osmolyte, or any combination thereof.

[0392] Without wishing to be bound by theory, an EOF may be tuned by altering the narrowest site of the lumen. For example, when mutating a protein nanopore, an EOF can be tuned by modifying the narrowest site of the lumen (e.g., introducing one or more amino acid mutations). A second region (e.g., constriction region) of a nanopore may contribute to its electrical resistance. By contributing (e.g., modulating) an electrical resistance of the nanopore, the second region (e.g., constriction region) may influence a nanopore’s ability to characterize one or more molecules. Thus, modifying a second region (e.g., constriction region) of a nanopore to modulate an EOF may affect a nanopore’s ability to characterize one or more molecules. One or more modifications to nanopores and/or monomers, as described herein, may enhance an EOF and/or improve single molecule analysis with the nanopore (e.g., engineered biological nanopore). As an example, nanopores comprising a geometry (e.g., an hourglass geometry or a conical geometry), it may be advantageous to enhance an EOF by modifying a second region (e.g., constriction region). Herein, it was found that ion selectivity (e.g., cation selectivity) of a nanopore (e.g., engineered biological nanopore) may be increased. The ion selectivity (e.g., cation selectivity) may be increased by one or more modifications to a second region (e.g., constriction region) and/or one or more modifications to a first region and/or third region. Without wishing to be bound by theory, introducing one or more negative charges to a first region adjacent to a second region (e.g., a constriction region) led to an engineered biological nanopore with increased ion selectivity (e.g., cation selectivity) and an enhanced EOF. One or more negative charges (e.g., negatively-charged amino acids) in one or more positions away from a second region (e.g., a constriction region, e.g., a narrowest region) of a nanopore may be beneficial as it may allow a constriction region (e.g., a neutral constriction region) available for characterizing an analyte.

[0393] In some cases, the engineered biological nanopore may generate an EOF that is greater than an EOF of a wild-type biological nanopore. The EOF of the engineered biological nanopore may be greater than an EOF of the wild-type biological nanopore due to a first region of a channel comprising a different charge as compared to that of the wild-type biological nanopore, and/or, a second region of the channel comprising a different charge as compared to that of the wild-type biological nanopore, a third region of a channel comprising a different charge as compared to that of the wild-type biological nanopore, or any combination thereof.

[0394] In some cases, the engineered biological nanopore may comprise a first region and/or third region with an increase in net negative charge compared to a first region and/or third region of a wild-type biological nanopore. In some cases, the engineered biological nanopore may comprise a first region and/or third region with a greater number of negatively charged amino acid residues than that of a first region and/or third region of a wild-type biological nanopore. In some cases, the engineered biological nanopore may comprise a first region and/or third region with a greater number of neutral charged amino acid residues than a first region and/or third region of a wild-type biological nanopore. In some cases, a net charge of a first and/or third region may be at least about 50% more negative as compared to a respective region of the wildtype biological nanopore. For example, a net charge of the first and/or third region may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or greater than about 95% more negative as compared to a respective region of the wild-type biological nanopore. As another example, a net charge of the first and/or third region may be at most about 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than about 20% more negative as compared to a respective region of the wild-type biological nanopore (e.g., another region adjacent to the constriction region of the wild-type biological nanopore). In some cases, the engineered biological nanopore described herein may comprise a first region and/or third region of the channel comprising a negative charge. In some cases, the engineered biological nanopore may comprise a second region with an increase in net neutral charge compared to a second region of a wild-type biological nanopore. In some cases, the engineered biological nanopore may comprise a second region with a greater number of negatively charged amino acid residues than that of a second region of a wild-type biological nanopore. In some cases, the engineered biological nanopore may comprise a second region with a greater number of neutral charged amino acid residues than a second region of a wild-type biological nanopore. In some cases, the engineered biological nanopore described herein may comprise a second region of the channel comprising a neutral charge. For example, a net charge of a second region may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or greater than about 95% more neutral as compared to a respective region of a wild-type biological nanopore. As another example, a net charge of the second region may be at most about 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than about 20% more neutral as compared to a respective region of the wild-type biological nanopore (e.g., a constriction region of the wild-type biological nanopore). As another example, a net charge of a second region may be at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or greater than about 95% more negative as compared to a respective region of a wild-type biological nanopore. As another example, a net charge of the second region may be at most about 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than about 20% more negative as compared to a respective region of the wild-type biological nanopore (e.g., a constriction region of the wild-type biological nanopore). In some cases, the negative charge of the first region and/or third region of the channel and the neutral charge of the second region of the channel may generate the EOF. The increase in negative charge of the first region and/or third region of the channel (e.g., adjacent to the constriction region) and/or an increase in negative charge of the second region may generate the EOF.

[0395] In some cases, a first region and/or third region may be modified. The first region and/or third region may be modified to be more net negative than a respective region of a wild-type nanopore (e.g., a wild-type biological nanopore). In some cases, the second region of the engineered biological nanopore may be modified. The second region may be modified to be more net neutral or more net negative than a respective region of a wild-type nanopore (e.g., a wild-type biological nanopore). For example, a pore (e.g., an engineered biological nanopore) described herein may comprise a first region and/or third region modified be more net negative and a second region modified to be more net neutral or more net negative than a respective region of a wild-type nanopore (e.g., a wild-type biological nanopore). A region (e.g., a second region) may have an amino acid composition comprising a plurality of amino acids. The plurality of amino acids may comprise one or more positively-charged amino acids, one or more neutral amino acids, one or more negatively-charged amino acids, or any combination thereof. A region (e.g., a second region) may be modified by introducing one or more amino acid mutations to the region (e.g., a second region). As an example, a region (e.g., a second region) may be modified to be more net negative as compared to a respective region of a wild-type nanopore by substituting one or more positively -charged amino acids and/or one or more neutral charged amino acids with one or more negatively-charged amino acids. As an example, a region (e.g., a second region) may be modified to be more net neutral as compared to a respective region of a wild-type nanopore by substituting one or more positively -charged amino acids and/or one or more negatively-charged amino acids with one or more neutral-charged amino acids. As another example, a region (e.g., a second region) may be modified to be more net negative as compared to a respective region of a wildtype nanopore by deleting one or more positively-charged amino acids and/or one or more neutral charged amino acids. As another example, a region (e.g., a second region) may be modified to be more net neutral as compared to a respective region of a wild-type nanopore by deleting a same number of positively-charged amino acids and negatively -charged amino acids. For example, if the region (e.g., a second region) has 10 amino acids with 4 positively -charged amino acids and 6 negatively -charged amino acids, the region may be modified to be more neutral by (i) deleting 2 negatively charged amino acids; (ii) substituting one or more positively-charged amino acids and/or one or more negatively -charged amino acids with one or more neutral- charged amino acids; (iii) adding 2 negatively -charged amino acids; or (iv) any combination thereof). One or more natural amino acids and/or non-natural amino acids may be introduced to modify the region to be more net negative as compared to a respective region of a wild-type nanopore. One or more natural amino acids and/or non-natural amino acids may be deleted to modify the region to be more net negative as compared to a respective region of a wild-type nanopore. The first region and/or third region modified to be more net negative and/or the second region modified to be more net neutral or more net negative may generate an EOF.

[0396] In some cases, a monomer of the nanopore can comprise a first portion, a second portion, a third portion, or any combinations thereof. A first portion of the monomer may correspond to a first region of the nanopore. A second portion of the monomer may correspond to a second region of the nanopore. A third portion of the monomer may correspond to a third region of the nanopore. A monomer described herein may be a modified monomer (e.g., an engineered monomer). The modified monomer can comprise one or more modifications (e.g., a plurality of modifications). The modifications can comprise one or more amino acid mutations. An unmodified monomer may be a wild-type monomer. An unmodified monomer (e.g., wild-type monomer) may comprise no modifications. In some cases, the engineered monomer may comprise a first portion and/or third portion with an increase in net negative charge compared to a first portion and/or third portion of a wild-type biological monomer. In some cases, the engineered monomer may comprise a first portion and/or third portion with a greater number of negatively charged amino acid residues than that of a first portion and/or third portion of a wild-type monomer. In some cases, the engineered monomer may comprise a first portion and/or third portion with a greater number of neutral charged amino acid residues than a first portion and/or third portion of a wild-type monomer. In some cases, the engineered monomer described herein may comprise a first portion and/or third portion of the channel comprising a negative charge. In some cases, the engineered monomer may comprise a second portion with an increase in net neutral charge compared to a second portion of a wild-type monomer. In some cases, the engineered monomer may comprise a second portion with a greater number of negatively charged amino acid residues than that of a second portion of a wild-type monomer. In some cases, the engineered monomer may comprise a second portion with a greater number of neutral charged amino acid residues than a second portion of a wild-type monomer. In some cases, the engineered monomer described herein may comprise a second portion of the channel comprising a neutral charge. In some cases, the negative charge of the first portion and/or third portion of the channel and the neutral charge of the second portion of the channel may generate the EOF.

[0397] The neutral charge of (i) the second region of the channel or (ii) the second portion (e.g., constriction-forming portion) of the engineered monomer may comprise a range of neutrality. For example, a second portion of a monomer may comprise two negatively-charged amino acid residues and mutating one amino acid residue to a neutral amino acid residue can result in a 50% increase in neutrality (e.g., neutral charge). In some cases, there may be a 100% increase in neutral charge if all non-neutral amino acid residues are mutated to neutral amino acid residues. The negative charge of (i) the second region of the channel or (ii) the second portion (e.g., constriction-forming portion) of the engineered monomer may comprise a range of negativity. For example, a second portion of a monomer may comprise two positively-charged amino acid residues and mutating one amino acid residue to a negatively-charged amino acid residue can result in a 50% increase in negativity (e.g., negative charge). In some cases, there may be a 100% increase in negative charge if all non-negative amino acid residues are mutated to negative amino acid residues. A similar range of negativity may be achieved in the first region and/or third region of the channel or (ii) the first portion and/or third portion of the engineered monomer.

[0398] In some cases, the negatively charged first region and/or third region of the channel adjacent to the neutral charged or negatively -charged second region (e.g., comprising the constriction region) of the nanopore can generate an EOF greater than an EOF generated by a wild-type pore (e.g., a pore that may not comprise the negatively charged first region of the channel adjacent to the neutral charged constriction region). An EOF may be generated between a difference of net ionic current flow between cations and anions. A cation can comprise a positively-charged ion, for example potassium (K⁺). An anion can comprise a negatively -charged ion, for example chlorine (C1‘).

[0399] In some cases, an EOF of the nanopore described herein comprising a negatively charged first region and/or third region of the channel adjacent to an increased neutrally -charged or negatively -charged second region (e.g., constriction region) 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 or a nanopore that does not comprise a negatively charged first region and/or third region of the channel adjacent to the increased neutrally -charged second portion (e.g., constriction region). In some cases, an EOF of the nanopore described herein comprising a negatively charged first region and/or third region of the channel adjacent to an increased neutrally -charged second region (e.g., constriction region) 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 or a nanopore that does not comprise a negatively charged first region and/or third region of the channel adjacent to the increased neutrally -charged second portion (e.g., constriction region).

[0400] In some cases, an EOF of the nanopore described herein comprising a negatively charged first region and/or third region of the channel adjacent to an increased neutrally -charged second region (e.g., constriction region) may comprise an EOF from about 1. 1 -fold to about 10-fold greater than an EOF generated by a wildtype nanopore or a nanopore that does not comprise a negatively charged first region and/or third region of the channel adjacent to the increased neutrally-charged second portion (e.g., constriction region). In some cases, an EOF of the nanopore described herein comprising a negatively charged first region and/or third region of the channel adjacent to an increased neutrally -charged second region (e.g., constriction region) may comprise an EOF from about 1.1-fold to about 1.2-fold, about 1.1-fold to about 1.3-fold, about 1.1-fold to about 1.4- fold, about 1. 1 -fold to about 1.5 -fold, about 1. 1 -fold to about 2-fold, about 1. 1 -fold to about 3 -fold, about 1.1- fold to about 4-fold, about 1.1-fold to about 5-fold, about 1.1-fold to about 8-fold, about 1.1-fold to about 10- fold, about 1.2-fold to about 1.3-fold, about 1.2-fold to about 1.4-fold, about 1.2-fold to about 1.5-fold, about

1.2-fold to about 2-fold, about 1.2-fold to about 3-fold, about 1.2-fold to about 4-fold, about 1.2-fold to about 5-fold, about 1.2-fold to about 8-fold, about 1.2-fold to about 10-fold, about 1.3-fold to about 1.4-fold, about

1.3 -fold to about 1.5 -fold, about 1.3 -fold to about 2-fold, about 1.3 -fold to about 3 -fold, about 1.3 -fold to about 4-fold, about 1.3-fold to about 5-fold, about 1.3-fold to about 8-fold, about 1.3-fold to about 10-fold, about

1.4-fold to about 1.5-fold, about 1.4-fold to about 2-fold, about 1.4-fold to about 3-fold, about 1.4-fold to about

4-fold, about 1.4-fold to about 5 -fold, about 1.4-fold to about 8-fold, about 1.4-fold to about 10-fold, about

1.5-fold to about 2-fold, about 1.5-fold to about 3-fold, about 1.5-fold to about 4-fold, about 1.5-fold to about

5-fold, about 1.5-fold to about 8-fold, about 1.5-fold to about 10-fold, about 2-fold to about 3-fold, about 2- fold to about 4-fold, about 2-fold to about 5-fold, about 2-fold to about 8-fold, about 2-fold to about 10-fold, about 3-fold to about 4-fold, about 3-fold to about 5-fold, about 3-fold to about 8-fold, about 3-fold to about 10-fold, about 4-fold to about 5 -fold, about 4-fold to about 8-fold, about 4-fold to about 10-fold, about 5 -fold to about 8-fold, about 5-fold to about 10-fold, or about 8-fold to about 10-fold greater than an EOF generated by a wild-type nanopore or a nanopore that does not comprise a negatively charged first region and/or third region of the channel adjacent to the increased neutrally -charged second portion (e.g., constriction region).

[0401] The second region of the channel (e.g., comprising the constriction region) may comprise a first entrance, a second entrance, or any combination thereof. In some cases, the negative charge of the first region and/or third region may be adjacent to a first entrance of the second region of the channel. The negative charge may be a negatively charged amino acid residue of the engineered biological nanopore (e.g., of a monomer of the engineered biological nanopore). The negative charge may be a ring of charge of the engineered biological nanopore. The negative charge of the first region and/or third region of the channel may be adjacent to the first entrance of the second region and/or the second entrance to the second region. For example, the negative charge may be on both sides of the second region of the channel. In some cases, the negative charge of the first region and/or third region of the channel may be at least about 0.001 nm, at least about 0.01 run, at least about 0.05 run, at least about 0. 1 nm, at least about 0.5 nm, at least about 1.0 nm, at least about 2.0 nm, at least about 3.0 nm, at least about 4.0 nm, at least about 5.0 nm, at least about 10 nm, or greater than about 10 nm from the first entrance and/or second entrance of the second region of the channel. In some cases, the negative charge of the first region and/or third region of the channel may be at most about 10 nm, at most about 5.0 nm, at most about 4.0 nm, at most about 3.0 nm, at most about 2.0 nm, at most about 1.0 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 from the first entrance and/or second entrance of the second region of the channel. In some cases, the negative charge of the first region and/or third region of the channel may be about 0.001 nm, 0.01 nm, 0.05 nm, 0. 1 nm, 0.5 nm, 1.0 nm, 2.0 nm, 3.0 nm, 4.0 nm, 5.0 nm, or 10 nm from the first entrance and/or second entrance of the second region of the channel.

[0402] An engineered biological nanopore described herein may comprise a second region of the channel with a greater neutrality (e.g., neutral charge) compared to a constriction region of a wild-type biological nanopore. An engineered biological nanopore may obtain a greater neutral charge in multiple methods. For example, a negatively-charged second region of a channel may be engineered to substitute neutral amino acid residues in place of negatively-charged amino acid residues, add (e.g., insert) positively-charged amino acid residues, delete negatively-charged amino acids, or any combination thereof to obtain a greater neutrally charged region. For example, a positively -charged second region of a channel may be engineered to substitute neutral amino acid residues in place of negatively -charged amino acid residues, add (e.g., insert) negatively-charged amino acid residues, delete positively-charged amino acids, or any combination thereof to obtain a greater neutrally charged region.

[0403] As another example, an amino acid residue of the second region of the channel, the first region of the channel, the third region of the channel, or any combination thereof, may be substituted for a non-natural amino acid. A “non-natural amino acid” can refer to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term “non- natural amino acid” can be “non-naturally encoded amino acid”, “unnatural amino acid”, “non-naturally- occurring amino acid”, and variously hyphenated and non-hyphenated versions thereof. The term “non- natural amino acid” may include, but is not limited to, amino acids which occur naturally by modification of a naturally encoded amino acid (e.g., the 20 common amino acids or pyrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of naturally -occurring amino acids that are not naturally-encoded include, but are not limited to, N- acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O- phosphotyrosine. Additionally, the term “non-natural amino acid” may comprise amino acids which do not occur naturally and may be obtained synthetically or may be obtained by modification of non-natural amino acids.

[0404] An engineered biological nanopore described herein may comprise a second region of the channel with a greater negative charge (e.g., an increased negative charge) compared to a constriction region of a wild-type biological nanopore. An engineered biological nanopore may obtain a greater negative charge in multiple methods. For example, a negatively-charged second region of a channel may be engineered by substituting positively-charged amino acid residues in place of negatively-charged amino acid residues, adding (e.g., inserting) negatively -charged amino acid residues and/or neutral charged residues, deleting positively-charged amino acids, or any combination thereof to achieve a negative charge, a more negative region as compared to a respective region of a wild-type pore, or a net negative charge. Increasing a net negative charge may comprise substituting one or more positively -charged amino acids, non-polar amino acids or aromatic amino acids at a second region (e.g., constriction region) and/or at a first region and/or third region (e.g., an adjacent region to the constriction region, e.g., one or more funnel regions) with one or more negatively-charged amino acids. As another example, a neutral-charged second region of a channel may be engineered by substituting negatively -charged amino acid residues and/or positively-charged amino acid residues in place of neutral charged residues, adding (e.g., inserting) neutral -charged amino acid residues, deleting negatively-charged amino acids and/or positively-charged amino acids, or any combination thereof to achieve a neutral charge, a more neutral region as compared to a respective region of a wild-type pore, or a net neutral charge. Without wishing to be bound by theory, a more negative second region of the channel may be achieved by mutating (e.g., adding and/or substituting) one or more amino acids of the monomer to negative amino acid residues (e.g., aspartic acid (D) and/or glutamic acid (E)). Without wishing to be bound by theory, the above methods may also be used to engineer a first portion of a monomer, a second portion of a monomer, a third portion of a monomer, or any combination thereof to have a more negative and/or more neutral charge.

[0405] In some cases, the engineered biological nanopores described herein may have one or more amino acids substituted for one or more negatively -charged amino acids. For example, one or more positively- charged amino acids, non-polar amino acids or aromatic amino acids at the first region, second region, third region, or any combination thereof, may be substituted with one or more negatively-charged amino acids. Negatively -charged amino acids may comprise aspartic acid (D) and/or glutamic acid (E). Positively -charged amino acids may comprise arginine (R), lysine (K), histidine (H), or any combination thereof. Aromatic amino acids may comprise phenylalanine (F), tryptophan (W), tyrosine (Y), or any combination thereof. Non-poler (e.g., neutrally-charged) amino acids may comprise alanine (A), asparagine (N), cysteine (C), glutamine (Q), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), or combinations thereof.

[0406] Without wishing to be bound by theory, a more neutral second region of the channel may be achieved by mutating one or more amino acids of the monomer to neutral amino acid residues (e.g., an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine (Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline (P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue). Without wishing to be bound by theory, a more neutral second region of the channel may be achieved by mutating one or more amino acids of the monomer to non-natural amino acid residues. Without wishing to be bound by theory, a more neutral second region of the channel may be achieved by mutating positively-charged amino acid residues and/or negatively-charged amino acid residues to obtain an equal number of positively-charged amino acid residues and negatively -charged amino acid residues in the second region. [0407] For example, a wild-type biological nanopore may comprise a second region of a channel with four negatively-charged amino acid residues. An engineered biological nanopore described herein may substitute the four negatively -charged amino acid residues with four neutral amino acid residues and increase the neutrality of the second region of the channel. As another example, a wild-type biological nanopore may comprise a second region of a channel with four negatively-charged amino acid residues. An engineered biological nanopore described herein may substitute two of the negatively -charged amino acid residues with two positively-charged amino acid residues to achieve a net neutral charge.

[0408] In some cases, one or more amino acids in a second region may be modified. For example, one or more amino acids in a second region may be modified to one or more neutral amino acid residues and/or one or more negatively -charged amino acid residues. As another example, two amino acids in a second region may be modified such that one amino acid is modified to a neutral amino acid residue and one amino acid is modified to a negatively-charged amino acid residue. In some cases, one or more amino acids in the first region may be mutated to one or more negatively-charged amino acid residues.

[0409] Modifications to one or more amino acids in a second region may be made along with modifications to one or more amino acids in a first region and/or third region. In some cases, when one or more amino acids in a second region may be modified to one or more neutral amino acids and/or one or more negatively- charged amino acids, then one or more amino acids in the first region and/or third region (e.g., adjacent region) may be mutated to one or more negatively-charged amino acids. As an example, two amino acids in the second region may be modified to negatively-charged amino acids and one amino acid in a first region may be modified to a negatively-charged amino acid. In some cases, at least about 1, 2, 3, 4, 5, or greater than about 5 amino acids in a second region may be modified to one or more neutral amino acids and/or one or more negatively -charged amino acids. In some cases, at most about 5, 4, 3, 2, 1, or less than about 1 amino acid in a second region may be modified to one or more neutral amino acids and/or one or more negatively- charged amino acids. In some cases, at least about 1, 2, 3, 4, 5, 10, or greater than about 10 amino acids in a second region may be modified to one or more neutral amino acids or one or more negatively-charged amino acids. In some cases, at most about 10, 5, 4, 3, 2, 1, or less than about 1 amino acid in a second region may be modified to one or more neutral amino acids or one or more negatively -charged amino acids.

[0410] In some cases, a plurality of amino acids in a second region may be modified to a plurality of neutral amino acids (e.g., in a ring of charge comprising the plurality of amino acids). In some cases, a plurality of amino acids in a first region may be mutated to a plurality of negatively-charged amino acids (e.g., in a ring of charge comprising the plurality of amino acids). In other cases, when a plurality of amino acids in a second region may be modified to a plurality of neutral amino acids, then a plurality of amino acids in a first region and/or third region (e.g., adjacent region) may be mutated to a plurality of negatively-charged amino acids. In some cases, a second region of the nanopore may comprise one or more amino acids modified to one or more amino acids comprising non-bulky side chains. For example, bulky amino acids with may comprise amino acids with aromatic groups and/or lengthy side chains. As another example, bulky amino acids may comprise those with large side chains (e.g., hydrophobic) that can impact protein structure and function. Non-limiting examples of bulky amino acids may be phenylalanine, tyrosine, tryptophan, isoleucine, leucine, and valine, methionine, histidine, threonine, or combinations thereof. It may be advantageous to minimize a number of bulky amino acids in a second region (e.g., constriction region) so an analyte may be captured or translocate through the pore. For example, in some cases, one or more amino acids in a second region (e.g., constriction region) may not be mutated to one or more bulky amino acids (e.g., phenylalanine, tyrosine, tryptophan, isoleucine, leucine, and valine, methionine, histidine, threonine, or combinations thereof).

[0411] In some cases, a net charge of the second region of the channel can be at least about 30% more neutral, at least about 40% more neutral, at least about 50% more neutral, at least about 60% more neutral, at least about 70% more neutral, at least about 80% more neutral, at least about 90% more neutral, or greater than 90% more neutral as compared to a constriction region of a wild-type biological nanopore. In some cases, a net charge of the second region of the channel can be at most about 90% more neutral, at most about 80% more neutral, at most about 70% more neutral, at most about 60% more neutral, at most about 50% more neutral, at most about 40% more neutral, at most about 30% more neutral, or less than about 30% more neutral as compared to a constriction region of a wild-type biological nanopore.

[0412] In some cases, a net charge of the second region of the channel can be from about 20% more neutral to about 90% more neutral as compared to a constriction region of a wild-type biological nanopore. In some cases, a net charge of the second region of the channel can be from about 20% more neutral to about 30% more neutral, about 20% more neutral to about 40% more neutral, about 20% more neutral to about 50% more neutral, about 20% more neutral to about 55% more neutral, about 20% more neutral to about 60% more neutral, about 20% more neutral to about 65% more neutral, about 20% more neutral to about 70% more neutral, about 20% more neutral to about 75% more neutral, about 20% more neutral to about 80% more neutral, about 20% more neutral to about 85% more neutral, about 20% more neutral to about 90% more neutral, about 30% more neutral to about 40% more neutral, about 30% more neutral to about 50% more neutral, about 30% more neutral to about 55% more neutral, about 30% more neutral to about 60% more neutral, about 30% more neutral to about 65% more neutral, about 30% more neutral to about 70% more neutral, about 30% more neutral to about 75% more neutral, about 30% more neutral to about 80% more neutral, about 30% more neutral to about 85% more neutral, about 30% more neutral to about 90% more neutral, about 40% more neutral to about 50% more neutral, about 40% more neutral to about 55% more neutral, about 40% more neutral to about 60% more neutral, about 40% more neutral to about 65% more neutral, about 40% more neutral to about 70% more neutral, about 40% more neutral to about 75% more neutral, about 40% more neutral to about 80% more neutral, about 40% more neutral to about 85% more neutral, about 40% more neutral to about 90% more neutral, about 50% more neutral to about 55% more neutral, about 50% more neutral to about 60% more neutral, about 50% more neutral to about 65% more neutral, about 50% more neutral to about 70% more neutral, about 50% more neutral to about 75% more neutral, about 50% more neutral to about 80% more neutral, about 50% more neutral to about 85% more neutral, about 50% more neutral to about 90% more neutral, about 55% more neutral to about 60% more neutral, about 55% more neutral to about 65% more neutral, about 55% more neutral to about 70% more neutral, about 55% more neutral to about 75% more neutral, about 55% more neutral to about 80% more neutral, about 55% more neutral to about 85% more neutral, about 55% more neutral to about 90% more neutral, about 60% more neutral to about 65% more neutral, about 60% more neutral to about 70% more neutral, about 60% more neutral to about 75% more neutral, about 60% more neutral to about 80% more neutral, about 60% more neutral to about 85% more neutral, about 60% more neutral to about 90% more neutral, about 65% more neutral to about 70% more neutral, about 65% more neutral to about 75% more neutral, about 65% more neutral to about 80% more neutral, about 65% more neutral to about 85% more neutral, about 65% more neutral to about 90% more neutral, about 70% more neutral to about 75% more neutral, about 70% more neutral to about 80% more neutral, about 70% more neutral to about 85% more neutral, about 70% more neutral to about 90% more neutral, about 75% more neutral to about 80% more neutral, about 75% more neutral to about 85% more neutral, about 75% more neutral to about 90% more neutral, about 80% more neutral to about 85% more neutral, about 80% more neutral to about 90% more neutral, or about 85% more neutral to about 90% more neutral as compared to a constriction region of a wild-type biological nanopore.

[0413] In some cases, a net charge of the second region of the channel can be at least about 30% more negative, at least about 40% more negative, at least about 50% more negative, at least about 60% more negative, at least about 70% more negative, at least about 80% more negative, at least about 90% more negative, or greater than 90% more negative as compared to a constriction region of a wild-type biological nanopore. In some cases, a net charge of the second region of the channel can be at most about 90% more negative, at most about 80% more negative, at most about 70% more negative, at most about 60% more negative, at most about 50% more negative, at most about 40% more negative, at most about 30% more negative, or less than about 30% more negative as compared to a constriction region of a wild-type biological nanopore.

[0414] A net charge of a second region of the channel of the engineered biological nanopore may be less cationic as compared to a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise less positively-charged amino acid residues than a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise more negatively-charged amino acid residues than a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise more neutral amino acid residues than a constriction region of a wild-type biological nanopore that comprises a net positive charge. A net charge of a second region of the channel of the engineered biological nanopore may be less anionic as compared to a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise more positively -charged amino acid residues than a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise less negatively -charged amino acid residues than a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise more neutral amino acid residues than a constriction region of a wild-type biological nanopore that comprises a net negative charge.

[0415] An engineered biological nanopore can be made more neutral (e.g., obtain a greater net neutral charge) depending on an amino acid composition of the wild-type biological nanopore. For example, a wildtype biological nanopore comprising a net positively-charged constriction region may have negatively- charged amino acid residues inserted to engineer a greater net neutral charge. For example, a wild-type biological nanopore comprising a net positively -charged constriction region may have neutral amino acid residues substituted in place of positively -charged residues to engineer a greater net neutral charge. For example, a wild-type biological nanopore comprising a net positively-charged constriction region may have positively-charged residues deleted to engineer a greater net neutral charge. For example, a wild-type biological nanopore comprising a net negatively-charged constriction region may have positively-charged amino acid residues inserted to engineer a greater net neutral charge. For example, a wild-type biological nanopore comprising a net negatively -charged constriction region may have neutral amino acid residues substituted in place of negatively -charged residues to engineer a greater net neutral charge. For example, a wild-type biological nanopore comprising a net negatively-charged constriction region may have negatively- charged residues deleted to engineer a greater net neutral charge.

[0416] A net charge of a second region of the channel of the engineered biological nanopore may be more anionic as compared to a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise less positively-charged amino acid residues than a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise more negatively-charged amino acid residues than a constriction region of a wild-type biological nanopore. For example, the second region of the channel may comprise less neutral amino acid residues and greater negatively-charged residues than a constriction region of a wild-type biological nanopore that comprises a net positive charge.

[0417] An engineered biological nanopore can be made more negative (e.g., obtain a greater net negative charge) depending on an amino acid composition of the wild-type biological nanopore. For example, a wildtype biological nanopore comprising a net neutrally -charged constriction region may have negatively- charged amino acid residues inserted to engineer a greater net negative charge. For example, a wild-type biological nanopore comprising a net positively -charged constriction region may have positively -charged amino acid residues deleted to engineer a greater net negative charge. For example, a wild-type biological nanopore comprising a net negatively -charged constriction region may have negatively-charged amino acid residues inserted to engineer a greater net negative charge.

[0418] An engineered monomer can be made more neutral (e.g., obtain a greater net neutral charge) depending on an amino acid composition of the wild-type monomer. For example, a wild-type monomer comprising a net positively -charged constriction-forming portion may have negatively -charged amino acid residues inserted to engineer a greater net neutral charge. For example, a wild-type monomer comprising a net positively-charged constriction-forming portion may have neutral amino acid residues substituted in place of positively-charged residues to engineer a greater net neutral charge. For example, a wild-type monomer comprising a net positively -charged constriction-forming portion may have positively-charged residues deleted to engineer a greater net neutral charge. For example, a wild-type monomer comprising a net negatively-charged constriction-forming portion may have positively-charged amino acid residues inserted to engineer a greater net neutral charge. For example, a wild-type monomer comprising a net negatively- charged constriction-forming portion may have neutral amino acid residues substituted in place of negatively-charged residues to engineer a greater net neutral charge. For example, a wild-type monomer comprising a net negatively-charged constriction-forming portion may have negatively -charged residues deleted to engineer a greater net neutral charge.

[0419] The second region of the nanopore (e.g., the engineered biological nanopore) may be more neutral as compared to a region of a wild-type biological nanopore. The second region of the nanopore (e.g., the engineered biological nanopore) may be more neutral as compared to a constriction region of a wild-type biological nanopore. For example, a more neutral second region of the nanopore (e.g., the engineered biological nanopore) may comprise a region with more neutral amino acid residues compared to that of a constriction region of a wild-type biological nanopore. A more neutral second region of the nanopore (e.g., the engineered biological nanopore) may comprise a region with a same number of positively-charged amino acid residues and negatively-charged amino acid residues compared to that of a constriction region of a wildtype biological nanopore. For example, a more neutral second region may comprise a region with ten positively-charged amino acid residues and ten negatively -charged amino acid residues, compared to a region (e.g., a constriction region) of a wild-type nanopore with a greater number of positively -charged amino acid residues than negatively-charged amino acid residues or with a greater number of negatively- charged amino acid residues than positively -charged amino acid residues.

[0420] The second portion of the monomer (e.g., the constriction-forming portion) may be more neutral as compared to a constriction-forming portion of a wild-type monomer. For example, a more neutral second portion of the monomer (e.g., the engineered biological monomer) may comprise a constriction-forming portion with more neutral amino acid residues compared to that of a constriction-forming portion of a wildtype biological monomer. A more neutral second portion of the monomer (e.g., the engineered biological monomer) may comprise a constriction-forming portion with a same number of positively-charged amino acid residues and negatively-charged amino acid residues compared to that of a constriction-forming portion of a wild-type biological monomer. For example, a more neutral constriction-forming portion may comprise a constriction-forming portion with ten positively -charged amino acid residues and ten negatively-charged amino acid residues, compared to a second portion (e.g., constriction-forming portion) of a wild-type monomer with a greater number of positively -charged amino acid residues than negatively -charged amino acid residues or with a greater number of negatively -charged amino acid residues than positively -charged amino acid residues.

[0421] The second region of the nanopore (e.g., the engineered biological nanopore) may be more negative as compared to a region of a wild-type biological nanopore. The second region of the nanopore (e.g., the engineered biological nanopore) may be more negative as compared to a constriction region of a wild-type biological nanopore. For example, a more neutral second region of the nanopore (e.g., the engineered biological nanopore) may comprise a region with more negatively -charged amino acid residues compared to that of a constriction region of a wild-type biological nanopore. A more negative second region of the nanopore (e.g., the engineered biological nanopore) may comprise a region with a greater number of negatively-charged amino acid residues compared to positively-charged amino acid residues and/or neutral amino acid residues, and compared to that of a constriction region of a wild-type biological nanopore. For example, a more negative second region may comprise a region with five positively-charged amino acid residues and/or neutrally charged amino acid residues, and ten negatively-charged amino acid residues, compared to a region (e.g., a constriction region) of a wild-type nanopore with a greater number of positively-charged amino acid residues than negatively-charged amino acid residues or with a greater number of neutrally-charged amino acid residues than negatively-charged amino acid residues. In some cases, a constriction region may be engineered so as to mutate amino acid residues to one or more neutral amino acid residues and one or more negative amino acid residues. For example, an engineered biological nanopore may have neutral and/or negatively-charged amino acid residues inserted into a constriction region. For example, an engineered biological nanopore may have neutral and/or negatively -charged amino acid residues substituted into a constriction region for positively-charged amino acid residues.

[0422] The second portion of the monomer (e.g., the constriction-forming portion) may be more negative as compared to a constriction-forming portion of a wild-type monomer. For example, a more negative second portion of the monomer (e.g., the engineered biological monomer) may comprise a constriction-forming portion with more negative amino acid residues compared to that of a constriction-forming portion of a wildtype biological monomer. A more negative second portion of the monomer (e.g., the engineered biological monomer) may comprise a constriction-forming portion with a greater number of negatively -charged amino acid residues compared to positively-charged amino acid residues and/or neutral amino acid residues, and compared to that of a constriction-forming portion of a wild-type biological monomer. For example, a more negative constriction-forming portion may comprise a constriction-forming portion with five positively- charged amino acid residues and ten negatively -charged amino acid residues, compared to a second portion (e.g., constriction-forming portion) of a wild-type monomer with a greater number of positively-charged amino acid residues than negatively-charged amino acid residues or with a greater number of negatively- charged amino acid residues than positively -charged amino acid residues. In some cases, a constrictionforming portion may be engineered so as to mutate amino acid residues to one or more neutral amino acid residues and one or more negative amino acid residues. For example, an engineered monomer may have neutral and/or negatively -charged amino acid residues inserted into a constriction-forming portion. For example, an engineered monomer may have neutral and/or negatively-charged amino acid residues substituted into a constriction-forming portion for positively -charged amino acid residues.

[0423] A first region and/or third region (e.g., the region adjacent to the second region) of the nanopore (e.g., the engineered biological nanopore) may be more negative compared to a region of a wild-type biological nanopore. The first region and/or third region (e.g., the region adjacent to the second region) of the nanopore (e.g., the engineered biological nanopore) may be more negative compared to a respective region of a wild-type biological nanopore (e.g., another region adjacent to a constriction region of a wild-type biological nanopore). For example, a more negative first region and/or third region of the nanopore (e.g., the engineered biological nanopore) may comprise a region with more negatively-charged amino acid residues compared to that of a respective region of a wild-type biological nanopore (e.g., another region adjacent to a constriction region of a wild-type biological nanopore). For example, a more negative first region and/or third region of the nanopore (e.g., the engineered biological nanopore) may comprise a region with less positively-charged amino acid residues compared to that of a respective region of a wild-type biological nanopore (e.g., another region adjacent to a constriction region of a wild-type biological nanopore).

[0424] In some cases, in an engineered biological nanopore described herein, if the second region of the channel may be more neutral or negative as compared to a constriction region of a wild-type biological nanopore, then the first region and/or third region of the engineered biological nanopore may be more negative as compared to another region adjacent to the constriction region of the wild-type biological nanopore. In some cases, in an engineered biological nanopore described herein, a second region of the channel may comprise a portion (e.g., a second portion) of a monomer. The second portion of the monomer may be more neutral and/or negative as compared to a portion of a monomer in a constriction region of a wild-type biological nanopore. The second portion of the monomer may be more negative as compared to a portion of a monomer in a constriction region of a wild-type biological nanopore. In some cases, in an engineered biological nanopore described herein, a first region of the channel may comprise a portion (e.g., a first portion) of a monomer. The first portion of the monomer may be more negative as compared to a portion of a monomer in a region adjacent to a constriction region of a wild-type nanopore. In some cases, in an engineered biological nanopore described herein, a third region of the channel may comprise a portion (e.g., a third portion) of a monomer. The third portion of the monomer may be more negative as compared to a portion of a monomer in a region adjacent to a constriction region of a wild-type nanopore.

[0425] In some cases, if a constriction-forming portion of an engineered monomer can be more neutral as compared to a constriction-forming portion of a monomer of a wild-type nanopore channel, then a first portion and/or third portion of the engineered monomer may be more negative as compared to a corresponding portion of a monomer of the wild-type biological nanopore. In some cases, if a constrictionforming portion of an engineered monomer can be more negative as compared to a constriction-forming portion of a monomer of a wild-type nanopore channel, then a first portion and/or third portion of the engineered monomer may be more negative as compared to a corresponding portion of a monomer of the wild-type biological nanopore.

[0426] In some cases, a second region of a channel of the nanopore (e.g., the engineered biological nanopore) may be at least about 30% more neutral, at least about 40% more neutral, at least about 50% more neutral, at least about 60% more neutral, at least about 70% more neutral, at least about 80% more neutral, at least about 85% more neutral, at least about 90% more neutral, at least about 95% more neutral, or greater than about 95% more neutral as compared to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore). In some cases, a second region of a channel of the nanopore (e.g., the engineered biological nanopore) may be at most about 95% more neutral, at most about 90% more neutral, at most about 85% more neutral, at most about 80% more neutral, at most about 70% more neutral, at most about 60% more neutral, at most about 50% more neutral, at most about 40% more neutral, at most about 30% more neutral, or less than about 30% more neutral as compared to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore). [0427] In some cases, a second region of a channel of the nanopore (e.g., the engineered biological nanopore) may be from about 20% more neutral to about 100% more neutral as compared to a constriction region of a wild-type biological nanopore. In some cases, a second region of a channel of the nanopore (e.g., the engineered biological nanopore) may be from about 20% more neutral to about 30% more neutral, about 20% more neutral to about 40% more neutral, about 20% more neutral to about 50% more neutral, about

20% more neutral to about 60% more neutral, about 20% more neutral to about 70% more neutral, about

20% more neutral to about 75% more neutral, about 20% more neutral to about 80% more neutral, about

20% more neutral to about 85% more neutral, about 20% more neutral to about 90% more neutral, about

20% more neutral to about 95% more neutral, about 20% more neutral to about 100% more neutral, about 30% more neutral to about 40% more neutral, about 30% more neutral to about 50% more neutral, about

30% more neutral to about 60% more neutral, about 30% more neutral to about 70% more neutral, about

30% more neutral to about 75% more neutral, about 30% more neutral to about 80% more neutral, about

30% more neutral to about 85% more neutral, about 30% more neutral to about 90% more neutral, about

30% more neutral to about 95% more neutral, about 30% more neutral to about 100% more neutral, about 40% more neutral to about 50% more neutral, about 40% more neutral to about 60% more neutral, about 40% more neutral to about 70% more neutral, about 40% more neutral to about 75% more neutral, about 40% more neutral to about 80% more neutral, about 40% more neutral to about 85% more neutral, about 40% more neutral to about 90% more neutral, about 40% more neutral to about 95% more neutral, about 40% more neutral to about 100% more neutral, about 50% more neutral to about 60% more neutral, about 50% more neutral to about 70% more neutral, about 50% more neutral to about 75% more neutral, about 50% more neutral to about 80% more neutral, about 50% more neutral to about 85% more neutral, about 50% more neutral to about 90% more neutral, about 50% more neutral to about 95% more neutral, about 50% more neutral to about 100% more neutral, about 60% more neutral to about 70% more neutral, about 60% more neutral to about 75% more neutral, about 60% more neutral to about 80% more neutral, about 60% more neutral to about 85% more neutral, about 60% more neutral to about 90% more neutral, about 60% more neutral to about 95% more neutral, about 60% more neutral to about 100% more neutral, about 70% more neutral to about 75% more neutral, about 70% more neutral to about 80% more neutral, about 70% more neutral to about 85% more neutral, about 70% more neutral to about 90% more neutral, about 70% more neutral to about 95% more neutral, about 70% more neutral to about 100% more neutral, about 75% more neutral to about 80% more neutral, about 75% more neutral to about 85% more neutral, about 75% more neutral to about 90% more neutral, about 75% more neutral to about 95% more neutral, about 75% more neutral to about 100% more neutral, about 80% more neutral to about 85% more neutral, about 80% more neutral to about 90% more neutral, about 80% more neutral to about 95% more neutral, about 80% more neutral to about 100% more neutral, about 85% more neutral to about 90% more neutral, about 85% more neutral to about 95% more neutral, about 85% more neutral to about 100% more neutral, about 90% more neutral to about 95% more neutral, about 90% more neutral to about 100% more neutral, or about 95% more neutral to about 100% more neutral as compared to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore).

[0428] In some cases, a second region of a channel of the nanopore (e.g., the engineered biological nanopore) may be at least about 30% more negative, at least about 40% more negative, at least about 50% more negative, at least about 60% more negative, at least about 70% more negative, at least about 80% more negative, at least about 85% more negative, at least about 90% more negative, at least about 95% more negative, or greater than about 95% more negative as compared to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore). In some cases, a second region of a channel of the nanopore (e.g., the engineered biological nanopore) may be at most about 95% more negative, at most about 90% more negative, at most about 85% more negative, at most about 80% more negative, at most about 70% more negative, at most about 60% more negative, at most about 50% more negative, at most about 40% more negative, at most about 30% more negative, or less than about 30% more negative as compared to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore).

[0429] In some cases, a first region and/or third region of a channel of the nanopore (e.g., the engineered biological nanopore) may be at least about 30% more negative, at least about 40% more negative, at least about 50% more negative, at least about 60% more negative, at least about 70% more negative, at least about 80% more negative, at least about 85% more negative, at least about 90% more negative, at least about 95% more negative, or greater than about 95% more negative as compared to a region adjacent to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore). In some cases, a first region and/or third region of a channel of the nanopore (e.g., the engineered biological nanopore) may be at most about 95% more negative, at most about 90% more negative, at most about 85% more negative, at most about 80% more negative, at most about 70% more negative, at most about 60% more negative, at most about 50% more negative, at most about 40% more negative, at most about 30% more negative, or less than about 30% more negative as compared to a region adjacent to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore).

[0430] In some cases, a first region and/or third region of a channel of the nanopore (e.g., the engineered biological nanopore) may be from about 20% more negative to about 100% more negative as compared to a respective region of a wild-type biological nanopore (e.g., a region adjacent to a constriction region of a wild-type biological nanopore). In some cases, a first region and/or third region of a channel of the nanopore (e.g., the engineered biological nanopore) may be from about 20% more negative to about 30% more negative, about 20% more negative to about 40% more negative, about 20% more negative to about 50% more negative, about 20% more negative to about 60% more negative, about 20% more negative to about 70% more negative, about 20% more negative to about 75% more negative, about 20% more negative to about 80% more negative, about 20% more negative to about 85% more negative, about 20% more negative to about 90% more negative, about 20% more negative to about 95% more negative, about 20% more negative to about 100% more negative, about 30% more negative to about 40% more negative, about 30% more negative to about 50% more negative, about 30% more negative to about 60% more negative, about 30% more negative to about 70% more negative, about 30% more negative to about 75% more negative, about 30% more negative to about 80% more negative, about 30% more negative to about 85% more negative, about 30% more negative to about 90% more negative, about 30% more negative to about 95% more negative, about 30% more negative to about 100% more negative, about 40% more negative to about 50% more negative, about 40% more negative to about 60% more negative, about 40% more negative to about 70% more negative, about 40% more negative to about 75% more negative, about 40% more negative to about 80% more negative, about 40% more negative to about 85% more negative, about 40% more negative to about 90% more negative, about 40% more negative to about 95% more negative, about 40% more negative to about 100% more negative, about 50% more negative to about 60% more negative, about 50% more negative to about 70% more negative, about 50% more negative to about 75% more negative, about 50% more negative to about 80% more negative, about 50% more negative to about 85% more negative, about 50% more negative to about 90% more negative, about 50% more negative to about 95% more negative, about 50% more negative to about 100% more negative, about 60% more negative to about 70% more negative, about 60% more negative to about 75% more negative, about 60% more negative to about 80% more negative, about 60% more negative to about 85% more negative, about 60% more negative to about 90% more negative, about 60% more negative to about 95% more negative, about 60% more negative to about 100% more negative, about 70% more negative to about 75% more negative, about 70% more negative to about 80% more negative, about 70% more negative to about 85% more negative, about 70% more negative to about 90% more negative, about 70% more negative to about 95% more negative, about 70% more negative to about 100% more negative, about 75% more negative to about 80% more negative, about 75% more negative to about 85% more negative, about 75% more negative to about 90% more negative, about 75% more negative to about 95% more negative, about 75% more negative to about 100% more negative, about 80% more negative to about 85% more negative, about 80% more negative to about 90% more negative, about 80% more negative to about 95% more negative, about 80% more negative to about 100% more negative, about 85% more negative to about 90% more negative, about 85% more negative to about 95% more negative, about 85% more negative to about 100% more negative, about 90% more negative to about 95% more negative, about 90% more negative to about 100% more negative, or about 95% more negative to about 100% more negative as compared to a respective region of a wild-type biological nanopore (e.g., a region adjacent to a constriction region of a wild-type biological nanopore). [0431] In some cases, a first region or a third region may comprise at least one amino acid that is mutated to exhibit an increased net negative charge. In some cases, a first region and/or a third region may comprise a plurality of amino acids that are mutated to exhibit an increased net negative charge. For example, a first region may comprise two amino acids that are mutated to exhibit an increased net negative charge and a third region may comprise two amino acids that are mutated to exhibit an increased net negative charge. In some cases, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, or greater than about 30 amino acids in a first region and/or third region may be modified to one or more amino acids that increase a net negative charge (e.g., one or more negatively- charged amino acids). In some cases, at most about 30, at most about 20, at most about 10, 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 amino acid in a first region and/or third region may be modified to one or more amino acids that increase a net negative charge (e.g., one or more negatively-charged amino acids).

[0432] A first region and/or a third region may comprise at least one amino acid that is mutated to exhibit an increased net negative charge as compared to a respective region of a wild-type biological nanopore. For example, a first region of an engineered biological nanopore described herein may have an amino acid 10 A from a second region (e.g., constriction region). A wild-type biological nanopore may have an amino acid at an identical position (e.g., 10 A from a constriction region). The amino acid in the engineered biological nanopore may be mutated to increase a net negative charge. Thus, the first region of the engineered biological nanopore comprises at least one amino acid that is mutated to exhibit an increased net negative charge as compared to a respective region of a wild-type biological nanopore. As another example, a first region and/or a third region may comprise at least one amino acid that is mutated to a negatively -charged amino acid to exhibit an increased net negative charge as compared to a respective region of a wild-type biological nanopore

[0433] The engineered biological nanopore described herein may comprise a high region of charge in a first region and/or third region of the channel (e.g., a region adjacent to the constriction region). The high region of charge may be 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, or greater than about 10 net unitary negative charges in the first region of the channel (e.g., the region adjacent to the constriction region). For example, the first region may comprise 10 negative charges and 5 positive charges, which would achieve net 5 unitary negative charges. The high region of charge may be obtained under system run condition (e.g., at a pH as charge can be dependent on pH). The high region of charge may be obtained under a pH of at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10. The high region of charge may be obtained under a pH of at most about 10, 9, 8, 7, 6, 5, 4, 3, or 2. The high region of charge (e.g., high region of negative charge) may not be in the second region of the channel (e.g., comprising the constriction region).

[0434] A constriction region may form from a net charge of the channel, the geometry of the channel, or any combinations thereof. Modifications to the net charge of the channel, the geometry of the channel, or any combination thereof, can modify a characteristic of a constriction region. A characteristic of a constriction region may be a placement, a location, a width, a charge, an amino acid composition, or combinations thereof. For example, a change to a geometry of the channel can change the width of a constriction region, or a change to a net charge of the channel can change the charge of a constriction region.

[0435] In some cases, a first region and/or third region of the channel of the engineered biological nanopore 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 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 cases coulombs, a first region and/or third region of the channel of the engineered biological nanopore 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.

[0436] In some cases, a first region and/or third region of the channel of the engineered biological nanopore comprises a net charge from about 2 to about 200 coulombs. In some cases, a nanopore channel comprises a net charge from at most about 200. In some cases, a first region and/or third region of the channel of the engineered biological nanopore 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.

[0437] In some cases coulombs, a nanopore channel comprises a net charge of about 2 coulombs, about 3 coulombs, about 4 coulombs, about 5 coulombs, about 10 coulombs, about 15 coulombs, about 20 coulombs, about 25 coulombs, about 30 coulombs, about 35 coulombs, about 40 coulombs, about 45 coulombs, about 50 coulombs, about 55 coulombs, about 60 coulombs, about 70 coulombs, about 80 coulombs, about 90 coulombs, about 100 coulombs, about 150 coulombs, about 200 coulombs.

[0438] In some cases, a first region and/or third region of the channel of the engineered biological nanopore can comprise a net negative charge to create the EOF. In some cases, the nanopore channel can comprise a net charge from about -20 to about +20 for a single subunit of an oligomeric nanopore. This net charge can be multiplied up to e.g. 5, 6, 7, 8, 9, 10 or more times in the final pore, thus it is possible to have >100 charges in the channel of the final nanopore which can comprise oligomeric pores, monomeric pores or fusion pores wherein all monomers are genetically fused into a single pore. [0439] The net charge of the first region and/or third region of the channel of the engineered biological nanopore described herein can be combined with any charge on the remainder of the nanopore protein. As an example, the net charge of the channel can be combined with a negative charge, or plurality of negative charges on the outside of the pore.

[0440] In some cases, the nanopore channel can comprise a net charge of at least about -150, at least about - 100, at least about -50, at least about -40, at least about -30, at least about -20, at least about -19, at least about -18, at least about -17, at least about -16, at least about -15, at least about -14, at least about -13, at least about -12, at least about -11, at least about -10, at least about -9, at least about -8, at least about -7, at least about -6, at least about -5, at least about -4, at least about -3, at least about -2, at least about -1, at least about 0, at least about +1, 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, at least about +30, at least about +40, at least about +50, at least about +100, at least about +150, or more than +150 for a single nanopore subunit. In some cases, the nanopore channel can comprise a net charge of at most about +150, at most about +100, at most about +50, at most about +40, at most about +30, 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, at most about +1, at most about 0, at most about -1, at most about -2, at most about -3, at most about -4, at most about -5, at most about -6, at most about -7, at most about -8, at most about -9, at most about -10, at most about -11, at most about - 12, at most about -13, at most about -14, at most about -15, at most about -16, at most about -17, at most about -18, at most about -19, at most about -20, at most about -30, at most about -40, at most about -50, at most about -100, at most about -150, or less than -150 for a single nanopore subunit. In some cases, the nanopore channel can comprise a net charge of about -150, about -100, about -50, about -40, about -30, about -20, about -19, about -18, about -17, about -16, about -15, about -14, about -13, about -12, about -11, about - 10, about -9, about -8, about -7, about -6, about -5, about -4, about -3, about -2, about -1, about 0, 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 +30, about +40, about +50, about +100, or about +150 for a single nanopore subunit.

[0441] In some cases, the engineered biological nanopore can comprise one or more monomers. A monomer of the one or more monomers may contain a first portion and at least a second portion. In some cases, a monomer of the engineered biological nanopore can comprise a first portion, a second portion, and a third portion. A first portion of the monomer may be adjacent to a first entrance of the second region of the channel (e.g., a first entrance of the constriction region). The first portion of the monomer may be adjacent to a second portion. A second portion of the monomer may comprise the second region of the channel. A second portion of the monomer may comprise a constriction-forming portion of the monomer (e.g., the engineered monomer). A third portion of the monomer may be adjacent to a second entrance of the second region of the channel (e.g., a second entrance of the constriction region). In some cases, a first portion of a monomer, a second portion of a monomer, a third portion of a monomer, or any combination thereof, may comprise at least one mutation.

[0442] Mechanisms of controlling or arranging the EOF can comprise genetic engineering (e.g. mutagenesis) of the channel of the nanopore to alter the steric and/or the electrostatic conditions. The mutagenesis may in turn adjust the specificity for translocating one ion over another. For example, the net charge of the inner channel of the nanopore can be increased so as to electrostatically limit the flux of one of the ions from one direction across the nanopore, while retaining/enhancing the flux of the oppositely charged ion 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, narrowing the channel dimensions, or any combination thereof.

[0443] The nanopores, systems, and/or methods disclosed herein allow for modifying (e.g., adding, substituting, and/or removing) charges around (e.g., a portion or portion adjacent to) a constriction-forming portion of a monomer to enhance EOF. For example, if (i) a constriction-forming portion of a monomer or (ii) a constriction region in a wild-type pore provides two negative charges, the charges may be modified by substituting one or more neutral amino acid residues, non-natural amino acid residues, or any combination thereof. For example, if (i) a constriction-forming portion of a monomer or (ii) a constriction region in a wild-type pore provides two positive charges, the charges may be modified by substituting one or more negatively-charged amino acid residues, non-natural amino acid residues (e.g., negatively-charged nonnatural amino acid residues), or any combination thereof. As another example, a (i) constriction-forming portion of a monomer, or (ii) a constriction region of a wild-type pore may be neutralized by substituting one or more negative amino acid residues for one or more neutral amino acid residues. In some cases, (i) a constriction-forming portion of a monomer, or (ii) a constriction region of a wild-type pore may be neutralized by inserting one or more positively-charged amino acid residues into a negatively-charged constriction region comprising one or more negative amino acid residues. In some cases, (i) a constrictionforming portion of a monomer, or (ii) a constriction region of a wild-type pore may have an increased net negative charge by inserting one or more negatively -charged amino acid residues into a positively-charged and/or neutral charged constriction region comprising one or more positive amino acid residues and/or neutral charged amino acid residues.

[0444] The nanopores, systems, and/or methods described herein may comprise a neutralized constriction. An EOF may be enhanced by increasing a region of charge in an area adjacent to the constriction region (e.g., a first region and/or third region of a channel). For example, if one or more negative charges are neutralized (e.g., by substitution of neutral amino acid residues) in a constriction region of the nanopore, an EOF may be enhanced by increasing a region of net negative charge in an area adjacent to the constriction region (e.g., a first region and/or third region of a channel). As an example, a second region (e.g., a constriction region) may be modified to be more net neutral as compared to a respective region of a wildtype nanopore by substituting one or more negatively -charged amino acids and/or one or more positively- charged amino acids with one or more neutral -charged amino acids. As another example, a second region (e.g., a constriction region) may be modified to be more net neutral as compared to a respective region of a wild-type nanopore by deleting a same number of negatively-charged amino acids and positively -charged amino acids. For example, if the region (e.g., a second region) has 10 amino acids with 4 negatively-charged amino acids and 6 positively-charged amino acids, the region may be modified to be more neutral by (i) deleting one or more positively charged amino acids; (ii) substituting one or more negatively -charged amino acids and/or one or more positively -charged amino acids with one or more neutral -charged amino acids; (iii) adding one or more negatively-charged amino acids; or (iv) any combination thereof). One or more natural amino acids and/or non-natural amino acids may be introduced to modify the region to be more net neutral as compared to a respective region of a wild-type nanopore. One or more natural amino acids and/or nonnatural amino acids may be deleted to modify the region to be more net neutral as compared to a respective region of a wild-type nanopore. The net negative region of charge in the first region and/or third region of the channel may be increased by mutating one or more amino acid residues in the first region and/or third region of the channel. The mutations may comprise substituting positively-charged amino acid residues and/or neutral amino acid residues for negatively-charged amino acid residues, inserting negatively-charged amino acid residues, deleting positively-charged amino acid residues, or any combination thereof.

[0445] An EOF may be enhanced by increasing a portion of charge in a constriction-forming portion (e.g., second portion) of a monomer. For example, if one or more negative charges are neutralized (e.g., by substitution of neutral amino acid residues and/or insertion of positively -charged amino acid residues) in a constriction-forming portion of the monomer, an EOF may be enhanced by increasing a region of net negative charge in an area adjacent to the constriction-forming portion of the monomer (e.g., a first portion and/or third portion of the monomer). As an example, a second portion (e.g., a constriction-forming portion) may be modified to be more net neutral as compared to a respective portion of a wild-type monomer by substituting one or more negatively -charged amino acids and/or one or more positively-charged amino acids with one or more neutral-charged amino acids. As another example, a second portion (e.g., a constrictionforming portion) may be modified to be more net neutral as compared to a respective portion of a wild-type monomer by deleting a same number of negatively-charged amino acids and positively-charged amino acids. For example, if the portion (e.g., a second portion) has 10 amino acids with 4 negatively -charged amino acids and 6 positively-charged amino acids, the portion may be modified to be more neutral by (i) deleting one or more positively charged amino acids; (ii) substituting one or more negatively-charged amino acids and/or one or more positively-charged amino acids with one or more neutral-charged amino acids; (iii) adding one or more negatively -charged amino acids; or (iv) any combination thereof). One or more natural amino acids and/or non-natural amino acids may be introduced to modify the second portion (e.g., the constrictionforming portion) to be more net neutral as compared to a respective portion of a wild-type monomer. One or more natural amino acids and/or non-natural amino acids may be deleted to modify the second portion (e.g., the constriction-forming portion) to be more net neutral as compared to a respective portion of a wild-type monomer. The net negative region of charge in the first portion and/or third portion of the monomer may be increased by mutating one or more amino acid residues in the first portion and/or third portion of the monomer. The mutations may comprise substituting positively-charged amino acid residues and/or neutral amino acid residues for negatively-charged amino acid residues, inserting negatively -charged amino acid residues, deleting positively-charged amino acid residues, or any combination thereof.

[0446] An EOF may be enhanced by increasing a negative charge (e.g., net negative charge) of a constriction region of a channel. A constriction region of a wild-type biological nanopore may not have a net negative charge, for example a constriction region of a pore may comprise a net neutral charge or net positive charge. By increasing a net negative charge, a stronger EOF may be generated and/or a cation selectivity of the pore may be increased. For example, a net negative charge of a constriction region of an engineered biological nanopore may be increased by substituting one or more positively -charged amino acid residues, neutral amino acid residues, or any combination thereof, with one or more negatively-charged amino acid residues. A net negative charge of a constriction region of an engineered biological nanopore may be increased by deleting positively-charged amino acid residues. A net negative charge of a constriction region of an engineered biological nanopore may be increased by inserting negatively -charged amino acid residues. [0447] A negative charge may be partly or fully established in a constriction region of an engineered biological nanopore described herein. For example, if the constriction region comprises two amino acid residues of a positive charge and/or neutral charge, a partly increased (e.g., less than 100% increased) negative charge may comprise (i) substituting one positively-charged amino acid residue or neutral amino acid residue with a negatively-charged amino acid residue, (ii) deleting one positively-charged amino acid residue or neutral amino acid residue, or (iii) inserting a negatively-charged amino acid residue. As another example, if the constriction region comprises two amino acid residues of a positive charge and/or neutral charge, a fully increased (e.g., 100% increased) negative charge may comprise (i) substituting the two positively-charged amino acid residue and/or neutral amino acid residue with two negatively -charged amino acid residue, (ii) deleting the two positively -charged amino acid residue and/or neutral amino acid residue, or (iii) inserting four or more negatively -charged amino acid residues.

[0448] An EOF may be enhanced by increasing a negative charge (e.g., net negative charge) of a second portion of a monomer (e.g., a constriction-forming portion of the monomer). A constriction-forming portion of the monomer may not have a net negative charge, for example a constriction-forming portion of the monomer may comprise a net neutral charge or net positive charge. By increasing a net negative charge, a stronger EOF may be generated and/or a cation selectivity of the pore may be increased. For example, a net negative charge of a constriction-forming portion of an engineered monomer may be increased by substituting one or more positively-charged amino acid residues, neutral amino acid residues, or any combination thereof, with one or more negatively -charged amino acid residues. A net negative charge of a constriction-forming portion of an engineered monomer may be increased by deleting positively-charged amino acid residues. A net negative charge of a constriction-forming portion of an engineered monomer may be increased by inserting negatively-charged amino acid residues.

[0449] A negative charge may be partly or fully established in a constriction-forming portion of an engineered monomer described herein. For example, if the constriction-forming portion comprises two amino acid residues of a positive charge and/or neutral charge, a partly increased (e.g., less than 100% increased) negative charge may comprise (i) substituting one positively-charged amino acid residue or neutral amino acid residue with a negatively-charged amino acid residue, (ii) deleting one positively-charged amino acid residue or neutral amino acid residue, or (iii) inserting a negatively -charged amino acid residue. As another example, if the constriction-forming portion comprises two amino acid residues of a positive charge and/or neutral charge, a fully increased (e.g., 100% increased) negative charge may comprise (i) substituting the two positively-charged amino acid residue and/or neutral amino acid residue with two negatively-charged amino acid residue, (ii) deleting the two positively -charged amino acid residue and/or neutral amino acid residue, or (iii) inserting four or more negatively -charged amino acid residues.

[0450] The mutations may be made to any number of monomers of the engineered biological nanopore. In some cases, a monomer of the engineered biological nanopore may comprise one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more mutations). In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, or more monomers of the engineered biological nanopore may comprise one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more mutations). Without wishing to be bound by theory, a greater EOF may be achieved with a neutralized constriction region (e.g., one or more neutral amino acid residues) and a rings of negative charges in a first region of the channel (e.g., adjacent to the constriction region). The rings of negative charges may comprise monomers of the engineered biological nanopore with at least about 1, 2, 3, 4, 5, 6, 7, 8, or more mutations, wherein the mutations comprise introduction (e.g., substitution and/or insertion) of negatively-charged amino acids.

[0451] In some cases, a portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer. A respective portion can be an identical portion of a wild-type monomer as compared to the portion of a monomer (e.g., an engineered monomer) described herein (e.g., an engineered biological monomer). A portion (e.g., a second portion) may have an amino acid composition comprising a plurality of amino acids. The plurality of amino acids may comprise one or more negatively-charged amino acids, one or more neutral amino acids, one or more positively-charged amino acids, or any combination thereof. A portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) may be modified by introducing one or more amino acid mutations to the portion (e.g., a second portion). In some cases, a portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) may be modified by introducing one or more amino acid mutations to a monomer (e.g., a wild-type monomer) comprising a portion that corresponds that that portion. As an example, a portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer by substituting one or more positively-charged amino acids and/or one or more neutral charged amino acids with one or more negatively-charged amino acids. As another example, a portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer by deleting one or more positively-charged amino acids and/or one or more neutral charged amino acids. As another example, a portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer by adding one or more negatively -charged amino acids to the portion. As another example, if a portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) has a net positive charge, the portion may be modified to be more net negative as compared to a respective portion of a wildtype monomer by substituting one or more positively-charged amino acids with one or more neutral charged amino acids and/or one or more negatively-charged amino acids. As another example, if the portion (e.g., a second portion) of a monomer (e.g., an engineered monomer) has a greater number of positively-charged amino acid residues compared to a number of negatively-charged amino acids and one or more neutral charged amino acids, the portion may still be modified to be more net negative as compared to a respective portion of a wild-type monomer by (i) substituting one or more positively-charged amino acids and/or one or more neutral charged amino acids with one or more negatively-charged amino acids; (ii) deleting one or more negatively-charged amino acids and/or one or more neutral charged amino acids; (iii) adding one or more negatively-charged amino acids; (iv) or any combination thereof.

[0452] One or more natural amino acids and/or non-natural amino acids may be introduced to modify the portion of a monomer (e.g., an engineered monomer) to be more net negative as compared to a respective portion of a wild-type monomer. For example, one or more negative natural amino acids may be aspartic acid (D) and/or glutamic acid (E). Negative non-natural amino acids can comprise an amino acid with a carboxylate group (COO-). One or more natural amino acids and/or non-natural amino acids may be deleted to modify the portion of a monomer (e.g., an engineered monomer) to be more net negative as compared to a respective portion of a wild-type monomer. These modifications may modify a second portion of a monomer (e.g., an engineered monomer) such that the second portion exhibits an increased net negative charge as compared to a respective portion of a wild-type biological monomer. [0453] In other cases, a second portion of a monomer (e.g., constriction-forming portion) may be modified to be more net neutral as compared to a respective region of a wild-type nanopore. Modifying the second portion of the monomer (e.g., constriction-forming portion) to be more net neutral may result from introduction of one or more amino acid modifications to the second portion. For example, substituting one or more negatively-charged amino acid residues and/or one or more positively-charged amino acid residues to one or more neutral-charged amino acid residues may modify a second portion to be more net positive as compared to a respective region of a wild-type nanopore. One or more natural or non-natural amino acids may be substituted for one or more negatively-charged amino acid residues and/or one or more positively- charged amino acid residues. For example, a natural neutral amino acid may be substituted in a second portion of a monomer. A second portion of a monomer may be modified to be more neutral by substituting an aspartic acid (D) residue to an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine (Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline (P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue. In other cases, a second portion of a monomer may be modified to be more neutral by substituting a glutamic acid (E) residue to an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine (Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline

(P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue. A second portion of a monomer may be modified to be more neutral by substituting an arginine (R) residue to an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine

(Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline (P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue. A second portion of a monomer may be modified to be more neutral by substituting a histidine (H) residue to an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine (Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline (P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue. A second portion of a monomer may be modified to be more neutral by substituting a lysine (K) residue to an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine (Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline (P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue. In other cases, deleting one or more negatively-charged amino acid residues and/or one or more positively -charged amino acid residues may also modify a second portion to be more net neutral as compared to a respective region of a wild-type nanopore. One or more natural negatively-charged amino acid residues and/or one or more natural positively -charged amino acid residues may be deleted to make a second portion of a monomer (e.g., an engineered monomer) more neutral. One or more non-natural negatively-charged amino acid residues and/or one or more natural positively-charged amino acid residues may be deleted to make a second portion of a monomer (e.g., an engineered monomer) more neutral. These modifications may modify a second portion of a monomer (e.g., an engineered monomer) such that the second portion exhibits an increased net neutral charge as compared to a respective portion of a wild-type biological monomer.

[0454] In some cases, a constriction-forming portion of an engineered monomer may be at least about 30% more neutral, at least about 40% more neutral, at least about 50% more neutral, at least about 60% more neutral, at least about 70% more neutral, at least about 80% more neutral, at least about 90% more neutral, or greater than 90% more neutral as compared to a constriction-forming portion of a wild-type monomer. In some cases, a constriction-forming portion of an engineered monomer may be at most about 90% more neutral, at most about 80% more neutral, at most about 70% more neutral, at most about 60% more neutral, at most about 50% more neutral, at most about 40% more neutral, at most about 30% more neutral, or less than about 30% more neutral as compared to a constriction-forming portion of a wild-type monomer. In some cases, a constriction-forming portion of an engineered monomer may be from about 20% more neutral to about 90% more neutral as compared to a constriction-forming portion of a wild-type monomer. In some cases, a constriction-forming portion of an engineered monomer may be from about 20% more neutral to about 30% more neutral, about 20% more neutral to about 40% more neutral, about 20% more neutral to about 50% more neutral, about 20% more neutral to about 55% more neutral, about 20% more neutral to about 60% more neutral, about 20% more neutral to about 65% more neutral, about 20% more neutral to about 70% more neutral, about 20% more neutral to about 75% more neutral, about 20% more neutral to about 80% more neutral, about 20% more neutral to about 85% more neutral, about 20% more neutral to about 90% more neutral, about 30% more neutral to about 40% more neutral, about 30% more neutral to about 50% more neutral, about 30% more neutral to about 55% more neutral, about 30% more neutral to about 60% more neutral, about 30% more neutral to about 65% more neutral, about 30% more neutral to about 70% more neutral, about 30% more neutral to about 75% more neutral, about 30% more neutral to about 80% more neutral, about 30% more neutral to about 85% more neutral, about 30% more neutral to about 90% more neutral, about 40% more neutral to about 50% more neutral, about 40% more neutral to about 55% more neutral, about 40% more neutral to about 60% more neutral, about 40% more neutral to about 65% more neutral, about 40% more neutral to about 70% more neutral, about 40% more neutral to about 75% more neutral, about 40% more neutral to about 80% more neutral, about 40% more neutral to about 85% more neutral, about 40% more neutral to about 90% more neutral, about 50% more neutral to about 55% more neutral, about 50% more neutral to about 60% more neutral, about 50% more neutral to about 65% more neutral, about 50% more neutral to about 70% more neutral, about 50% more neutral to about 75% more neutral, about 50% more neutral to about 80% more neutral, about 50% more neutral to about 85% more neutral, about 50% more neutral to about 90% more neutral, about 55% more neutral to about 60% more neutral, about 55% more neutral to about 65% more neutral, about 55% more neutral to about 70% more neutral, about 55% more neutral to about 75% more neutral, about 55% more neutral to about 80% more neutral, about 55% more neutral to about 85% more neutral, about 55% more neutral to about 90% more neutral, about 60% more neutral to about 65% more neutral, about 60% more neutral to about 70% more neutral, about 60% more neutral to about 75% more neutral, about 60% more neutral to about 80% more neutral, about 60% more neutral to about 85% more neutral, about 60% more neutral to about 90% more neutral, about 65% more neutral to about 70% more neutral, about 65% more neutral to about 75% more neutral, about 65% more neutral to about 80% more neutral, about 65% more neutral to about 85% more neutral, about 65% more neutral to about 90% more neutral, about 70% more neutral to about 75% more neutral, about 70% more neutral to about 80% more neutral, about 70% more neutral to about 85% more neutral, about 70% more neutral to about 90% more neutral, about 75% more neutral to about 80% more neutral, about 75% more neutral to about 85% more neutral, about 75% more neutral to about 90% more neutral, about 80% more neutral to about 85% more neutral, about 80% more neutral to about 90% more neutral, or about 85% more neutral to about 90% more neutral as compared to a constriction-forming portion of a wild-type monomer.

[0455] In some cases, a constriction-forming portion of an engineered monomer may be at least about 30% more negative, at least about 40% more negative, at least about 50% more negative, at least about 60% more negative, at least about 70% more negative, at least about 80% more negative, at least about 90% more negative, or greater than 90% more negative as compared to a constriction-forming portion of a wild-type monomer. In some cases, a constriction-forming portion of an engineered monomer may be at most about 90% more negative, at most about 80% more negative, at most about 70% more negative, at most about 60% more negative, at most about 50% more negative, at most about 40% more negative, at most about 30% more negative, or less than about 30% more negative as compared to a constriction-forming portion of a wild-type monomer. In some cases, a constriction-forming portion of an engineered monomer may be from about 20% more negative to about 90% more negative as compared to a constriction-forming portion of a wild-type monomer.

[0456] One or more mutations may be introduced to modify a charge of a first portion, second portion, third portion, or any combination thereof. In some cases, a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer. A respective portion can be an identical portion of a wild-type monomer as compared to the portion of a monomer (e.g., an engineered monomer) described herein (e.g., an engineered biological monomer). A portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) may have an amino acid composition comprising a plurality of amino acids. The plurality of amino acids may comprise one or more negatively-charged amino acids, one or more neutral amino acids, one or more positively-charged amino acids, or any combination thereof. A portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) may be modified by introducing one or more amino acid mutations to the portion (e.g., a first portion, a second portion, a third portion, or any combination thereof). In some cases, a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) may be modified by introducing one or more amino acid mutations to a monomer (e.g., a wild-type monomer) comprising a portion that corresponds to that portion. As an example, a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer by substituting one or more positively-charged amino acids and/or one or more neutral charged amino acids with one or more negatively -charged amino acids. As another example, a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer by deleting one or more positively-charged amino acids and/or one or more neutral charged amino acids. As another example, a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) may be modified to be more net negative as compared to a respective portion of a wild-type monomer by adding one or more negatively -charged amino acids to the portion. As another example, if a portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) has a net negative charge, the portion may be modified to be more net negative as compared to a respective portion of a wild-type monomer by substituting one or more positively-charged amino acids with one or more neutral charged amino acids and/or one or more negatively-charged amino acids. As another example, if the portion (e.g., a first portion, a second portion, a third portion, or any combination thereof) of a monomer (e.g., an engineered monomer) has a greater number of negatively -charged amino acid residues compared to a number of positively -charged amino acids and one or more neutral charged amino acids, the portion may still be modified to be more net negative as compared to a respective portion of a wild-type monomer by (i) substituting one or more positively-charged amino acids and/or one or more neutral charged amino acids with one or more negatively-charged amino acids; (ii) deleting one or more positively -charged amino acids and/or one or more neutral charged amino acids; (iii) adding one or more negatively -charged amino acids; (iv) or any combination thereof. One or more natural amino acids and/or non-natural amino acids may be introduced to modify the portion of a monomer (e.g., an engineered monomer) to be more net negative as compared to a respective portion of a wild-type monomer. One or more natural amino acids and/or non-natural amino acids may be deleted to modify the portion of a monomer (e.g., an engineered monomer) to be more net negative as compared to a respective portion of a wild-type monomer. [0457] In some cases, a portion of an engineered monomer that forms the channel region adjacent to the constriction region (e.g., a first portion and/or third portion of the engineered monomer) may be at least about 30% more negative, at least about 40% more negative, at least about 50% more negative, at least about 60% more negative, at least about 70% more negative, at least about 80% more negative, at least about 90% more negative, or greater than 90% more negative as compared to a portion of a wild-type monomer that forms the channel region adjacent to the constriction region of a wild-type biological nanopore. In some cases, a portion of an engineered monomer that forms the channel region adjacent to the constriction region (e.g., a first portion and/or third portion of the engineered monomer) may be at most about 90% more negative, at most about 80% more negative, at most about 70% more negative, at most about 60% more negative, at most about 50% more negative, at most about 40% more negative, at most about 30% more negative, or less than about 30% more negative as compared to a portion of a wild-type monomer that forms the channel region adjacent to the constriction region of a wild-type biological nanopore.

[0458] In some cases, a portion of an engineered monomer that forms the channel region adjacent to the constriction region (e.g., a first portion and/or third portion of the engineered monomer) may be from about 20% more negative to about 90% more negative as compared to a portion of a wild-type monomer that forms the channel region adjacent to the constriction region of a wild-type biological nanopore. In some cases, a portion of an engineered monomer that forms the channel region adjacent to the constriction region (e.g., a first portion and/or third portion of the engineered monomer) may be from about 20% more negative to about 30% more negative, about 20% more negative to about 40% more negative, about 20% more negative to about 50% more negative, about 20% more negative to about 55% more negative, about 20% more negative to about 60% more negative, about 20% more negative to about 65% more negative, about 20% more negative to about 70% more negative, about 20% more negative to about 75% more negative, about 20% more negative to about 80% more negative, about 20% more negative to about 85% more negative, about 20% more negative to about 90% more negative, about 30% more negative to about 40% more negative, about 30% more negative to about 50% more negative, about 30% more negative to about 55% more negative, about 30% more negative to about 60% more negative, about 30% more negative to about 65% more negative, about 30% more negative to about 70% more negative, about 30% more negative to about 75% more negative, about 30% more negative to about 80% more negative, about 30% more negative to about 85% more negative, about 30% more negative to about 90% more negative, about 40% more negative to about 50% more negative, about 40% more negative to about 55% more negative, about 40% more negative to about 60% more negative, about 40% more negative to about 65% more negative, about 40% more negative to about 70% more negative, about 40% more negative to about 75% more negative, about 40% more negative to about 80% more negative, about 40% more negative to about 85% more negative, about 40% more negative to about 90% more negative, about 50% more negative to about 55% more negative, about 50% more negative to about 60% more negative, about 50% more negative to about 65% more negative, about 50% more negative to about 70% more negative, about 50% more negative to about 75% more negative, about 50% more negative to about 80% more negative, about 50% more negative to about 85% more negative, about 50% more negative to about 90% more negative, about 55% more negative to about 60% more negative, about 55% more negative to about 65% more negative, about 55% more negative to about 70% more negative, about 55% more negative to about 75% more negative, about 55% more negative to about 80% more negative, about 55% more negative to about 85% more negative, about 55% more negative to about 90% more negative, about 60% more negative to about 65% more negative, about 60% more negative to about 70% more negative, about 60% more negative to about 75% more negative, about 60% more negative to about 80% more negative, about 60% more negative to about 85% more negative, about 60% more negative to about 90% more negative, about 65% more negative to about 70% more negative, about 65% more negative to about 75% more negative, about 65% more negative to about 80% more negative, about 65% more negative to about 85% more negative, about 65% more negative to about 90% more negative, about 70% more negative to about 75% more negative, about 70% more negative to about 80% more negative, about 70% more negative to about 85% more negative, about 70% more negative to about 90% more negative, about 75% more negative to about 80% more negative, about 75% more negative to about 85% more negative, about 75% more negative to about 90% more negative, about 80% more negative to about 85% more negative, about 80% more negative to about 90% more negative, or about 85% more negative to about 90% more negative as compared to a portion of a wild-type monomer that forms the channel region adjacent to the constriction region of a wild-type biological nanopore.

[0459] In some cases, an ion-selective nanopore (e.g., an ion-selective MspA, MspA paralog or homolog porin) can have a neutral constriction area. In other cases, the ion-selective nanopore (e.g., an ion-selective MspA, MspA paralog or homolog porin) can have a negatively-charged constriction area. In other cases, the ion-selective nanopore (e.g., an ion-selective MspA, MspA paralog or homolog porin) can have a neutral- charged constriction area. In other cases, the ion-selective nanopore (e.g., an ion-selective MspA, MspA paralog or homolog porin) can have a combination of neutral-charged amino acid residues and negatively- charged amino acid residues in the constriction area.

[0460] In some cases, a nanopore described herein may comprise a monomer 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 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 of a wild-type monomer originating from Mycobacterium smegmatis (e.g., MspA). In some cases, a nanopore described herein may comprise a monomer comprising an amino acid sequence with 100% sequence identity to an amino acid sequence of a wild-type monomer originating from Mycobacterium smegmatis (e.g., MspA).

[0461] In some cases, a nanopore described herein may comprise a monomer 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 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: 1. In some cases, a nanopore described herein may comprise a monomer comprising an amino acid sequence with at most about 99.9%, at most about 99.5%, at most about 99%, at most about 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 94%, at most about 93%, at most about 92%, at most about 91%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, or less than about 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a nanopore described herein comprises a monomer comprising an amino acid sequence from about 70% to about 97% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a nanopore described herein comprises a monomer comprising an amino acid sequence from about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 91%, about 70% to about 92%, about 70% to about 93%, about 70% to about 94%, about 70% to about 95%, about 70% to about 96%, about 70% to about 97%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 91%, about 75% to about 92%, about 75% to about 93%, about 75% to about 94%, about 75% to about 95%, about 75% to about 96%, about 75% to about 97%, about 80% to about 85%, about 80% to about 90%, about 80% to about 91%, about 80% to about 92%, about 80% to about 93%, about 80% to about 94%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 85% to about 90%, about 85% to about 91%, about 85% to about 92%, about 85% to about 93%, about 85% to about 94%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 90% to about 91%, about 90% to about 92%, about 90% to about 93%, about 90% to about 94%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 91% to about 92%, about 91% to about 93%, about 91% to about 94%, about 91% to about 95%, about 91% to about 96%, about 91% to about 97%, about 92% to about 93%, about 92% to about 94%, about 92% to about 95%, about 92% to about 96%, about 92% to about 97%, about 93% to about 94%, about 93% to about 95%, about 93% to about 96%, about 93% to about 97%, about 94% to about 95%, about 94% to about 96%, about 94% to about 97%, about 95% to about 96%, about 95% to about 97%, or about 96% to about 97% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a nanopore described herein comprises a monomer comprising an amino acid sequence as set forth in SEQ ID NO: 1.

[0462] In some cases, a nanopore (e.g., a biological nanopore) described herein can be an engineered biological nanopore. The nanopore may comprise one or more mutations. The mutation can comprise an insertion, a substitution, a deletion, or combinations thereof. The mutation can comprise a substitution (e.g., a change from one amino acid residue to another amino acid residue). The mutation may be in at least one monomer of the nanopore. In some cases, the nanopore comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some cases, a monomer of the engineered biological nanopore comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. In some cases, two or more monomers of the engineered biological nanopore each comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations. An engineered nanopore disclosed herein may comprise a plurality of monomers disclosed herein.

[0463] In some cases, a nanopore described herein may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitution in an amino acid sequence set forth in SEQ ID NO: 1. In some cases, a nanopore described herein may comprise 10 or fewer amino acid (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) substitutions in an amino acid sequence set forth in SEQ ID NO: 1. In some cases, a nanopore described herein may comprise about one (e.g., 0, 1) amino acid substitution in an amino acid sequence as set forth in SEQ ID NO: 1.

[0464] In some cases, a mutation may be in a constriction region of a nanopore. In some cases, a mutation may be in a channel region of the nanopore. In some cases, a nanopore can comprise one or more mutations in a constriction region, a channel region, or any combination thereof. In some cases, a nanopore described herein may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10) mutations in a constriction region. In some cases, a nanopore described herein may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10) mutations in a first region and/or third region of a channel. In some cases, a nanopore described herein may have at least one mutation in a constriction region and no mutation in a channel region. In some cases, a nanopore described herein may have at least one mutation in a channel region and no mutation in a constriction region.

[0465] In some cases, the constriction region may comprise one or more neutral charges. For example, a constriction region may comprise one or more neutral amino acid residues. A neutral amino acid residue can comprise those amino acid residues described herein. The constriction region may comprise at least one neutral amino acid residue and one or more positively -charged amino acid residues, negatively -charged amino acid residues, or any combination thereof.

[0466] In some cases, the constriction region may comprise one or more negative charges. For example, a constriction region may comprise one or more negative amino acid residues. A negative amino acid residue can comprise those amino acid residues described herein. The constriction region may comprise at least one negative amino acid residue and one or more neutral-charged amino acid residues, non-natural amino acid residues, or any combination thereof.

[0467] In some cases, the constriction region or one or more constriction-forming portions of the one or more monomers of the engineered biological nanopore may comprise at least about 1 neutral amino acid residue, at least about 2 neutral amino acid residues, at least about 3 neutral amino acid residues, at least about 4 neutral amino acid residues, at least about 5 neutral amino acid residues, at least about 6 neutral amino acid residues, at least about 7 neutral amino acid residues, at least about 8 neutral amino acid residues, at least about 9 neutral amino acid residues, at least about 10 neutral amino acid residues, at least about 15 neutral amino acid residues, at least about 20 neutral amino acid residues, or greater than about 20 neutral amino acid residues. In some cases, the constriction region or one or more constriction-forming portions of the one or more monomers of the engineered biological nanopore may comprise at most about 20 neutral amino acid residue, at most about 15 neutral amino acid residues, at most about 10 neutral amino acid residues, at most about 9 neutral amino acid residues, at most about 8 neutral amino acid residues, at most about 7 neutral amino acid residues, at most about 6 neutral amino acid residues, at most about 5 neutral amino acid residues, at most about 4 neutral amino acid residues, at most about 3 neutral amino acid residues, at most about 2 neutral amino acid residues, at most about 1 neutral amino acid residues, or less than about 1 neutral amino acid residues.

[0468] In some cases, the constriction region or one or more constriction-forming portions of the one or more monomers of the engineered biological nanopore may comprise from about 1 neutral amino acid residue to about 20 neutral amino acid residues. In some cases, the constriction region or one or more constrictionforming portions of the one or more monomers of the engineered biological nanopore may comprise from about 1 neutral amino acid residue to about 2 neutral amino acid residues, about 1 neutral amino acid residue to about 3 neutral amino acid residues, about 1 neutral amino acid residue to about 4 neutral amino acid residues, about 1 neutral amino acid residue to about 5 neutral amino acid residues, about 1 neutral amino acid residue to about 6 neutral amino acid residues, about 1 neutral amino acid residue to about 7 neutral amino acid residues, about 1 neutral amino acid residue to about 8 neutral amino acid residues, about 1 neutral amino acid residue to about 9 neutral amino acid residues, about 1 neutral amino acid residue to about 10 neutral amino acid residues, about 1 neutral amino acid residue to about 15 neutral amino acid residues, about 1 neutral amino acid residue to about 20 neutral amino acid residues, about 2 neutral amino acid residues to about 3 neutral amino acid residues, about 2 neutral amino acid residues to about 4 neutral amino acid residues, about

2 neutral amino acid residues to about 5 neutral amino acid residues, about 2 neutral amino acid residues to about 6 neutral amino acid residues, about 2 neutral amino acid residues to about 7 neutral amino acid residues, about 2 neutral amino acid residues to about 8 neutral amino acid residues, about 2 neutral amino acid residues to about 9 neutral amino acid residues, about 2 neutral amino acid residues to about 10 neutral amino acid residues, about 2 neutral amino acid residues to about 15 neutral amino acid residues, about 2 neutral amino acid residues to about 20 neutral amino acid residues, about 3 neutral amino acid residues to about 4 neutral amino acid residues, about 3 neutral amino acid residues to about 5 neutral amino acid residues, about 3 neutral amino acid residues to about 6 neutral amino acid residues, about 3 neutral amino acid residues to about 7 neutral amino acid residues, about 3 neutral amino acid residues to about 8 neutral amino acid residues, about

3 neutral amino acid residues to about 9 neutral amino acid residues, about 3 neutral amino acid residues to about 10 neutral amino acid residues, about 3 neutral amino acid residues to about 15 neutral amino acid residues, about 3 neutral amino acid residues to about 20 neutral amino acid residues, about 4 neutral amino acid residues to about 5 neutral amino acid residues, about 4 neutral amino acid residues to about 6 neutral amino acid residues, about 4 neutral amino acid residues to about 7 neutral amino acid residues, about 4 neutral amino acid residues to about 8 neutral amino acid residues, about 4 neutral amino acid residues to about 9 neutral amino acid residues, about 4 neutral amino acid residues to about 10 neutral amino acid residues, about 4 neutral amino acid residues to about 15 neutral amino acid residues, about 4 neutral amino acid residues to about 20 neutral amino acid residues, about 5 neutral amino acid residues to about 6 neutral amino acid residues, about 5 neutral amino acid residues to about 7 neutral amino acid residues, about 5 neutral amino acid residues to about 8 neutral amino acid residues, about 5 neutral amino acid residues to about 9 neutral amino acid residues, about 5 neutral amino acid residues to about 10 neutral amino acid residues, about 5 neutral amino acid residues to about 15 neutral amino acid residues, about 5 neutral amino acid residues to about 20 neutral amino acid residues, about 6 neutral amino acid residues to about 7 neutral amino acid residues, about 6 neutral amino acid residues to about 8 neutral amino acid residues, about 6 neutral amino acid residues to about 9 neutral amino acid residues, about 6 neutral amino acid residues to about 10 neutral amino acid residues, about 6 neutral amino acid residues to about 15 neutral amino acid residues, about 6 neutral amino acid residues to about 20 neutral amino acid residues, about 7 neutral amino acid residues to about 8 neutral amino acid residues, about 7 neutral amino acid residues to about 9 neutral amino acid residues, about 7 neutral amino acid residues to about 10 neutral amino acid residues, about 7 neutral amino acid residues to about 15 neutral amino acid residues, about 7 neutral amino acid residues to about 20 neutral amino acid residues, about 8 neutral amino acid residues to about 9 neutral amino acid residues, about 8 neutral amino acid residues to about 10 neutral amino acid residues, about 8 neutral amino acid residues to about 15 neutral amino acid residues, about 8 neutral amino acid residues to about 20 neutral amino acid residues, about 9 neutral amino acid residues to about 10 neutral amino acid residues, about 9 neutral amino acid residues to about 15 neutral amino acid residues, about 9 neutral amino acid residues to about 20 neutral amino acid residues, about 10 neutral amino acid residues to about 15 neutral amino acid residues, about 10 neutral amino acid residues to about 20 neutral amino acid residues, or about 15 neutral amino acid residues to about 20 neutral amino acid residues.

[0469] In some cases, the constriction region or one or more constriction-forming portions of the one or more monomers of the engineered biological nanopore may comprise at least about 1 negatively-charged amino acid residue, at least about 2 negatively -charged amino acid residues, at least about 3 negatively-charged amino acid residues, at least about 4 negatively-charged amino acid residues, at least about 5 negatively -charged amino acid residues, at least about 6 negatively-charged amino acid residues, at least about 7 negatively- charged amino acid residues, at least about 8 negatively-charged amino acid residues, at least about 9 negatively-charged amino acid residues, at least about 10 negatively-charged amino acid residues, at least about 15 negatively-charged amino acid residues, at least about 20 negatively-charged amino acid residues, or greater than about 20 negatively-charged amino acid residues. In some cases, the constriction region or one or more constriction-forming portions of the one or more monomers of the engineered biological nanopore may comprise at most about 20 negatively -charged amino acid residue, at most about 15 negatively-charged amino acid residues, at most about 10 negatively-charged amino acid residues, at most about 9 negatively -charged amino acid residues, at most about 8 negatively -charged amino acid residues, at most about 7 negatively- charged amino acid residues, at most about 6 negatively-charged amino acid residues, at most about 5 negatively-charged amino acid residues, at most about 4 negatively -charged amino acid residues, at most about 3 negatively-charged amino acid residues, at most about 2 negatively-charged amino acid residues, at most about 1 negatively-charged amino acid residues, or less than about 1 negatively-charged amino acid residues. [0470] There may be a distance between an amino acid (e.g., a modified amino acid) in one region of a nanopore described herein (e.g., an engineered biological nanopore) and another amino acid (another modified amino acid) in another region of the nanopore. For example, a mutated amino acid in a first region may be separated by a distance from a mutated amino acid in a second region. In some cases, an amino acid (e.g., a modified amino acid) in one region of a nanopore described herein (e.g., an engineered biological nanopore) may be separated by a distance from another amino acid (another modified amino acid) in the same region. As another example, a mutated amino acid in a first region may be separated by a distance from another mutated amino acid in the first region.

[0471] A mutated amino acid in a first region or third region may be at most about 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4.5 nm, 4 nm, 3.5 nm, 3 nm, 2.5 nm, 2 nm, 1.5 nm, 1 nm, 0.5 nm, or less than about 0.5 nm away from a mutated amino acid in a second region. A mutated amino acid in a first region or third region may be at least about 0.5 nm, 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, or greater than about 10 nm away from a mutated amino acid in a second region. In some cases, a mutated amino acid in a first region or third region may be separated by a distance from a mutated amino acid in a narrowest region of the nanopore. The narrowest region of the nanopore may be in a second region. The narrowest region of the nanopore may comprise a dimension (e.g., a C(alpha)-C(alpha) diameter) of at most about 10 nm, 5 nm, 4.5 nm, 4 nm, 3.5 nm, 3 nm, 2.5 nm, 2 nm, 1.5 nm, 1 nm, 0.5 nm, or less than about 0.5 nm.

[0472] As described herein, a first region can comprise a first ring of charge. The first ring of charge may be formed by an assembly of monomers of the nanopore. An assembly of first portions of a plurality of monomers may form a first region in which there may be a first ring of charge. In some cases, the first ring of charge can comprise at least one mutated amino acid residue. The mutated amino acid residue in the first ring of charge may be separated by a distance from another amino acid residue (e.g., mutated amino acid residue) in another region (e.g., a second region). For example, the first ring of charge comprising a mutated amino acid in the first region may be at most about 15 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4.5 nm, 4 nm, 3.5 nm, 3 nm, 2.5 nm, 2 nm, 1.5 nm, 1 nm, 0.5 nm, or less than about 0.5 nm away from a mutated amino acid in a second region. As another example, the first ring of charge comprising a mutated amino acid in the first region may be at least about 0.5 nm, 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, or greater than about 15 nm away from a mutated amino acid in a second region.

[0473] In some cases, the second region can comprise a second ring of charge. An assembly of second portions of a plurality of monomers may form a second region in which there may be a second ring of charge. In some cases, the second ring of charge can comprise at least one mutated amino acid residue. The mutated amino acid residue in the first ring of charge may be separated by a distance from another amino acid residue (e.g., mutated amino acid residue) in a second ring of charge (e.g., a ring of charge in the second region). For example, the first ring of charge comprising a mutated amino acid in the first region may be at most about 15 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4.5 nm, 4 nm, 3.5 nm, 3 nm, 2.5 nm, 2 nm, 1.5 nm, 1 nm, 0.5 nm, or less than about 0.5 nm away from a second ring of charge comprising a mutated amino acid. As another example, the first ring of charge comprising a mutated amino acid in the first region may be at least about 0.5 nm, 1 nm, 1.5 nm, 2 nm, 2.5 nm, 3 nm, 3.5 nm, 4 nm, 4.5 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 15 nm, or greater than about 15 nm away from a second ring of charge comprising a mutated amino acid.

[0474] The nanopore may comprise a distance between a negatively charged amino acid residue (e.g., the negatively charged amino acid residue of the first region and/or third region of the channel) and a neutrally charged amino acid residue (e.g., the neutrally charged amino acid residue of the second region of the channel) and/or negatively-charged amino acid residue (e.g., the negatively charged amino acid residue of the second region of the channel). The distance may be between two points of a monomer of the engineered biological nanopore. In some cases, (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue of the first portion and/or third portion and the neutrally charged amino acid residue of the second portion; and/or (iii) a distance (e.g., smallest distance) between the negatively charged amino acid residue of the first portion and/or third portion and the negatively charged amino acid residue of the second portion may be at least about 0. 1 nm, at least about 0.5 nm, at least about 1.0 nm, at least about 1.5 nm, at least about 2.0 nm, at least about 2.5 nm, at least about 3.0 nm, at least about 3.5 nm, at least about 4.0 nm, at least about 4.5 nm, at least about 5.0 nm, at least about 6.0 nm, at least about 7.0 nm, at least about 8.0 nm, or greater than about 8.0 nm. In some cases, (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue of the first portion and the neutrally charged amino acid residue of the second portion; and/or (iii) a distance (e.g., smallest distance) between the negatively charged amino acid residue of the first portion and/or third portion and the negatively charged amino acid residue of the second portion may be at most about 8.0 nm, at most about 7.0 nm, at most about 6.0 nm, 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 rim, at most about 2.0 run, at most about 1.5 run, at most about 1.0 run, at most about 0.5 run, at most about 0. 1 run, or less than about 0. 1 run.

[0475] In some cases, (i) the distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) the distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be from about 0. 1 nm to about 8 nm. In some cases, (i) the distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) the distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be from about 0. 1 nm to about 0.5 nm, about 0. 1 nm to about 1 nm, about 0. 1 nm to about

1.5 nm, about 0. 1 nm to about 2 nm, about 0. 1 nm to about 2.5 nm, about 0. 1 nm to about 3 nm, about 0. 1 nm to about 3.5 nm, about 0. 1 nm to about 4 nm, about 0. 1 nm to about 4.5 nm, about 0. 1 nm to about 5 nm, about 0. 1 nm to about 8 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 4.5 nm, about 0.5 nm to about 5 nm, about 0.5 nm to about 8 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 4.5 nm, about 1 nm to about 5 nm, about 1 nm to about 8 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 4.5 nm, about 1.5 nm to about 5 nm, about 1.5 nm to about 8 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 4.5 nm, about 2 nm to about 5 nm, about 2 nm to about 8 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 4.5 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about 8 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about 4.5 nm, about 3 nm to about 5 nm, about 3 nm to about 8 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 4.5 nm, about 3.5 nm to about 5 nm, about 3.5 nm to about 8 nm, about 4 nm to about 4.5 nm, about 4 nm to about 5 nm, about 4 nm to about 8 nm, about 4.5 nm to about 5 nm, about 4.5 nm to about 8 nm, or about 5 nm to about 8 nm.

[0476] In some cases, the distance comprising (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be at least about 0. 1 A, at least about 0.5 A, at least about 1 A, at least about 5 A, at least about 10 A, at least about 15 A, at least about 20 A, at least about 25 A, at least about 30 A, at least about 35 A, at least about 40 A, at least about 45 A, at least about 50 A, or greater than about 50 A. In some cases, the distance comprising (i) a distance (e.g., smallest distance) between a mutated residue in a first portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be at most about 50 A, 45 A, 40 A, 35 A, 30 A, 25 A, 20 A, 15 A, 10 A, 5 A, 1 A, 0.5 A, 0. 1 A, or less than about 0. 1 A.

[0477] In some cases, the distance comprising (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue of the first portion and/or third portion and the negatively charged amino acid residue of the constriction region of the nanopore may be at least about 0. 1 A, at least about 0.5 A, at least about 1 A, at least about 5 A, at least about 10 A, at least about 15 A, at least about 20 A, at least about 25 A, at least about 30 A, at least about 35 A, at least about 40 A, at least about 45 A, at least about 50 A, or greater than about 50 A. In some cases, the distance comprising (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be at most about 50 A, 45 A, 40 A, 35 A, 30 A, 25 A, 20 A, 15 A, 10 A, 5 A, 1 A, 0.5 A, 0. 1 A, or less than about 0. 1 A.

[0478] In some cases, (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be from about 0. 1 A to about 100 A. In some cases, (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be from at most about 100 A. In some cases, (i) a distance (e.g., smallest distance) between a mutated residue in a first portion and/or third portion of the monomer and another mutated residue of the second portion of the monomer; or (ii) a distance (e.g., smallest distance) between the negatively charged amino acid residue and the neutrally charged amino acid residue of the nanopore may be from about 0. 1 A to about 0.5 A, about 0. 1 A to about 1 A, about 0. 1 A to about 5 A, about 0. 1 A to about 10 A, about 0. 1 A to about 15 A, about 0. 1 A to about 20 A, about 0. 1 A to about 25 A, about 0. 1 A to about 30 A, about 0. 1 A to about 40 A, about 0. 1 A to about 50 A, about 0. 1 A to about 100 A, about 0.5 A to about 1 A, about 0.5 A to about 5 A, about 0.5 A to about 10 A, about 0.5 A to about 15 A, about 0.5 A to about 20 A, about 0.5 A to about 25 A, about 0.5 A to about 30 A, about 0.5 A to about 40 A, about 0.5 A to about 50 A, about 0.5 A to about 100 A, about 1 A to about 5 A, about 1 A to about 10 A, about 1 A to about 15 A, about 1 A to about 20 A, about 1 A to about 25 A, about 1 A to about 30 A, about 1 A to about 40 A, about 1 A to about 50 A, about 1 A to about 100 A, about 5 A to about 10 A, about 5 A to about 15 A, about 5 A to about 20 A, about 5 A to about 25 A, about 5 A to about 30 A, about 5 A to about 40 A, about 5 A to about 50 A, about 5 A to about 100 A, about 10 A to about 15 A, about 10 A to about 20 A, about 10 A to about 25 A, about 10 A to about 30 A, about 10 A to about 40 A, about 10 A to about 50 A, about 10 A to about 100 A, about 15 A to about 20 A, about 15 A to about 25 A, about 15 A to about 30 A, about 15 A to about 40 A, about 15 A to about 50 A, about 15 A to about 100 A, about 20 A to about 25 A, about 20 A to about 30 A, about 20 A to about 40 A, about 20 A to about 50 A, about 20 A to about 100 A, about 25 A to about 30 A, about 25 A to about 40 A, about 25 A to about 50 A, about 25 A to about 100 A, about 30 A to about 40 A, about 30 A to about 50 A, about 30 A to about 100 A, about 40 A to about 50 A, about 40 A to about 100 A, or about 50 A to about 100 A.

[0479] In some cases, a mutated amino acid in a first portion or third portion may be at most about 15 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or less than about 1 nm away from at least one mutated amino acid in the second portion. In some cases, a mutated amino acid in a first portion or third portion may be at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm away from at least one mutated amino acid in the second portion. In some cases, at least one mutated amino acid (e.g., in a ring of charge) in the first portion or third portion may be at most at most about 15 nm, at most about 10 nm, at most about 9 nm, at most about 8 nm, at most about 7 nm, at most about 6 nm, at most about 5.5 nm, at most about 5 nm, at most about 4.5 nm, at most about 4 nm, at most about 3.5 nm, at most about 3 nm, at most about 2.5 nm, at most about 2 nm, at most about 1.5 nm, at most about 1 nm, or less than about 1 nm away from the at least one mutated amino acid (e.g., in a second ring of charge) in the second portion. In some cases, at least one mutated amino acid (e.g., in a ring of charge) in the first portion or third portion may be at least about 1 nm, at least about 1.5 nm, at least about 2 nm, at least about 2.5 nm, at least about 3 nm, at least about 3.5 nm, at least about 4 nm, at least about 4.5 nm, at least about 5 nm, at least about 5.5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 15 nm, or greater than about 15 nm away from the at least one mutated amino acid (e.g., in a second ring of charge) in the second portion. [0480] As shown in FIG. IB, mutated amino acid residues may be at a number of locations in an engineered biological nanopore (e.g., an engineered monomer) as described herein. One or more mutated amino acid residues may be at a first region adjacent to a constriction (104). One or more mutated amino acid residues may be at another region adjacent to a constriction (106). One or more mutated amino acid residues may be at a constriction region (105). In some cases, an engineered biological nanopore (e.g., an engineered monomer of the biological nanopore) may comprise one or more amino acid residues at a first region adjacent to a constriction (104), at another region adjacent to a constriction (106), at a constriction region (105), or any combination thereof. An area of charge may be above a constriction region (104) which may be closer to a first entrance of a nanopore (101). An area of charge may be within a constriction region (105). An area of charge may be below a constriction region (106) which may be closer to a second entrance of a nanopore (102). The nanopore can have a first entrance (101) with a diameter (107) and a second entrance (102) with a diameter (108). The constriction region has a diameter (109).

[0481] A mutation of a nanopore described herein can comprise a substitution of one amino acid for another amino acid. In some cases, the mutation can comprise a substitution of a non-neutral amino acid residue (e.g., a negatively charged amino acid residue) for a neutral amino acid residue. In some cases, the constriction region comprises a mutation of a non-neutral amino acid residue (e.g., a negatively charged amino acid residue) for a neutral amino acid residue. In some cases, the constriction region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10) mutations of non-neutral amino acid residues (e.g., negatively charged amino acid residues) for neutral amino acid residues. A neutral net charge of the constriction region may result from one or more mutations of non-neutral amino acid residues (e.g., positively charged amino acid residues or negatively charged amino acid residues) for neutral amino acid residues. For example, mutation of a non- neutral amino acid residue (e.g., a positively charged amino acid residue or a negatively charged amino acid residue) to a neutral amino acid residue may decrease a net positive charge or a net negative charge of a constriction region. In some cases, the mutation can comprise a substitution of a negatively-charged amino acid residue for a neutral amino acid residue and/or positively -charged amino acid residue. In some cases, the constriction region comprises a mutation of a neutral amino acid residue and/or a positively charged amino acid residue for a negatively-charged amino acid residue. In some cases, the constriction region comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10) insertions or substitutions of negatively- charged amino acid. A negative net charge of the constriction region may result from one or more mutations of negatively-charged amino acid residues for neutral amino acid residues and/or positively-charged amino acid residues. For example, insertion of a negatively-charged amino acid residue and/or substitution of a negatively-charged amino acid residue for a neutral amino acid residue and/or positive amino acid residue may increase a net negative charge of a constriction region.

[0482] In some cases, a nanopore (e.g., an engineered EOF nanopore) described herein may comprise an aspartic acid (D) residue mutated to an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine (Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline (P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue. In some cases, a nanopore (e.g., an engineered EOF nanopore) described herein may comprise a glutamate (glutamic acid) (E) residue mutated to an alanine (A) residue, an asparagine (N) residue, a cysteine (C) residue, a glutamine (Q) residue, a glycine (G) residue, an isoleucine (I) residue, a leucine (L) residue, a methionine (M) residue, a phenylalanine (F) residue, a proline (P) residue, a serine (S) residue, a threonine (T) residue, a tryptophan (W) residue, a tyrosine (Y) residue, or a valine (V) residue. [0483] In some cases, a nanopore (e.g., engineered biological nanopore) described herein may comprise a non-natural amino acid. The engineered biological nanopore may comprise one or more amino acid analogs, one or more amino acid mimetics that function in a manner similar to the naturally occurring amino acids, or any combination thereof. In some cases, the nanopore may be comprised of a protein (e.g., monomer) as set forth in Table 1.

Table 1. Sequences of monomers.

[0484] In Table 1, the underlined residues of SEQ ID NO: 1 designate D90, D91, and D93. The underlined residues of SEQ ID NO: 4 designate Y51, N55, and F56. The underlined residues of SEQ ID NO: 5 designate N15, N17, A20, L23, and N24.

[0485] In some cases, an engineered biological nanopore may be a cation-selective nanopore. The engineered monomer of the engineered biological nanopore can comprise a negative charge at one or more amino acid residues in the amino acid resides at positions 82-91, 103-111, or any combinations thereof within those ranges as according to the wild-type amino acid sequence as set forth in SEQ ID NO: 1. The engineered monomer of the engineered biological nanopore can comprise one or more negatively-charged amino acid residues in the amino acid resides at positions 82-91, 103-111, or any combinations thereof within those ranges as according to the wild-type amino acid sequence as set forth in SEQ ID NO: 1. The engineered monomer of the engineered biological nanopore can comprise one or more neutrally -charged amino acid residues in the amino acid resides at positions 82-91, 103-111, or any combinations thereof within those ranges as according to the wild-type amino acid sequence as set forth in SEQ ID NO: 1. FIG. 2A shows an example of a MspA nanopore comprising a constriction region and two channel regions on either side of the constriction. The MspA pore can be an octameric nanopore (e.g., comprising 8 monomers). As shown in FIG. 2B, a MspA monomer can comprise amino acid residues at positions 83, 88, 90, 91, 105, 108, or any combination thereof. The wild-type MspA monomer can comprise amino acid residues threonine at position 83 (T83), leucine at position 88 (L88), aspartic acid at position 90 (D90), aspartic acid at position 91 (D91), serine at position 103 (S103), isoleucine at position 105 (1105), asparagine at position 108 (N108), or any combination thereof. In some cases, each monomer of the MspA nanopore may comprise those amino acid residues.

[0486] In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position 90, 91, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position D90 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position D90 and D91 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. Positions D90, D91, or any combination thereof may be located at a second region of the channel (e.g., the constriction region).

[0487] In some cases, the engineered monomer of the engineered biological nanopore described herein comprises a mutation (e.g., a substitution mutation) at position D90 to a neutral amino acid residue (an alanine residue, an asparagine residue, a cysteine residue, a glutamine residue, a glycine residue, an isoleucine residue, a leucine residue, a methionine residue, a phenylalanine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue, a valine residue, or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises a mutation (e.g., a substitution mutation) at position D90 to a neutral amino acid residue, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises a mutation (e.g., a substitution mutation) at position D90 to an amidic amino acid residue (e.g., an asparagine residue, a glutamine residue, or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises 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: 1.

[0488] In some cases, the engineered monomer of the engineered biological nanopore described herein comprises a mutation (e.g., a substitution mutation) at position D91 to a neutral amino acid residue (an alanine residue, an asparagine residue, a cysteine residue, a glutamine residue, a glycine residue, an isoleucine residue, a leucine residue, a methionine residue, a phenylalanine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, a tyrosine residue, a valine residue, or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises a mutation (e.g., a substitution mutation) at position D91 to a neutral amino acid residue, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises a mutation (e.g., a substitution mutation) at position D91 to an amidic amino acid residue (e.g., an asparagine residue, a glutamine residue, or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises a mutation (e.g., a substitution mutation) at position D91 to an asparagine residue, wherein the mutation comprises D9 IN and wherein the residue numbering corresponds to SEQ ID NO: 1.

[0489] In some cases, the engineered monomer of the engineered biological nanopore described herein comprises two or more mutations (e.g., two or more substitution mutations) at positions D90 and D91 to neutral amino acid residues (alanine residues, asparagine residues, cysteine residues, glutamine residues, glycine residues, isoleucine residues, leucine residues, methionine residues, phenylalanine residues, proline residues, serine residues, threonine residues, tryptophan residues, tyrosine residues, valine residues, or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises two or more mutations (e.g., two or more substitution mutations) at positions D90 and D91 to neutral amino acid residues, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises two or more mutations (e.g., two or more substitution mutations) at positions D90 and D91 to amidic amino acid residues (e.g., asparagine residues, glutamine residues, or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the second region of the channel (e.g., the constriction region) comprises two or more mutations (e.g., two or more substitution mutations) at positions D90 and D91 to asparagine residues, wherein the mutations comprise D90N and D91N and wherein the residue numbering corresponds to SEQ ID NO: 1.

[0490] In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position 83, 88, 103, 105, 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position S103 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position 1105 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position N 108 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position T83 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position L88 of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at position 103, 1105, N108, T83, L88, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. Position S103, 1105, N108, T83, L88, or any combination thereof may be located at a first region of the channel of the nanopore (e.g., engineered biological nanopore) described herein. In some cases, S103 can be located at a third region of the channel of the nanopore (e.g., engineered biological nanopore) described herein.

[0491] In some cases, the engineered monomer of the engineered biological nanopore described herein comprises a mutation (e.g., a substitution mutation) in one or more monomers at position S103, 1105, N108, T83, L88, or any combination thereof, to a negatively charged amino acid residue (e.g., an aspartic acid (D) residue, a glutamic acid residue (E), or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the first region of the channel comprises a mutation (e.g., a substitution mutation) in one or more monomers at position S103, 1105, N108, T83, L88, or any combination thereof, to a negatively charged amino acid residue, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the first region of the channel comprises a mutation (e.g., a substitution mutation) in one or more monomers at position S 103 to a glutamic acid residue, wherein the mutation comprises S 103E and wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the first region of the channel comprises a mutation (e.g., a substitution mutation) in one or more monomers at position 1105 to a glutamic acid residue, wherein the mutation comprises I105E and wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the first region of the channel comprises a mutation (e.g., a substitution mutation) in one or more monomers at position N 108 to a glutamic acid residue, wherein the mutation comprises N 108E and wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the first region of the channel comprises a mutation (e.g., a substitution mutation) in one or more monomers at position T83 to a glutamic acid residue, wherein the mutation comprises T83E and wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the first region of the channel comprises a mutation (e.g., a substitution mutation) in one or more monomers at position L88 to a glutamic acid residue, wherein the mutation comprises L88E and wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, the first region of the channel comprises a mutation (e.g., a substitution mutation) in one or more monomers, wherein the mutation comprises S103E, I105E, N108E, L88E, T83E, or any combination thereof, and wherein the residue numbering corresponds to SEQ ID NO: 1.

[0492] In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position 83, 88, 90, 91, 103, 105, 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at position 90 and position 83, 88, 103, 105, 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at position 91 and position 83, 88, 103, 105, 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 1. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise mutations at positions 90 and 91, and a mutation at position 83, 88, 103, 105, 108, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 1.

[0493] In some cases, an engineered monomer of the engineered biological nanopore described herein comprises a mutation at a first region and/or third region of a channel comprising a mutation of one or more monomers at position T83, L88, S103, 1105, N108, or any combination thereof, and a mutation at a second region of a channel (e.g., a constriction region) comprising a mutation of one or more monomers at position D90, D91, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, an engineered monomer of the engineered biological nanopore described herein comprises a mutation at a first region and/or third region of a channel comprising a mutation of one or more monomers at position T83E, L88E, S 103E, I105E, N108E, or any combination thereof, and a mutation at a second region of a channel (e.g., a constriction region) comprising a mutation of one or more monomers at position D90N, D91N, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, an engineered monomer of the engineered biological nanopore described herein comprises a mutation at a first region and/or third region of a channel (e.g., a first portion and/or third portion of a monomer) comprising S 103E and a mutation of one or more monomers at position D90N, D9 IN, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, an engineered monomer of the engineered biological nanopore described herein comprises a mutation at a first region and/or third region of a channel (e.g., a first portion and/or third portion of a monomer) comprising I105E and a mutation of one or more monomers at position D90N, D91N, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, an engineered monomer of the engineered biological nanopore described herein comprises a mutation at a first region and/or third region of a channel (e.g., a first portion and/or third portion of a monomer) comprising N108E and a mutation of one or more monomers at position D90N, D91N, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, an engineered monomer of the engineered biological nanopore described herein comprises a mutation at a first region and/or third region of a channel comprising T83E and a mutation of one or more monomers at position D90N, D91N, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, an engineered monomer of the engineered biological nanopore described herein comprises a mutation at a first region and/or third region of a channel (e.g., a first portion and/or third portion of a monomer) comprising L88E and a mutation of one or more monomers at position D90N, D91N, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 1. In some cases, an engineered monomer of the engineered biological nanopore described herein may comprise mutations at positions D90, D91, T83, L88, S 103, 1105, N108, or any combination thereof. In some cases, an engineered monomer of the engineered biological nanopore described herein may comprise mutations at positions D90, D91, D93, T83, L88, S103, 1105, N108, or any combination thereof. In some cases, an engineered monomer of the engineered biological nanopore described herein may comprise mutations at positions D90, D91, D93, A96, T83, L88, S103, 1105, N108, or any combination thereof.

[0494] In some cases, an engineered monomer of the engineered biological nanopore may be a cationselective nanopore. An engineered monomer of a engineered biological nanopore described herein can comprise a negative charge, neutral charge, or any combination thereof at one or more amino acid residues in the amino acid resides at positions 39-69, or any combinations thereof within those ranges as according to the wild-type amino acid sequence as set forth in SEQ ID NO: 4. An engineered monomer of a engineered biological nanopore described herein can comprise one or more negatively -charged amino acid residues in the amino acid resides at positions 39-69, or any combinations thereof within those ranges as according to the wild-type amino acid sequence as set forth in SEQ ID NO: 4. An engineered monomer of a engineered biological nanopore described herein can comprise one or more neutrally-charged amino acid residues in the amino acid resides at positions 39-69, or any combinations thereof within those ranges as according to the wild-type amino acid sequence as set forth in SEQ ID NO: 4. An engineered monomer of a engineered biological nanopore described herein can comprise a negative charge, neutral charge, or any combination thereof at one or more amino acid residues in the amino acid resides at positions 15-27, or any combinations thereof within those ranges as according to the wild -type amino acid sequence as set forth in SEQ ID NO: 5. An engineered monomer of a engineered biological nanopore described herein can comprise one or more negatively-charged amino acid residues in the amino acid resides at positions 15-27, or any combinations thereof within those ranges as according to the wild -type amino acid sequence as set forth in SEQ ID NO: 5. An engineered monomer of a engineered biological nanopore described herein can comprise one or more neutrally -charged amino acid residues in the amino acid resides at positions 15-27, or any combinations thereof within those ranges as according to the wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0495] FIG. 4 shows an example of a CsgG pore comprising a constriction region and two channel regions on either side of the constriction. The CsgG pore can be an oligomeric nanopore (e.g., comprising two or more monomers). The CsgG pore may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more monomers. FIG. 5 shows an example of a CsgG/F pore. The CsgG/F pore may comprise a CsgG nanopore with a channel region (501) and constriction region (502). The pore may further comprise a CsgF protein (e.g., a CsgF peptide adapter). The CsgF peptide adapter (503) may be at a second entrance (e.g., a trans entrance of the CsgG pore. Addition of the CsgF peptide adapter may create another channel (504). The CsgF peptide adapter (503) can comprise CsgF proteins. In some cases, CsgG may be a primary nanopore embedded in a membrane and CsgF may be a partner accessory protein and/or peptide (e.g., a peptide adapter). For example, the CsgF may dock with the CsgG pore and insert inside an entrance (e.g., a trans entrance) of the channel. [0496] A CsgG monomer can comprise amino acid residues at position 39, 40, 42, 43, 44, 45, 48, 51, 55, 56, 58, 62, 65, 69, or any combinations thereof. The wild-type CsgG monomer can comprise amino acid residues tyrosine at position 39 (Y39), asparagine at position 40 (N40), glutamine at position 42 (Q42), aspartic acid at position 43 (D43), glutamic acid at position 44 (E44), phenylalanine at position 48 (F48), tyrosine at position 51 (Y51), asparagine at position 55 (N55), phenylalanine at position 56 (F56), threonine at position 58 (T58), glutamine at position 62 (Q62), threonine at position 65 (T65), valine at position 69 (V69), or any combination thereof. In some cases, each monomer of the CsgG nanopore may comprise those amino acid residues. In some cases, each monomer of the CsgG nanopore may not comprise those amino acid residues. Positions Y51, N55, F56, or any combination thereof may be at a constriction-forming portion of a CsgG monomer. A CsgF protein (e.g., a CsgF proteinaceous adapter protein) may comprise amino acid residues at position 15, 17, 20, 23, 24, 1 , or any combinations thereof. The wild-type CsgF protein can comprise amino acid residues asparagine at position 15 (N15), asparagine at position 17 (N17), alanine at position 20 (A20), leucine at position 23 (L23), asparagine at position 24 (N24), glutamine at position 1 (Q27), or any combination thereof.

[0497] In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position 39, 40, 42, 43, 44, 45, 48, 51, 55, 56, 58, 62, 65, 69, or any combinations thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some cases, a monomer of a nanopore (e.g., engineered biological nanopore) described herein may comprise a mutation at a position 15, 17, 20, 23, 24, l, or any combinations thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5.

[0498] In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation (e.g., a substitution, insertion, deletion, or any combination thereof) at a position Y51, N55, F56, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. Positions Y51, N55, F56, or any combination thereof may be located at a second region of the channel of the engineered biological nanopore (e.g., at a constriction-forming portion of the monomer). In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation at position Y51, N55, F56, F48, F58, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 4. In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation (e.g., a substitution mutation) at position Y51, N55, F56, F48, F58, or any combination thereof, to a negatively charged amino acid residue (e.g., an aspartic acid (D) residue, a glutamic acid residue (E), or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation (e.g., a substitution mutation) at position Y51, N55, F56, F48, F58, or any combination thereof, to a neutrally-charged amino acid residue, wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, the engineered monomer may comprise a mutation (e.g., insertion, substitution, deletion, or any combination thereof) at position Y51, N55, F56, F48, F58, or any combination thereof, to a neutrally-charged amino acid residue, negatively -charged amino acid residue, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 4.

[0499] In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position Y51 to an aspartic acid residue, an asparagine residue, or a glutamic acid residue, wherein the mutation comprises Y51N, Y51D, or Y51E, and wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position Y51 to a hydrophilic amino acid residue (e.g., an asparagine residue, glutamine residue, serine residue, or threonine residue), wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position Y51 to a neutral residue, wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position N55 to an aspartic acid residue or a glutamic acid residue, wherein the mutation comprises N55D or N55E, and wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position N55 to a hydrophilic amino acid residue (e.g., an asparagine residue, glutamine residue, serine residue, or threonine residue), wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position N55 to a neutral residue, wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position F56 to an aspartic acid residue, an asparagine residue, or a glutamic acid residue, wherein the mutation comprises F56N, F56D, or F56E, and wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position F56 to a hydrophilic amino acid residue (e.g., an asparagine residue, glutamine residue, serine residue, or threonine residue), wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position F56 to a neutral residue, wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position F48 to an aspartic acid residue or a glutamic acid residue, wherein the mutation comprises F48D or F48E, and wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position F48 to a hydrophilic amino acid residue (e.g., an asparagine residue, glutamine residue, serine residue, or threonine residue), wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position F48 to a neutral residue, wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position T58 to an aspartic acid residue or a glutamic acid residue, wherein the mutation comprises T58D or T58E, and wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position T58 to a hydrophilic amino acid residue (e.g., an asparagine residue, glutamine residue, serine residue, or threonine residue), wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position T58 to a neutral residue, wherein the residue numbering corresponds to SEQ ID NO: 4. In some cases, an engineered biological nanopore described herein comprises one or more monomers comprising any mutation or combination of mutations as set forth in Table 7.

[0500] In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation (e.g., a substitution, insertion, deletion, or any combination thereof) at a position N17, A20, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation (e.g., a substitution, insertion, deletion, or any combination thereof) at a position N15, N17, A20, L23, N24, Q27, or any combination thereof, of a wild-type amino acid sequence as set forth in SEQ ID NO: 5. In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation (e.g., a substitution mutation) at position N15, N17, A20, L23, N24, Q27, or any combination thereof, to a negatively charged amino acid residue (e.g., an aspartic acid (D) residue, a glutamic acid residue (E), or any combination thereof), wherein the residue numbering corresponds to SEQ ID NO: 5. In some cases, a monomer of a nanopore (e.g., an engineered monomer of an engineered biological nanopore) described herein may comprise a mutation (e.g., a substitution mutation) at position N15, N17, A20, L23, N24, Q27, or any combination thereof, to a neutrally-charged amino acid residue, wherein the residue numbering corresponds to SEQ ID NO: 5. In some cases, the engineered monomer may comprise a mutation (e.g., insertion, substitution, deletion, or any combination thereof) at position N15, N17, A20, L23, N24, Q27, or any combination thereof, to a neutrally -charged amino acid residue, negatively-charged amino acid residue, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 5.

[0501] In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position N15 to an aspartic acid residue, wherein the mutation comprises N15D, and wherein the residue numbering corresponds to SEQ ID NO: 5. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position N17 to an aspartic acid residue, wherein the mutation comprises N17D, and wherein the residue numbering corresponds to SEQ ID NO: 5. In some cases, an engineered biological nanopore described herein may comprise a mutation (e.g., a substitution mutation) at one or more monomers at position A20 to an aspartic acid residue, wherein the mutation comprises A20D, and wherein the residue numbering corresponds to SEQ ID NO: 5.

[0502] In some cases, an engineered biological nanopore described herein may comprise a protein comprising an amino acid sequence as set forth in SEQ ID NO: 4 and/or a protein comprising an amino acid sequence as set forth in SEQ ID NO: 5. In some cases, an engineered biological nanopore described herein may comprise a protein comprising an amino acid sequence as set forth in SEQ ID NO: 4 and/or a protein comprising one or more amino acid mutations in an amino acid sequence as set forth in SEQ ID NO: 5. In some cases, an engineered biological nanopore described herein may comprise a protein comprising one or more amino acid mutations in an amino acid sequence as set forth in SEQ ID NO: 4 and/or a protein comprising an amino acid sequence as set forth in SEQ ID NO: 5. In some cases, an engineered biological nanopore described herein may comprise a protein comprising one or more amino acid mutations an amino acid sequence as set forth in SEQ ID NO: 4 and/or a protein comprising one or more amino acid mutation in an amino acid sequence as set forth in SEQ ID NO: 5. In some cases, an engineered biological nanopore described herein may comprise a protein comprising one or more mutations at position Y51, N55, F56, F48, F58, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 4, and/or a protein comprising one or more mutations at position N15, N17, A20, L23, N24, Q27, or any combination thereof, wherein the residue numbering corresponds to SEQ ID NO: 5. In some cases, an engineered biological nanopore described herein comprises one or more monomers comprising any mutation or combination of mutations as set forth in T able 8.

[0503] In some cases, (i) a first portion and/or a third portion of an engineered monomer described herein, or (ii) a first region (e.g., adjacent to the constriction region) and/or a third region (e.g., adjacent to the constriction region) of the engineered biological nanopore may comprise at least about 1, 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, or greater than about 15 amino acid mutations. In some cases, (i) a first portion and/or a third portion of an engineered monomer described herein, or (ii) a first region (e.g., adjacent to the constriction region) and/or a third region (e.g., adjacent to the constriction region) of the engineered biological nanopore may comprise 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, at most about 1, or less than about 1 amino acid mutations. In some cases, (i) a first portion and/or a third portion of an engineered monomer described herein, or (ii) a first region (e.g., adjacent to the constriction region) and/or a third region (e.g., adjacent to the constriction region) of the engineered biological nanopore may comprise at least about 1, 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, or greater than about 15 amino acid mutations to negatively- charged amino acid residues. In some cases, , (i) a first portion and/or a third portion of an engineered monomer described herein, or (ii) a first region (e.g., adjacent to the constriction region) and/or a third region (e.g., adjacent to the constriction region) of the engineered biological nanopore may comprise 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, at most about 1, or less than about 1 amino acid mutations to negatively-charged amino acid residues.

[0504] In some cases, (i) a second portion (e.g., constriction-forming portion) of an engineered monomer described herein, or (ii) a second region of the channel (e.g., comprising the constriction region) of the engineered biological nanopore may comprise at least about 1, 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, or greater than about 15 amino acid mutations. In some cases, (i) a second portion (e.g., constriction-forming portion) of an engineered monomer described herein, or (ii) a second region of the channel (e.g., comprising the constriction region) of the engineered biological nanopore may comprise 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, at most about 1, or less than about 1 amino acid mutations. In some cases, (i) a second portion (e.g., constriction-forming portion) of an engineered monomer described herein, or (ii) a second region of the channel (e.g., comprising the constriction region) of the engineered biological nanopore may comprise at least about 1, 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, or greater than about 15 amino acid mutations to neutrally-charged amino acid residues. In some cases, (i) a second portion (e.g., constriction-forming portion) of an engineered monomer described herein, or (ii) a second region of the channel (e.g., comprising the constriction region) of the engineered biological nanopore may comprise 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, at most about 1, or less than about 1 amino acid mutations to neutrally -charged amino acid residues.

[0505] In some cases, if at least one amino acid residue in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g., constriction-forming portion) of a monomer may be mutated, then at least one amino acid residue in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated. In some cases, if at least one amino acid residue in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g., constriction-forming portion) of a monomer may be mutated to a negatively-charged amino acid residue, then at least one amino acid residue in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated to a negatively -charged amino acid residue and/or a neutrally -charged amino acid residue. In some cases, if at least two amino acid residues in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g., constriction-forming portion) of a monomer may be mutated, then at least one amino acid residue in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated. In some cases, if at least two amino acid residues in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g., constriction-forming portion) of a monomer may be mutated to negatively-charged amino acid residues, then at least one amino acid residue in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated to a negatively-charged amino acid residue and/or a neutrally-charged amino acid residue. In some cases, if at least one amino acid residue in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g., constriction-forming portion) of a monomer may be mutated, then at least two amino acid residues in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated. In some cases, if at least one amino acid residue in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g. , constriction-forming portion) of a monomer may be mutated to a negatively-charged amino acid residue, then at least two amino acid residues in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated to negatively -charged amino acid residues and/or neutrally -charged amino acid residues. In some cases, if at least two amino acid residues in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g., constriction-forming portion) of a monomer may be mutated, then at least two amino acid residues in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated. In some cases, if at least two amino acid residues in (i) a second region of an engineered biological nanopore, or (ii) a second portion (e.g., constriction-forming portion) of a monomer may be mutated to negatively-charged amino acid residues, then at least two amino acid residues in (i) a first region and/or third region of an engineered biological nanopore, or (ii) a first portion and/or third portion of the monomer may be mutated to negatively -charged amino acid residues and/or neutrally-charged amino acid residues.

[0506] In some cases, only a first region and/or third region of an engineered biological nanopore described herein may be mutated. In some cases, only a first portion and/or a third portion of an engineered monomer described herein may be mutated. In some cases, only a second region (e.g., comprising a constriction region) of an engineered biological nanopore described herein may be mutated. In some cases, only a second portion (e.g., constriction-forming portion) of an engineered monomer described herein may be mutated.

[0507] A distance may exist between two mutated amino acid residues of a monomer of a nanopore (e.g., engineered biological nanopore) described herein. In some cases, the distance may be characterized as the closest distance between two mutated amino acid residues (e.g., an amino acid residue of a first portion of a channel and a second portion of a channel). In some cases, a distance between closest mutated amino acid residues can be 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, or greater than about 5 nm. In some cases, a distance between closest mutated amino acid residues can be 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.

[0508] In some cases, a distance between closest mutated amino acid residues can be from about 0. 1 nm to about 8 nm. In some cases, a distance between closest mutated amino acid residues can be from at least about 0. 1 nm. In some cases, a distance between closest mutated amino acid residues can be from at most about 8 nm. In some cases, a distance between closest mutated amino acid residues can be from about 0. 1 nm to about 0.5 nm, about 0. 1 nm to about 1 nm, about 0. 1 nm to about 1.5 nm, about 0. 1 nm to about 2 nm, about 0. 1 nm to about 2.5 nm, about 0. 1 nm to about 3 nm, about 0. 1 nm to about 3.5 nm, about 0.1 nm to about 4 nm, about 0. 1 nm to about 4.5 nm, about 0. 1 nm to about 5 nm, about 0. 1 nm to about 8 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 4.5 nm, about 0.5 nm to about 5 nm, about 0.5 nm to about 8 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 4.5 nm, about 1 nm to about 5 nm, about 1 nm to about 8 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 4.5 nm, about 1.5 nm to about 5 nm, about 1.5 nm to about 8 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 4.5 nm, about 2 nm to about 5 nm, about 2 nm to about 8 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 4.5 nm, about 2.5 nm to about 5 nm, about 2.5 nm to about 8 nm, about 3 nm to about 3.5 nm, about 3 nm to about 4 nm, about 3 nm to about

4.5 nm, about 3 nm to about 5 nm, about 3 nm to about 8 nm, about 3.5 nm to about 4 nm, about 3.5 nm to about 4.5 nm, about 3.5 nm to about 5 nm, about 3.5 nm to about 8 nm, about 4 nm to about 4.5 nm, about 4 nm to about 5 nm, about 4 nm to about 8 nm, about 4.5 nm to about 5 nm, about 4.5 nm to about 8 nm, or about 5 nm to about 8 nm. [0509] In some cases, an EOF may be modulated by inducing a strong net flow of ions in one direction across the nanopore (e.g., engineered biological nanopore). In some cases, the EOF can be controlled by inducing a strong net flow of ion ions in one direction across the nanopore (e.g., engineered biological nanopore). The EOF can be controlled by modifying of the nanopore (e.g., engineered biological nanopore), applying specific electrolyte asymmetries and concentrations, or any combination thereof. Modulating a net charge of a channel (e.g., lumen) of a nanopore (e.g., engineered biological nanopore) may generate a strong EOF. Modulating a net charge of a channel (e.g., lumen) of a nanopore (e.g., engineered biological nanopore) may generate a weak EOF.

[0510] One or more mutations of the first region of the channel, the second region of the channel (e.g., the constriction region), or any combination thereof may modulate an ion selectivity of a nanopore (e.g., engineered biological nanopore) described herein. The ion selectivity may comprise a potassium selectivity (e.g., P(K⁺/Glu')). Mutating a non-neutral amino acid residue (e.g., a positively charge amino acid residue or negatively charged amino acid residue) to a neutral residue in a nanopore (e.g., engineered biological nanopore) may reduce an ion selectivity for positive ions, an analyte (e.g., biopolymer) comprising a positive charge, or any combination thereof. Mutating a non-neutral amino acid residue (e.g., a positively charge amino acid residue or negatively charged amino acid residue) to a neutral residue in a nanopore (e.g., engineered biological nanopore) may reduce an ion selectivity for negative ions, an analyte (e.g., biopolymer) comprising a negative charge, or any combination thereof. The increased neutral charge of the channel of the nanopore (e.g., a second portion of the nanopore) may inhibit and/or prevent charged ion species from translocating to the nanopore. A combination of one or more mutated neutral residues of a second portion of a channel (e.g., a constriction region) with one or more mutated negatively charged residues of a first portion and/or third portion of channel may increase an ion selectivity for positive ions (e.g., increase a cation selectivity). The one or more mutated negatively charged residues may provide a strong net negative charge to increase an ion selectivity for positive ions, analytes (e.g., biopolymers) comprising a positive charge, or any combination thereof. Mutating a nonnegative amino acid residue (e.g., a positively charge amino acid residue or neutrally charged amino acid residue) to a negatively -charged residue in a nanopore (e.g., engineered biological nanopore) may increase an ion selectivity for cations, an analyte (e.g., biopolymer) comprising a positive charge, or any combination thereof. Mutating a non-negative amino acid residue (e.g., a positively charge amino acid residue or neutrally charged amino acid residue) to a negatively-charged residue in a nanopore (e.g., engineered biological nanopore) may reduce an ion selectivity for negative ions, an analyte (e.g., biopolymer) comprising a negative charge, or any combination thereof. The increased negative charge of the channel of the nanopore (e.g., a second portion of the nanopore) may improve an ability of charged cation species from translocating to the nanopore. A combination of one or more mutated negatively -charged residues of a second portion of a channel (e.g., a constriction region) with one or more mutated negatively charged residues of a first portion and/or third portion of channel may increase an ion selectivity for positive ions (e.g., increase a cation selectivity). The one or more mutated negatively charged residues may provide a strong net negative charge to increase an ion selectivity for positive ions, analytes (e.g., biopolymers) comprising a positive charge, or any combination thereof.

[0511] In some cases, one or more mutations to a first and/or third region of a nanopore and a second region of a nanopore may provide an additive effect to an enhanced EOF and/or an enhanced ion selectivity. A cation selectivity and/or an EOF may be increased to a greater degree in an engineered biological nanopore comprising one or more mutated amino acid residues in a first and/or third region of a nanopore and a second region of a nanopore, compared to a cation selectivity and/or an EOF of a nanopore that comprises a mutated amino acid residue in only the first and/or third region or the second region. For example, mutation(s) of amino acid residues to negatively -charged residues in both the first and/or third region of a nanopore and a second region of the nanopore (e.g., to increase a net negative charge) may result in a greater cation-selectivity and/or EOF compared to a nanopore with mutation(s) of amino acid residues to negatively -charged residues in only the first and/or third region of the nanopore. For example, mutation(s) of amino acid residues to negatively- charged residues in both the first and/or third region of a nanopore and a second region of the nanopore (e.g., to increase a net negative charge) may result in a greater cation-selectivity and/or EOF compared to a nanopore with mutation(s) of amino acid residues to negatively-charged residues in only the second region of the nanopore.

[0512] In some cases, one or more mutations to a first and/or third portion of a monomer and a second portion (e.g., constriction-forming portion) of a monomer may provide an additive effect to an enhanced EOF and/or an enhanced ion selectivity. A cation selectivity and/or an EOF may be increased to a greater degree in an engineered biological nanopore comprising one or more mutated amino acid residues in a first and/or third portion of a monomer and a second portion of a monomer, compared to a cation selectivity and/or an EOF of a nanopore that comprises a mutated amino acid residue in only the first and/or third portion or the second portion. For example, mutation(s) of amino acid residues to negatively-charged residues in both the first and/or third portion of a monomer and a second portion of the monomer may result in a greater cation-selectivity and/or EOF compared to a nanopore with mutation(s) of amino acid residues to negatively-charged residues in only the first and/or third portion of the monomer. For example, mutation(s) of amino acid residues to negatively-charged residues in both the first and/or third portion of a monomer and a second portion of the monomer (e.g., to increase a net negative charge) may result in a greater cation-selectivity and/or EOF compared to a nanopore with mutation(s) of amino acid residues to negatively -charged residues in only the second portion of the monomer.

[0513] In some cases, a mutated nanopore (e.g., engineered biological nanopore) described herein may have an ion selectivity P(+)/P(-) of at least about 0.1, at least about 0.5, 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 4.5, at least about 5.0, or greater than about 5.0 under an applied voltage difference across the membrane. In some cases, a mutated nanopore (e.g., engineered biological nanopore) described herein may have an ion selectivity P(+)/P(-) of at most about 5.0, at most about 4.5, 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 0.5, at most about 0. 1, or less than about 0. 1 under an applied voltage difference across the membrane.

[0514] In some cases, a mutated nanopore (e.g., engineered biological nanopore) described herein may have an ion selectivity P(+)/P(-) from about 0.1 to about 5.5 under an applied voltage difference across the membrane. In some cases, a mutated nanopore (e.g., engineered biological nanopore) described herein may have an ion selectivity P(+)/P(-) from 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 3.5, about 0. 1 to about 4, about 0. 1 to about 4.5, about 0.1 to about 5, about 0. 1 to about 5.5, 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 3.5, about 0.5 to about 4, about 0.5 to about 4.5, about 0.5 to about 5, about 0.5 to about 5.5, 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 3.5, about 1 to about 4, about 1 to about 4.5, about 1 to about 5, about 1 to about 5.5, about 1.5 to about 2, about 1.5 to about 2.5, about 1.5 to about 3, about 1.5 to about

3.5, about 1.5 to about 4, about 1.5 to about 4.5, about 1.5 to about 5, about 1.5 to about 5.5, about 2 to about

2.5, about 2 to about 3, about 2 to about 3.5, about 2 to about 4, about 2 to about 4.5, about 2 to about 5, about 2 to about 5.5, about 2.5 to about 3, about 2.5 to about 3.5, about 2.5 to about 4, about 2.5 to about 4.5, about 2.5 to about 5, about 2.5 to about 5.5, about 3 to about 3.5, about 3 to about 4, about 3 to about 4.5, about 3 to about 5, about 3 to about 5.5, about 3.5 to about 4, about 3.5 to about 4.5, about 3.5 to about 5, about 3.5 to about 5.5, about 4 to about 4.5, about 4 to about 5, about 4 to about 5.5, about 4.5 to about 5, about 4.5 to about

5.5, or about 5 to about 5.5 under an applied voltage difference across the membrane.

[0515] In some cases, one or more amino acid mutations of an engineered biological nanopore described herein may increase an ion selectivity P(+)/P(-) (e.g., P(K+/C1-)) relative to a wild-type nanopore that does not have the one or more amino acid mutations. For example, the one or more amino acid mutations of an engineered monomer described herein (e.g., one or more amino acid mutations to increase a net negative charge) in a portion (e.g., a first portion, second portion, third portion, or combination thereof) may increase an ion selectivity P(+)/P(-) (e.g., P(K+/C1-)) relative to a wild-type monomer that does not have the one or more amino acid mutations. For example, an ion selectivity (e.g., cation-selectivity) may be increased in a nanopore (e.g., an engineered biological nanopore) comprising one or more modifications of a second region (e.g., one or more modifications to make a second region more net neutral and/or net negative). As another example, the ion selectivity (e.g., cation-selectivity) may be increased in a nanopore (e.g., an engineered biological nanopore) comprising one or more modifications of a second region (e.g., one or more modifications to make a second region more net neutral or net negative) and one or more modifications in a first region and/or third region (e.g., one or more modifications to make a first region and/or third region more net negative). The nanopore may be a cation-selective nanopore. In some cases, the nanopore comprising one or more modifications of a second region (e.g., a constriction region) may be a cation-selective nanopore. In some cases, the cation-selective nanopore may be a nanopore comprising one or more modifications of a second region and at least one modification of a first region or third region. In some cases, the cation-selective nanopore may be a nanopore comprising one or more modifications of a second region and at least two modifications of a first region and/or third region.

[0516] The engineered biological nanopore described herein may have an ion selectivity P(+)/P(-) (e.g., P(K+/C1-)) of at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, 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. The engineered biological nanopore described herein may have an ion selectivity P(+)/P(-) (e.g., P(K+/C1-)) 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 1.9, at most about 1.8, at most about 1.7, at most about 1.6, at most about 1.5, at most about 1.4, at most about 1.3, or less than about 1.3. The engineered biological nanopore described herein may have an ion selectivity P(+)/P(-) (e.g., P(K+/C1-)) 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, or greater than about 1.5. The engineered biological nanopore described herein may have an ion selectivity P(+)/P(-) (e.g., P(K+/C1- )) of 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.

[0517] Improving sensing properties may comprise increasing the cation-selectivity and/or increasing EOF of the nanopore. The increase in cation selectivity and/or increase in EOF may be at least about 10%, at least about 15%, at least about 18%, at least about 20%, at least about 23%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 140%, at least about 150%, at least about 170%, at least about 180%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, or greater than about 700% as compared to a wild-type nanopore that does not have an increased net neutral charge or increased net negative charge of amino acid residues in the second region (e.g., constriction region) of the engineered biological nanopore.

[0518] The increase in cation selectivity and/or increase in EOF may be at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 350%, at most about 300%, at most about 250%, at most about 200%, at most about 180%, at most about 170%, at most about 150%, at most about 140%, at most about 125%, at most about 100%, at most about 90%, at most about 80%, at most about 700%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 18%, at most about 15%, at most about 10%, or less than about 10% as compared to a wild -type nanopore that does not have an increased net neutral charge or increased net negative charge of amino acid residues in the second region (e.g., constriction region) of the engineered biological nanopore.

[0519] The increase in cation selectivity and/or increase in EOF may be at least about 10%, at least about 15%, at least about 18%, at least about 20%, at least about 23%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 140%, at least about 150%, at least about 170%, at least about 180%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, or greater than about 700% as compared to a wild-type nanopore that does not have an increased net negative charge of amino acid residues in the first and/or third region (e.g., adjacent to the constriction region) of the engineered biological nanopore. [0520] The increase in cation selectivity and/or increase in EOF may be at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 350%, at most about 300%, at most about 250%, at most about 200%, at most about 180%, at most about 170%, at most about 150%, at most about 140%, at most about 125%, at most about 100%, at most about 90%, at most about 80%, at most about 700%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 18%, at most about 15%, at most about 10%, or less than about 10% as compared to a wild -type nanopore that does not have an increased net negative charge of amino acid residues in the first and/or third region (e.g., adjacent to the constriction region) of the engineered biological nanopore.

[0521] Improving sensing properties may comprise increasing the cation-selectivity and/or increasing EOF of the nanopore. The increase in cation selectivity and/or increase in EOF may be at least about 10%, at least about 15%, at least about 18%, at least about 20%, at least about 23%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 140%, at least about 150%, at least about 170%, at least about 180%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, or greater than about 700% as compared to a wild-type nanopore that does not have an increased net neutral charge or increased net negative charge of amino acid residues in the second portion (e.g., constriction-forming portion) of a monomer of the engineered biological nanopore.

[0522] The increase in cation selectivity and/or increase in EOF may be at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 350%, at most about 300%, at most about 250%, at most about 200%, at most about 180%, at most about 170%, at most about 150%, at most about 140%, at most about 125%, at most about 100%, at most about 90%, at most about 80%, at most about 700%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 18%, at most about 15%, at most about 10%, or less than about 10% as compared to a wild -type nanopore that does not have an increased net neutral charge or increased net negative charge of amino acid residues in the second portion (e.g., constriction-forming portion) of a monomer of the engineered biological nanopore.

[0523] The increase in cation selectivity and/or increase in EOF may be at least about 10%, at least about 15%, at least about 18%, at least about 20%, at least about 23%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 140%, at least about 150%, at least about 170%, at least about 180%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, at least about 600%, at least about 700%, or greater than about 700% as compared to a wild-type nanopore that does not have an increased net negative charge of amino acid residues in the first and/or third portion (e.g., adjacent to the constriction-forming portion) of a monomer of the engineered biological nanopore.

[0524] The increase in cation selectivity and/or increase in EOF may be at most about 700%, at most about 600%, at most about 500%, at most about 400%, at most about 350%, at most about 300%, at most about 250%, at most about 200%, at most about 180%, at most about 170%, at most about 150%, at most about 140%, at most about 125%, at most about 100%, at most about 90%, at most about 80%, at most about 700%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 18%, at most about 15%, at most about 10%, or less than about 10% as compared to a wild -type nanopore that does not have an increased net negative charge of amino acid residues in the first and/or third portion (e.g., adjacent to the constriction-forming portion) of a monomer of the engineered biological nanopore.

Analytes

[0525] The analyte may be obtained, isolated or extracted from any organism or microorganism. For instance, it is 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 analyte may be obtained from a plant e.g. a cereal, legume, ornamental plant, fruit or vegetable, or part thereof including tubers, roots and bulbs.

[0526] The analyte can be produced inside (animal) cells such that it can be extracted from cells for characterization by the disclosed methods. The analyte may comprise the products of cellular expression of a plasmid in a (microbial) host cell. In some cases the analyte is secreted from cells.

[0527] The analyte can be provided as an impure mixture of one or more analytes and one or more (proteinaceous) impurities. For example, the analyte may be a full length protein and impurities may comprise fragments of the analyte. Impurities may comprise truncated forms of the analyte which are distinct from the “analytes” for characterization in the disclosed methods. Impurities may also comprise analyte s other than the analytes e.g. which may be co-purified from a cell culture or obtained from a sample.

[0528] A analyte may comprise any combination of any amino acids, amino acid analogs and modified amino acids (i.e. amino acid derivatives). Amino acids (and derivatives, analogs etc) in the analyte can be distinguished by their physical size and charge. The amino acids/derivatives/analogs can be naturally occurring or artificial.

[0529] In some cases, the analyte is modified. In some cases, the analyte is modified by a leader construct according to the disclosed methods. In some cases, the disclosed methods are for characterizing modifications in the analyte. In one aspect, one or more of the amino acids/derivatives/analogs in the analyte is post- translationally modified. Any one or more post-translational modifications may be present in the analyte. Typical post-translational modifications can include, but are not limited to, modification with a hydrophobic group, modification with a cofactor, addition of a chemical group, glycation (the non-enzymatic attachment of a sugar), biotinylation, deamidation, acetylation, ubiquitination, phosphorylation, sumolation, methylation, and PEGylation. Post-translational modifications can also be non-natural, for instance are chemical modifications introduced in a laboratory for biotechnological or biomedical purposes. This allows for monitoring the levels of post-translational modifications of the laboratory-derived peptide, polypeptide or protein as compared to the natural counterpart. As such, the methods disclosed herein can be used to detect the presence, absence, extent or number of positions of post-translational modifications in a polypeptide.

[0530] The nanopores, methods, and/or systems described herein can be readily designed to detect any analyte (or multiple analytes) of interest. The invention can be advantageously used to detect a label-free analyte. The nanopores described herein can capture a wide range of particles in a similar size range. Examples include inorganic particles (e.g. gold beads), polymeric particles such as plastics/beads/dendrimers, or oligomeric particles (e.g. micelles, liposomes and other fatty droplets).

[0531] In one embodiment, the invention provides a method for detecting an analyte/antigen selected from the group consisting of a biopolymer, a protein, a polypeptide, a protein assembly, a protein/DNA assembly, saccharide (e.g., polysaccharide), lipid, lipid membrane, lipid particle, bacterium, virus capsid, virus particle, dendrimer, polymer, inorganic particle, oligomeric particle, non-nucleic acid based polymer analyte, or any combination thereof. In some cases, the analyte can be a nucleic acid analyte. In some cases, the analyte may not be a nucleic acid analyte. A non-nucleic acid analyte may be an analyte that comprises a protein, polypeptide, or peptide coupled to a nucleic acid.

[0532] In some cases, an analyte may be of synthetic origin, semi-synthetic origin, or biological origin. For example, the analyte may be a biopolymer. The biopolymer may comprise one or more peptide units, one or more saccharide units, one or more nucleic acid units, one or more water-soluble plastic monomers, or any combination thereof. In some cases, the analyte may be one or more proteinaceous polymers, one or more non- nucleic acid based polymers, one or more nucleic acid-peptide conjugates, or any combination thereof.

[0533] The nanopores, methods, and systems of the present disclosure can be very suitable for the analysis of a complex sample, e.g. a solution comprising a mixture of components including one or more target analytes and one or more unwanted analytes. For example, the sample can be a complex sample comprising a mixture of proteins. In some cases, the sample comprises a (diluted) clinical sample. In some cases, the sample can be a bodily fluid or sample, such as whole blood, plasma, blood serum, urine, feces, saliva, cerebrospinal fluid, nasopharyngeal swab, breast milk, sputum, or any combination thereof. In another aspect, the sample comprises (diluted) complex media. In some cases, a sample can be obtained from a healthy subject. In some cases, a sample can be obtained from a subject with a disease or condition.

[0534] In one embodiment, the target analyte can be a clinically relevant analyte, for example a clinically relevant protein or fragment thereof. In a specific embodiment, the target analyte can be a cytokine, an inflammation marker (e.g. C-reactive protein) or a cell metabolite. In some cases, the cytokine molecule may comprise interleukin-2 (IL-2) or a functional variant thereof, interleukin-7 (IL-7) or a functional variant thereof, interleukin- 12 (IL-12) or a functional variant thereof, interleukin- 15 (IL-15) or a functional variant thereof, interleukin- 18 (IL- 18) or a functional variant thereof, interleukin-21 (IL-21) or a functional variant thereof, or interferon gamma or a functional variant thereof, or any combination thereof. In some cases, the analyte can be a protein, for example selected from the group consisting of a folded/native protein, a protein biomarker, a pathogenic protein, a cell surface protein.

[0535] The present invention can be particularly suitable for detecting protein targets covering a very wide range of masses and dimensions, from very small proteins and peptides to very large proteins and complexes. In some cases, the analyte can comprise at least about 2 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids, at least about 250 amino acids, at least about 300 amino acids, at least about 350 amino acids, at least about 400 amino acids, at least about 450 amino acids, at least about 500 amino acids, at least about 600 amino acids, at least about 700 amino acids, at least about 800 amino acids, at least about 900 amino acids, at least about 1000 amino acids, at least about 2000 amino acids, at least about 3000 amino acids, at least about 4000 amino acids, at least about 5000 amino acids, at least about 6000 amino acids, at least about 7000 amino acids, at least about 8000 amino acids, at least about 9000 amino acids, at least about 10000 amino acids, at least about 20000 amino acids, at least about 30000, at least about 34000 amino acids, or greater than about 34000 amino acids in length. In some cases, the analyte can be at most about 34000 amino acids, at most about 30000 amino acids, at most about 20000 amino acids, at most about 10000 amino acids, at most about 9000 amino acids, at most about 8000 amino acids, at most about 7000 amino acids, at most about 6000 amino acids, at most about 5000 amino acids, at most about 4000 amino acids, at most about 3000 amino acids, at most about 2000 amino acids, at most about 1000 amino acids, at most about 900 amino acids, at most about 800 amino acids, at most about 700 amino acids, at most about 600 amino acids, at most about 500 amino acids, at most about 450 amino acids, at most about 400 amino acids, at most about 350 amino acids, at most about 300 amino acids, at most about 250 amino acids, at most about 30000 amino acids, at most about 30000 amino acids, at most about 200 amino acids, at most about 150 amino acids, at most about 100 amino acids, at most about 90 amino acids, at most about 80 amino acids, at most about 70 amino acids, at most about 60 amino acids, 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 15 amino acids, at most about 10 amino acids, at most about 5 amino acids, at most about 2 amino acids, or less than about 2 amino acids in length.

[0536] In some cases, the analyte can be from about 2 amino acids to about 1,000 amino acids in length. In some cases, the analyte can be from about 2 amino acids to about 10 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 200 amino acids, about 2 amino acids to about 300 amino acids, about 2 amino acids to about 400 amino acids, about 2 amino acids to about 500 amino acids, about 2 amino acids to about 600 amino acids, about 2 amino acids to about 700 amino acids, about 2 amino acids to about 800 amino acids, about 2 amino acids to about 900 amino acids, about 2 amino acids to about 1,000 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 1,000 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 1,000 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 1,000 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 1,000 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 1,000 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 1,000 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 1,000 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 1,000 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 1,000 amino acids, or about 900 amino acids to about 1,000 amino acids in length.

[0537] In some cases, the analyte can be from about 1,000 amino acids to about 34,000 amino acids in length. In some cases, the analyte can be from about 1,000 amino acids to about 2,500 amino acids, about 1,000 amino acids to about 5,000 amino acids, about 1,000 amino acids to about 7,500 amino acids, about 1,000 amino acids to about 10,000 amino acids, about 1,000 amino acids to about 15,000 amino acids, about 1,000 amino acids to about 20,000 amino acids, about 1,000 amino acids to about 25,000 amino acids, about 1,000 amino acids to about 30,000 amino acids, about 1,000 amino acids to about 34,000 amino acids, about 2,500 amino acids to about 5,000 amino acids, about 2,500 amino acids to about 7,500 amino acids, about 2,500 amino acids to about 10,000 amino acids, about 2,500 amino acids to about 15,000 amino acids, about 2,500 amino acids to about 20,000 amino acids, about 2,500 amino acids to about 25,000 amino acids, about 2,500 amino acids to about 30,000 amino acids, about 2,500 amino acids to about 34,000 amino acids, about 5,000 amino acids to about 7,500 amino acids, about 5,000 amino acids to about 10,000 amino acids, about 5,000 amino acids to about 15,000 amino acids, about 5,000 amino acids to about 20,000 amino acids, about 5,000 amino acids to about 25,000 amino acids, about 5,000 amino acids to about 30,000 amino acids, about 5,000 amino acids to about 34,000 amino acids, about 7,500 amino acids to about 10,000 amino acids, about 7,500 amino acids to about 15,000 amino acids, about 7,500 amino acids to about 20,000 amino acids, about 7,500 amino acids to about 25,000 amino acids, about 7,500 amino acids to about 30,000 amino acids, about 7,500 amino acids to about 34,000 amino acids, about 10,000 amino acids to about 15,000 amino acids, about 10,000 amino acids to about 20,000 amino acids, about 10,000 amino acids to about 25,000 amino acids, about 10,000 amino acids to about 30,000 amino acids, about 10,000 amino acids to about 34,000 amino acids, about 15,000 amino acids to about 20,000 amino acids, about 15,000 amino acids to about 25,000 amino acids, about 15,000 amino acids to about 30,000 amino acids, about 15,000 amino acids to about 34,000 amino acids, about 20,000 amino acids to about 25,000 amino acids, about 20,000 amino acids to about 30,000 amino acids, about 20,000 amino acids to about 34,000 amino acids, about 25,000 amino acids to about 30,000 amino acids, about 25,000 amino acids to about 34,000 amino acids, or about 30,000 amino acids to about 34,000 amino acids in length.

[0538] In some cases, the analyte can be about 2 amino acids, about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 60 amino acids, about 70 amino acids, about 80 amino acids, about 90 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 350 amino acids, about 400 amino acids, about 450 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids, about 2000 amino acids, about 3000 amino acids, about 4000 amino acids, about 5000 amino acids, about 6000 amino acids, about 7000 amino acids, about 8000 amino acids, about 9000 amino acids, about 10000 amino acids, about 20000 amino acids, about 30000, or about 34000 amino acids in length. [0539] In some cases, the analyte may comprise a mass. In some cases, 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 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 cases, 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.

[0540] In some cases, the analyte may comprise a mass from about 1 kDa to about 100 kDa. In some cases, 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 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.

[0541] In some cases, the analyte may comprise a mass from about 100 kDa to about 4,000 kDa. In some cases, 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.

[0542] In some cases, 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.

Systems

[0543] In some aspects, the present disclosure provides a sensor system comprising any nanopore described herein. 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 cases, 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 cases, one side (e.g., a first side) of a fluid filled chamber can be a cis side and another side (e.g., a second side) of a fluid filled chamber can be a trans side. In some cases, the nanopore can be a conical shaped proteinaceous nanopore. In some cases, the nanopore can be a cylindrical shaped proteinaceous nanopore. In some cases, 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).

[0544] In another aspect, the present disclosure provides a system comprising (i) a fluidic chamber. The system may comprise a membrane. The membrane may comprise an engineered biological nanopore. The membrane may separate the fluid chamber. The fluid chamber may be separated into a first side (e.g., a cis side) and/or a second side (e.g., a trans side). The engineered biological nanopore may be an engineered biological nanopore described herein. For example, the engineered biological nanopore may comprise a channel. The channel may comprise a first region. The channel may comprise a second region. The second region may have a constriction region. The first region of the engineered biological nanopore may be modified. The first region may be modified to be more net negative than a respective region of a wild-type biological nanopore. The second region of the engineered biological nanopore may be modified. The second region may be modified to be more net neutral than a respective region of a wild-type biological nanopore. The second region may be modified to be more net negative than a respective region of a wild-type biological nanopore. The first region of the channel may be adjacent to the second region of the channel. The engineered biological nanopore (e.g., the engineered biological nanopore of the system) may be configured to contact a biopolymer. The biopolymer may be any biopolymer described herein.

[0545] In another aspect, the present disclosure provides a system comprising (i) a fluidic chamber. The system may comprise a membrane. The membrane may comprise an engineered biological nanopore. The membrane may separate the fluid chamber. The fluid chamber may be separated into a first side (e.g., a cis side) and/or a second side (e.g., a trans side). The engineered biological nanopore may be an engineered biological nanopore described herein. For example, the engineered biological nanopore may comprise a channel. The channel may comprise a first region. The channel may comprise a second region. The second region may have a constriction region. The first region of the channel may be adjacent to the second region of the channel. The first region of the channel may have a negative charge. The second region of the channel may have a neutral charge. The first region of the channel may be adjacent to the second region of the channel. The engineered biological nanopore (e.g., the engineered biological nanopore of the system) may be configured to contact a biopolymer. The biopolymer may be any biopolymer described herein.

[0546] In another aspect, the present disclosure provides a system comprising (i) a fluidic chamber. The system may comprise a membrane. The membrane may comprise an engineered biological nanopore. The membrane may separate the fluid chamber. The fluid chamber may be separated into a first side (e.g., a cis side) and/or a second side (e.g., a trans side). The engineered biological nanopore may be an engineered biological nanopore described herein. For example, the engineered biological nanopore may comprise a channel. The channel may comprise a first region. The channel may comprise a second region. The second region may have a constriction region. The first region of the channel may be adjacent to the second region of the channel. The first region of the channel may have a negative charge. The second region of the channel may have a net charge. The net charge of the second region of the channel may be at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wildtype biological nanopore). The engineered biological nanopore (e.g., the engineered biological nanopore of the system) may be configured to contact a biopolymer. The biopolymer may be any biopolymer described herein [0547] 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 cases, 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 cases, a membrane of a nanopore system described herein may 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, 900, 1000, 5000, 10000, or greater than about 10000 nanopores. In some cases, a membrane of a nanopore system described herein may comprise at most about 10000, 5000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nanopore. In some cases, a membrane of a nanopore system described herein may comprise 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.

[0548] 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 cases, the membrane may comprise multiple layers. In some cases, the membrane may be functionalized. In some cases, the membrane may be functionalized with a thiol group, a peptide, a nucleic acid, 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.

[0549] In some cases, a membrane of a system described herein may comprise a thickness. In some cases, 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 greaterthan about 150 nm thick. In some cases, a membrane comprise a thickness from about 0.5 nm to about 100 nm. In some cases, 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. [0550] 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. The conductive liquid medium in the chambers of the nanopore system can have a wide range of ionic contents well known in the art, typically from 0.05 M to > 3 M. A wide range of salts can be used, such as NaCl and KC1. Suitable solutions include 150 mM NaCl, 50 mM Tris-HCl, pH 7.5. In some cases, a salt, ion, osmolyte, or electrolyte concentration on the 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 cases, a salt, ion, osmolyte, or electrolyte concentration on the 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.

[0551] In some cases, a salt, ion, osmolyte, or electrolyte concentration on the cis side can be from about 0.01 M to about 5 M. In some cases, a salt, ion, osmolyte, or electrolyte concentration on the 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. [0552] In some cases, a salt, ion, osmolyte, or electrolyte concentration on the 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.

[0553] The solution or solutions may have a pH of at least about 1, at least about 2, at least about 3, at least about 3.8, at least about 4, at least about 4.5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 10.5 at least about 11, at least about 12, at least about 13, or greater than about 13 that can be employed. The solution or solutions may have a pH of at most about 13, at most about 12, at most about 11, at most about 10.5, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 4.5, at most about 4, at most about 3.8, at most about 3, at most about 2, at most about 1, or less than about 1 that can be employed.

[0554] The solution or solutions may have a pH from about 1 to about 13 that can be employed. The solution or solutions may have a pH from about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 11, about 1 to about 12, about 1 to about 13, about 2 to about 3, about 2 to about 4, 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 11, about 2 to about 12, about 2 to about 13, about 3 to about 4, 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 11, about 3 to about 12, about 3 to about 13, 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 11, about 4 to about 12, about 4 to about 13, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 11, about 6 to about 12, about 6 to about 13, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 11, about 7 to about 12, about 7 to about 13, about 8 to about 9, about 8 to about 10, about 8 to about 11, about 8 to about 12, about 8 to about 13, about 9 to about 10, about 9 to about 11, about 9 to about 12, about 9 to about 13, about 10 to about 11, about 10 to about 12, about 10 to about 13, about 11 to about 12, about 11 to about 13, or about 12 to about 13 that can be employed.

[0555] The solution or solutions may have a pH of about 1, about 2, about 3, about 3.8, about 4, about 4.5, about 6, about 7, about 8, about 9, about 10, about 10.5 about 11, about 12, or about 13 that can be employed. [0556] The first side and second side may be symmetric or asymmetric. A wide range of pH and temperature conditions can be used, for example in the range of pH 3-11, 10-80 °C, for example at about room temperature or at about 37 °C. In some cases, a cis chamber and/or a trans chamber may have a pH of at least about 1, at least about 2, at least about 3, at least about 3.8, at least about 4, at least about 4.5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 10.5 at least about 11, at least about 12, at least about 13, or greater than about 13. In some cases, a first side and/or second side may have a pH of at most about 13, at most about 12, at most about 11, at most about 10.5, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 4.5, at most about 4, at most about 3.8, at most about 3, at most about 2, at most about 1, or less than about 1. In some cases, a cis chamber and/or a trans chamber may have a pH from about 1 to about 13 that can be employed. In some cases, a first side and/or second side may have a pH from about 1 to about 2, about 1 to about 3, about 1 to about 4, about 1 to about 6, about 1 to about 7, about 1 to about 8, about 1 to about 9, about 1 to about 10, about 1 to about 11, about 1 to about 12, about 1 to about 13, about 2 to about 3, about 2 to about 4, 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 11, about 2 to about 12, about 2 to about 13, about 3 to about 4, 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 11, about 3 to about 12, about 3 to about 13, 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 11, about 4 to about 12, about 4 to about 13, about 6 to about 7, about 6 to about 8, about 6 to about 9, about 6 to about 10, about 6 to about 11, about 6 to about 12, about 6 to about 13, about 7 to about 8, about 7 to about 9, about 7 to about 10, about 7 to about 11, about 7 to about 12, about 7 to about 13, about 8 to about 9, about 8 to about 10, about 8 to about 11, about 8 to about 12, about 8 to about 13, about 9 to about 10, about 9 to about 11, about 9 to about 12, about 9 to about 13, about 10 to about 11, about 10 to about 12, about 10 to about 13, about 11 to about 12, about 11 to about 13, or about 12 to about 13 that can be employed.

[0557] In some cases, a first side and/or second side may have a temperature of at least about 5 °C, at least about 10 °C, at least about 15 °C, at least about 20 °C, at least about 25 °C, at least about 30 °C, at least about 35 °C, at least about 40 °C, at least about 45 °C, at least about 50 °C, at least about 60 °C, at least about 70 °C, at least about 80 °C, or greater than about 80 °C. In some cases, a first side and/or second side may have a temperature of at most about 80 °C, at most about 70 °C, at most about 60 °C, at most about 50 °C, at most about 45 °C, at most about 40 °C, at most about 35 °C, at most about 30 °C, at most about 25 °C, at most about 20 °C, at most about 15 °C, at most about 10 °C, at most about 5 °C, or less than about 5 °C. In some cases, a first side and/or second side may have a temperature from about 5 °C to about 80 C. In some cases, a first side and/or second side may have a temperature from about 5 °C to about 10 °C, about 5 °C to about 15 °C, about 5 °C to about 20 °C, about 5 °C to about 25 °C, about 5 °C to about 30 °C, about 5 °C to about 35 °C, about 5 °C to about 40 °C, about 5 °C to about 50 °C, about 5 °C to about 60 °C, about 5 °C to about 70 °C, about 5 °C to about 80 °C, about 10 °C to about 15 °C, about 10 °C to about 20 °C, about 10 °C to about 25 °C, about 10 °C to about 30 °C, about 10 °C to about 35 °C, about 10 °C to about 40 °C, about 10 °C to about 50 °C, about 10 °C to about 60 °C, about 10 °C to about 70 °C, about 10 °C to about 80 °C, about 15 °C to about 20 °C, about 15 °C to about 25 °C, about 15 °C to about 30 °C, about 15 °C to about 35 °C, about 15 °C to about 40 °C, about 15 °C to about 50 °C, about 15 °C to about 60 °C, about 15 °C to about 70 °C, about 15 °C to about 80 °C, about 20 °C to about 25 °C, about 20 °C to about 30 °C, about 20 °C to about 35 °C, about 20 °C to about 40 °C, about 20 °C to about 50 °C, about 20 °C to about 60 °C, about 20 °C to about 70 °C, about 20 °C to about 80 °C, about 25 °C to about 30 °C, about 25 °C to about 35 °C, about 25 °C to about 40 °C, about 25 °C to about 50 °C, about 25 °C to about 60 °C, about 25 °C to about 70 °C, about 25 °C to about 80 °C, about 30 °C to about 35 °C, about 30 °C to about 40 °C, about 30 °C to about 50 °C, about 30 °C to about 60 °C, about 30 °C to about 70 °C, about

30 °C to about 80 °C, about 35 °C to about 40 °C, about 35 °C to about 50 °C, about 35 °C to about 60 °C, about

35 °C to about 70 °C, about 35 °C to about 80 °C, about 40 °C to about 50 °C, about 40 °C to about 60 °C, about

40 °C to about 70 °C, about 40 °C to about 80 °C, about 50 °C to about 60 °C, about 50 °C to about 70 °C, about

50 °C to about 80 °C, about 60 °C to about 70 °C, about 60 °C to about 80 °C, or about 70 °C to about 80 C.

[0558] In some cases, the system further comprises a translocase. As used herein, the term “protein translocase” or “translocase” (e.g., motor protein, unfoldase) can refer to a protein which can bind and/or translocate along an analyte through the chemical energy provided by NTP (nucleoside triphosphate) hydrolysis. The translocase may be able to unfold protein structure in the process. As used herein, the translocase may be able to move along an analyte to feed the analyte through the nanopore in sequential order. In some cases, the protein translocase is an NTP-driven unfoldase, such as an AAA+ unfoldase. See for example US2016/0032235 and Dougan et al. (FEBS Letters 529 (2002) 1873-3468) and Olivares et al. 2016, Nature Reviews Microbiology Vol. 14, pg. 33-44.

[0559] In some cases, members of the AAA+ superfamily have been identified in all organisms studied to date. They are involved in a wide range of cellular events. In bacteria, representatives of this superfamily are involved in functions as diverse as transcription and protein degradation and play an important role in the protein quality control network. Often, they may employ a common mechanism to mediate an ATP-dependent unfolding/disassembly of protein-protein or DNA-protein complexes. In an increasing number of examples, it appears that the activities of these AAA+ proteins may be modulated by a group of otherwise unrelated proteins, called adaptor proteins. In some cases, the translocase comprises an ATP-driven unfoldase. In some cases, the translocase comprises an NTP-driven unfoldase. In some cases, the translocase comprises an AAA+ enzyme. In some cases, the AAA+ enzyme is selected from the group consisting of ClpX, ClpA, Pan, LON, VAT, AMA, 854, MBA, SAMP, ClpC, ClpE, HsIU, ClpY, LonA, LonB, FtsH, Mpa, Cpa, Mspl, SecA, and functional homologs, orthologs, paralogs thereof.

[0560] In some cases, the nanopore system can have a cis-to-trans electro-osmotic flow, or vice versa, which creates a drag on the particles dispersed in the solution (independent of their charge) that is often termed an electro-osmotic force (EOF). The EOF arises 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 (Chinappi et al., 2020, ACS Nano, 14, 11, pg. 15816-15828), which imposes a significant force on any molecules within the flux. Electroosmosis can either compete or cooperate with electrophoresis (EPF).

[0561] In some cases, the nanopore system has a cis-to-trans electro-osmotic flow with an EOF that dominates over EPF. A dominant cis-to-trans EOF enables capture and translocation of complex and charged analyte s against EPF acting in a trans-to-cis direction. This selected high and dominant EOF is believed to capture and retain the analyte in the nanopore. The EOF pulls on the analyte and/or the translocase directly, pulling the analyte through the nanopore and in turn through transferred force keeps the translocase pinned to the top of the nanopore, whereupon the translocase can then control the translocation of the analyte through the nanopore. Without an EOF, the entire complex may be ejected in cases where the charge on the analyte section within the nanopore was of a polarity such that the net EPF repelled the analyte from the trans side-to-the cis side. Alternatively, in cases where the analyte has net neutral charge (i.e. a balance of negative and positive residues) or no charged residues, without EOF the analyte may most likely become stuck in the nanopore, while the translocase above the nanopore may continue to move along the analyte and away from the top of the nanopore, thus no longer controlling the movement of the analyte in any manner. Analytes can have a diversity of positive, negative and neutral sections, it is not possible to control translocation without a dominant EOF.

[0562] In some cases, the cis side is meant to indicate the compartment of the sensor system to which analyte(s) is added and/or the nanopore is added in the case of a biologically derived nanopore (and assuming vectorial insertion as most nanopores have a selective insertion orientation based on which compartment they are inserted from). However, it is to be noted that the terms ‘ ’trans ’ ’ and ‘ ’cis ’ ’ are used herein as the common convention determined by electronics/voltage polarity at the trans electrode. For example, without wishing to be bound to any one type of electrical circuit as many options are possible, the cis chamber is at ground and the applied transmembrane potential is given as the potential on the trans side i.e. the trans potential minus the cis potential. A positive current is one in which positive charge (e.g. K⁺ ions) moves through the nanopore from the trans to the cis side, or negative charge (e.g. Cl ions) from the cis to the trans side (see e.g. Maglia et al. Methods Enzymol. 2010; 475: 591-623).

[0563] In some cases, the direction of the EOF may be dependent on the polarity of the applied voltage and the relative conditions in the cis and trans compartments in combination with any ion selectivity of the nanopore. Further, the present disclosure teaches that the direction of the EOF (be it cis-lo-trans or trans-lo- cis) dictates the direction of the net forces acting to translocate the analyte across the nanopore, and thus dictates to which side the analyte and translocase are added within the context of the methods described herein. Thus, for example, it is also possible within the context of the present disclosure to add the analyte to the trans side of the membrane to enable trans-to-cis threading for a system where the EOF is created in a trans-lo-cis direction. A person of skill will also understand that while it is conventional to insert biological nanopores from the cis compartment, it will also be possible to insert them from the trans compartment, and that both orientations of the nanopore relative to the EOF can be employed.

[0564] The ion-selectivity (the specificity for translocating one ion species over another) is reflected in the GHK flux equation by the ion-permeability of each ion species.

[0565] The ion selectivity of a nanopore system can be quantified by methods of measuring the currentvoltage (I-V) relationship under asymmetric electrolyte conditions. Under asymmetric electrolyte conditions, a net flow of ions will occur when no voltage is applied (V_m = 0 mV). However, when a specific reversal potential (IT) is applied, the flux of positive and negative ions is equal in magnitude and direction and no net current is measured across the system, enabling the GHK flux equation to be solved at 0 pA for both species of ions to discover the ion-selectivity ratio, as set forth in Formula I.

[0566] P(x+) and P(Y-) can denote the permeability of the nanopore system for cation species X and anion species Y respectively. [«y-] 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 (known and tabulated for most electrolytes (Lide, D. R., 2003, CRC handbook of chemistry and physics, 84th edition, Handb. Chem. Phys. 53, 2616)). The latter is to correct for the presence of other ions in concentrated electrolyte solutions. The empirical ion-selectivity ratio (P(X+/P(Y-)) can inserted back into the GHK flux equations in combination with experimental measurements of ionic current versus applied voltage (I-V curves) for a nanopore 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 nanopore system of the present disclosure, and then used in the GHK flux equations to determine the underlying ionic current flows for nanopore systems containing a mixtures of two or more ion species (e.g. asymmetric salts types).

[0567] In some cases, 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 cases, 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.

[0568] In some cases, 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 cases, 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.

[0569] In some cases, 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.

[0570] In some cases, the applied voltage across the membrane can be at least about 1 mV, at least about 5 mV, at least about 10 mV, at least about 20 mV, at least about 30 mV, at least about 40 mV, at least about 50 mV, at least about 60 mV, at least about 70 mV, at least about 80 mV, at least about 90 mV, at least about 100 mV, at least about 150 mV, at least about 200 mV, at least about 250 mV, at least about 300 mV, at least about 350 mV, at least about 400 mV, at least about 450 mV, at least about 500 mV, at least about 600 mV, at least about 700 mV, at least about 800 mV, at least about 900 mV, at least about 1000 mV, or greater than about 1000 mV in magnitude. In some cases, the applied voltage across the membrane can be at least about 1000 mV, at most about 900 mV, at most about 800 mV, at most about 700 mV, at most about 600 mV, at most about 500 mV, at most about 450 mV, at most about 400 mV, at most about 350 mV, at most about 300 mV, at most about 250 mV, at most about 200 mV, at most about 150 mV, at most about 100 mV, at most about 90 mV, at most about 80 mV, at most about 70 mV, at most about 60 mV, at most about 50 mV, at most about 40 mV, at most about 30 mV, at most about 20 mV, at most about 10 mV, at most about 5 mV, at most about 1 mV, or less than about 1 mV in magnitude.

[0571] In some cases, the applied voltage across the membrane can be from about 1 mV to about 100 mV in magnitude. In some cases, the applied voltage across the membrane can be from about 1 mV to about 5 mV, about 1 mV to about 10 mV, about 1 mV to about 20 mV, about 1 mV to about 30 mV, about 1 mV to about 40 mV, about 1 mV to about 50 mV, about 1 mV to about 60 mV, about 1 mV to about 70 mV, about 1 mV to about 80 mV, about 1 mV to about 90 mV, about 1 mV to about 100 mV, about 5 mV to about 10 mV, about 5 mV to about 20 mV, about 5 mV to about 30 mV, about 5 mV to about 40 mV, about 5 mV to about 50 mV, about 5 mV to about 60 mV, about 5 mV to about 70 mV, about 5 mV to about 80 mV, about 5 mV to about 90 mV, about 5 mV to about 100 mV, about 10 mV to about 20 mV, about 10 mV to about 30 mV, about 10 mV to about 40 mV, about 10 mV to about 50 mV, about 10 mV to about 60 mV, about 10 mV to about 70 mV, about 10 mV to about 80 mV, about 10 mV to about 90 mV, about 10 mV to about 100 mV, about 20 mV to about 30 mV, about 20 mV to about 40 mV, about 20 mV to about 50 mV, about 20 mV to about 60 mV, about 20 mV to about 70 mV, about 20 mV to about 80 mV, about 20 mV to about 90 mV, about 20 mV to about 100 mV, about 30 mV to about 40 mV, about 30 mV to about 50 mV, about 30 mV to about 60 mV, about 30 mV to about 70 mV, about 30 mV to about 80 mV, about 30 mV to about 90 mV, about 30 mV to about 100 mV, about 40 mV to about 50 mV, about 40 mV to about 60 mV, about 40 mV to about 70 mV, about 40 mV to about 80 mV, about 40 mV to about 90 mV, about 40 mV to about 100 mV, about 50 mV to about 60 mV, about 50 mV to about 70 mV, about 50 mV to about 80 mV, about 50 mV to about 90 mV, about 50 mV to about 100 mV, about 60 mV to about 70 mV, about 60 mV to about 80 mV, about 60 mV to about 90 mV, about 60 mV to about 100 mV, about 70 mV to about 80 mV, about 70 mV to about 90 mV, about 70 mV to about 100 mV, about 80 mV to about 90 mV, about 80 mV to about 100 mV, or about 90 mV to about 100 mV in magnitude.

[0572] In some cases, the applied voltage across the membrane can be from about 100 mV to about 1,000 mV in magnitude. In some cases, the applied voltage across the membrane can be from about 100 mV to about 150 mV, about 100 mV to about 200 mV, about 100 mV to about 250 mV, about 100 mV to about 300 mV, about 100 mV to about 400 mV, about 100 mV to about 500 mV, about 100 mV to about 600 mV, about 100 mV to about 700 mV, about 100 mV to about 800 mV, about 100 mV to about 900 mV, about 100 mV to about 1,000 mV, about 150 mV to about 200 mV, about 150 mV to about 250 mV, about 150 mV to about 300 mV, about 150 mV to about 400 mV, about 150 mV to about 500 mV, about 150 mV to about 600 mV, about 150 mV to about 700 mV, about 150 mV to about 800 mV, about 150 mV to about 900 mV, about 150 mV to about 1,000 mV, about 200 mV to about 250 mV, about 200 mV to about 300 mV, about 200 mV to about 400 mV, about 200 mV to about 500 mV, about 200 mV to about 600 mV, about 200 mV to about 700 mV, about 200 mV to about 800 mV, about 200 mV to about 900 mV, about 200 mV to about 1,000 mV, about 250 mV to about 300 mV, about 250 mV to about 400 mV, about 250 mV to about 500 mV, about 250 mV to about 600 mV, about 250 mV to about 700 mV, about 250 mV to about 800 mV, about 250 mV to about 900 mV, about 250 mV to about 1,000 mV, about 300 mV to about 400 mV, about 300 mV to about 500 mV, about 300 mV to about 600 mV, about 300 mV to about 700 mV, about 300 mV to about 800 mV, about 300 mV to about 900 mV, about 300 mV to about 1,000 mV, about 400 mV to about 500 mV, about 400 mV to about 600 mV, about 400 mV to about 700 mV, about 400 mV to about 800 mV, about 400 mV to about 900 mV, about 400 mV to about 1,000 mV, about 500 mV to about 600 mV, about 500 mV to about 700 mV, about 500 mV to about 800 mV, about 500 mV to about 900 mV, about 500 mV to about 1,000 mV, about 600 mV to about 700 mV, about 600 mV to about 800 mV, about 600 mV to about 900 mV, about 600 mV to about 1,000 mV, about 700 mV to about 800 mV, about 700 mV to about 900 mV, about 700 mV to about 1,000 mV, about 800 mV to about 900 mV, about 800 mV to about 1,000 mV, or about 900 mV to about 1,000 mV in magnitude.

[0573] In some cases, the applied voltage across the membrane can be about 1 mV, about 5 mV, about 10 mV, about 20 mV, about 30 mV, about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about 90 mV, about 100 mV, about 150 mV, about 200 mV, about 250 mV, about 300 mV, about 350 mV, about 400 mV, about 450 mV, about 500 mV, about 600 mV, about 700 mV, about 800 mV, about 900 mV, or about 1000 mV in magnitude.

[0574] In some cases, mechanisms of controlling the EOF by inducing a strong net flow of ions in one direction across the nanopore are known in the art. In some cases, the EOF can be controlled by inducing a strong net flow of ion ions in one direction across the nanopore. The EOF can be controlled by modifying of the nanopore, applying specific electrolyte asymmetries and concentrations, or any combination thereof.

[0575] In some cases, the nanopore is comprised in a membrane separating a fluidic chamber of a nanopore system into a first side (e.g., cis side) and a second side (e.g., a trans side). The term "membrane" is used herein in its conventional sense to refer to a thin, film-like structure that separates the chamber of the system into a first side (e.g., cis side) and a second side (e.g., a trans side). The membrane separating the first and second sides can comprise at least one nanopore or channel. Membranes can be classified into synthetic membranes and biological membranes.

[0576] In some cases, the membrane can be an amphiphilic layer. An amphiphilic layer can be a layer formed from amphiphilic molecules, such as phospholipids, which may have both at least one hydrophilic portion, and at least one lipophilic portion, hydrophobic portion, or any combination thereof. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles which form a monolayer can include, for example, block copolymers.

[0577] The sensor system can be advantageously integrated in a portable device comprising a plurality of sensor systems. In some cases, a portable device may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more of the sensor systems described herein. Each sensor system may be configured to characterize the same analyte. Sensor systems within a portable device may be configured to characterize different analytes. Analytes may differ on size, length, weight, pH, charge, chemical composition, or any combination thereof. For example, the system may be comprised in a point-of-care diagnostic medical devices, which are in vitro diagnostics used by health care professionals to obtain results rapidly near or at the site of a patient. These products can be useful to quickly determine a marker responsible for a certain disease, e.g., at a doctor's office or clinic. The device can be designed for performing an analytical method as herein disclosed. The device can be a portable device, a medical device, implant, single use device, or a disposable device. In one aspect, the device can be configured to allow for real-time detection of at least one analyte, for example a clinically relevant analyte. Real-time detection may comprise detecting an analyte within 2000, 1000, 750, 500, 250, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less than 10 milliseconds (ms) from applying the analyte to the device (e.g., applying the analyte to the sensor system). A clinically relevant analyte may comprise a protein or peptide sample from any disease or condition (e.g., an infectious disease, a cancer, or an autoimmune disease). A clinically relevant analyte may comprise a protein or peptide sample from a viral or bacterial pathogen.

[0578] The nanopore systems described herein may characterize a number of analytes. In some cases, a nanopore system described herein may characterize at least about 2 analytes, at least about 3 analytes, at least about 4 analytes, at least about 5 analytes, at least about 6 analytes, at least about 7 analytes, at least about 8 analytes, at least about 9 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 200 analytes, at least about 300 analytes, at least about 400 analytes, at least about 500 analytes, at least about 600 analytes, at least about 700 analytes, at least about 800 analytes, at least about 900 analytes, at least about 1000 analytes, at least about 1500 analytes, at least about 2000 analytes, at least about 2500 analytes, at least about 3000 analytes, at least about 3500 analytes, at least about 4000 analytes, at least about 4500 analytes, at least about 5000 analytes, at least about 5500 analytes, at least about 6000 analytes, at least about 6500 analytes, at least about 7000 analytes, at least about 7500 analytes, at least about 8000 analytes, at least about 8500 analytes, at least about 9000 analytes, at least about 9500 analytes, at least about 10000 analytes, or greater than about 10000 analytes. In some cases, a nanopore system described herein may characterize at most about 10000 analytes, at most about 9500 analytes, at most about 9000 analytes, at most about 8500 analytes, at most about 8000 analytes, at most about 7500 analytes, at most about 7000 analytes, at most about 6500 analytes, at most about 6000 analytes, at most about 5500 analytes, at most about 5000 analytes, at most about 4500 analytes, at most about 4000 analytes, at most about 3500 analytes, at most about 3000 analytes, at most about 2500 analytes, at most about 2000 analytes, at most about 1500 analytes, at most about 1000 analytes, at most about 900 analytes, at most about 800 analytes, at most about 700 analytes, at most about 600 analytes, at most about 500 analytes, at most about 400 analytes, at most about 300 analytes, at most about 200 analytes, at most about 100 analytes, at most about 90 analytes, at most about 80 analytes, at most about 70 analytes, at most about 60 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 9 analytes, at most about 8 analytes, at most about 7 analytes, at most about 6 analytes, at most about 5 analytes, at most about 4 analytes, at most about 3 analytes, at most about 2 analytes, or less than about 2 analytes.

[0579] In some cases, a nanopore system described herein may characterize from about 2 analytes to about 100 analytes may be characterized. In some cases, from about 2 analytes to about 5 analytes, about 2 analytes to about 10 analytes, about 2 analytes to about 20 analytes, about 2 analytes to about 30 analytes, about 2 analytes to about 40 analytes, about 2 analytes to about 50 analytes, about 2 analytes to about 60 analytes, about 2 analytes to about 70 analytes, about 2 analytes to about 80 analytes, about 2 analytes to about 90 analytes, about 2 analytes to about 100 analytes, about 5 analytes to about 10 analytes, about 5 analytes to about 20 analytes, about 5 analytes to about 30 analytes, about 5 analytes to about 40 analytes, about 5 analytes to about 50 analytes, about 5 analytes to about 60 analytes, about 5 analytes to about 70 analytes, about 5 analytes to about 80 analytes, about 5 analytes to about 90 analytes, about 5 analytes to about 100 analytes, about 10 analytes to about 20 analytes, about 10 analytes to about 30 analytes, about 10 analytes to about 40 analytes, about 10 analytes to about 50 analytes, about 10 analytes to about 60 analytes, about 10 analytes to about 70 analytes, about 10 analytes to about 80 analytes, about 10 analytes to about 90 analytes, about 10 analytes to about 100 analytes, about 20 analytes to about 30 analytes, about 20 analytes to about 40 analytes, about 20 analytes to about 50 analytes, about 20 analytes to about 60 analytes, about 20 analytes to about 70 analytes, about 20 analytes to about 80 analytes, about 20 analytes to about 90 analytes, about 20 analytes to about 100 analytes, about 30 analytes to about 40 analytes, about 30 analytes to about 50 analytes, about 30 analytes to about 60 analytes, about 30 analytes to about 70 analytes, about 30 analytes to about 80 analytes, about 30 analytes to about 90 analytes, about 30 analytes to about 100 analytes, about 40 analytes to about 50 analytes, about 40 analytes to about 60 analytes, about 40 analytes to about 70 analytes, about 40 analytes to about 80 analytes, about 40 analytes to about 90 analytes, about 40 analytes to about 100 analytes, about 50 analytes to about 60 analytes, about 50 analytes to about 70 analytes, about 50 analytes to about 80 analytes, about 50 analytes to about 90 analytes, about 50 analytes to about 100 analytes, about 60 analytes to about 70 analytes, about 60 analytes to about 80 analytes, about 60 analytes to about 90 analytes, about 60 analytes to about 100 analytes, about 70 analytes to about 80 analytes, about 70 analytes to about 90 analytes, about 70 analytes to about 100 analytes, about 80 analytes to about 90 analytes, about 80 analytes to about 100 analytes, or about 90 analytes to about 100 analytes.

[0580] In some cases, the system described herein can comprise a sensor or an array of sensors. The system can comprise an electrical energy source and two or more electrodes. In some cases, the system comprises a pair of electrodes. In some cases, an electrode may be disposed on a first side (e.g., a cis side) of the membrane of a sensor, and another electrode (e.g., a second electrode) may be disposed on a second side (e.g., a trans 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 applied potential can also cause an analyte, if charged, to translocate to the pore and reside in the pore. The applied potential can create an electrophoretic force (EPF), which can provide a driving force for an analyte to translocate to the pore in order to generate a change in signal. The sensor system 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 to a pore. These electrodes can be configured to measure the current across a membrane as an analyte translocates to a pore and reside in a pore (e.g., in a constriction region of a pore). The sensor system can be in electrical communication with a recording device to record measured signals. The system can be in electrical communication with a computer or a processor (e.g., a circuit, an integrated circuit, etc.), which can receive a signal from the sensor or the array of sensors, store the signals in digital form, and/or process the signal.

[0581] In some cases, the EOF can be employed in the nanopore system of the present disclosure. Where EOF has previously been employed in nanopore systems, it has most often been either in the trans-to-cis direction acting against a cis-to-trans EPF (slowing down the EPF driven translocation), or in the cis-to-trans direction in combination with a cis-to-trans EPF to aid translocation. [0582] In some cases, the translocation of the analyte through the nanopore occurs in the direction of the electro-osmotic force (EOF). In some cases, the translocation of the analyte through the nanopore occurs in the opposite direction of the electrophoretic force. In some cases, the translocation of the analyte through the nanopore occurs in the direction of the EOF and opposite the direction of the EPF.

[0583] In some cases, the EOF can be greater than the EPF. In some cases, the EOF is between about 0. 1% to about 500% greater than the EPF. In some cases, the EOF is 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% longer greater than the EPF.

[0584] In some cases, the EOF 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%, 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% greater than the EPF.

[0585] In some cases, the EOF 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% greater than the EPF.

[0586] In some cases, the EOF 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% greater than the EPF.

[0587] Alternatively, in some cases, the translocation of the analyte through the nanopore occurs in the direction of the EPF. In some cases, the translocation of the analyte through the nanopore occurs in the opposite direction of the EOF. In some cases, the translocation of the analyte through the nanopore occurs in the direction of the EPF and opposite the direction of the EOF.

[0588] Alternatively, in some cases, the EPF can be greater than the EOF. In some cases, the EPF can be greater than the EOF. In some cases, the EPF is between about 0. 1% to about 500% greater than the EOF. In some cases, the EPF is 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% longer greater than the EOF.

[0589] In some cases, the EPF 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%, 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% greater than the EOF.

[0590] In some cases, the EPF 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

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% greater than the EOF.

[0591] In some cases, the EPF 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

440%, about 450%, about 460%, about 470%, about 480%, about 490%, or about 500% greater than the EOF. [0592] In some cases, the translocation of the analyte through the nanopore occurs in the direction of the EOF. In some cases, the translocation of the analyte through the nanopore occurs in the direction of the EPF. In some cases, the translocation of the analyte through the nanopore occurs in the direction of the EOF and the direction of the EPF.

[0593] In some cases, a system described herein comprises at least one solution. In some cases, the first side of the system can comprise a first solution and the second side of the system can comprise a second solution. In some cases, the first solution and second solution may be the same. In some cases, the first solution and second solution may be different. In some cases, the first solution comprises a first concentration of a solute. The second solution can comprise a second concentration of the solute. In some cases, the first solution and second solution comprise the same solute. In some cases, the first solution and second solution comprise different solutes. The first solution can comprise a concentration of a first solute and the second solution can comprise a concentration of a second solute.

[0594] In some cases, the solute can comprise an ion. In some cases, the solute can comprise an osmolyte. In some cases, an osmolyte can comprise a non-ionic or a zwitterionic solute, (e.g., glycine betaine, glucose, sucrose, glycerol, PEGs, dextrans, or any combination thereof). In some cases, different ionic concentrations between the first and second sides of a system may result in high mobility ions and low mobility ions. Without wishing to be bound by theory, 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 to generate an EOF of the sensor system. [0595] Salt imbalances on two sides of a pore can create strong osmotic gradients. Specific osmolytes can be selected and balanced based on their osmolarity and their concentrations. In some cases, 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 to the pore. In some cases, 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 to the pore.

[0596] In some cases, 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 cases, 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.

[0597] In some cases, 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 cases, 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.

[0598] In some cases, 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.

[0599] In some cases, a salt, ion, osmolyte, or electrolyte concentration on the second side (e.g., trans 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 cases, a salt, ion, osmolyte, or electrolyte concentration on the second side (e.g., trans 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.

[0600] In some cases, a salt, ion, osmolyte, or electrolyte concentration on the second side (e.g., trans side) can be from about 0.01 M to about 5 M. In some cases, a salt, ion, osmolyte, or electrolyte concentration on the second side (e.g., trans 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.

[0601] In some cases, a salt, ion, osmolyte, or electrolyte concentration on the second side (e.g., trans 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. In some cases, the salt may be a non-halide salt. In some cases, the salt may not be a non-halide salt.

[0602] In some cases, there is no difference in salt, ion, or electrolyte concentrations between the first side and second side of the chamber. In some cases, a difference in salt, ion, or electrolyte concentrations between the cis and trans sides can be at least about 0.01 M, at least about 0.05, at least about 0. 10, at least about 0.20, at least about 0.30, at least about 0.40, at least about 0.50, at least about 0.60, at least about 0.70, at least about 0.80, at least about 0.90, at least about 1.00, at least about 1. 10, at least about 1.25, at least about 1.50, at least about 1.75, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5 M, or greater than about 5 M. In some embodiments, a difference in salt, ion, or electrolyte concentrations between the cis and trans sides 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.

[0603] In some cases, an engineered biological nanopore described herein may comprise one or more modifications (e.g., amino acid mutations) to enhance an EOF. The generated EOF may be strong enough to provide for one or more analytes to be captured by the engineered biological nanopore and/or be translocated through the engineered biological nanopore. In some cases, the strong EOF from the engineered biological nanopore described herein may be generated in a system with symmetric salt concentrations. In some cases, the strong EOF from the engineered biological nanopore described herein may be generated in a system with asymmetric salt concentrations.

Methods and Applications

[0604] 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, one or more ligands coupled to the biopolymer, or any combination thereof.

[0605] In another aspect of the present disclosure, provided herein is a method comprising: providing a nanopore system. The nanopore system may comprise a membrane comprising any nanopore described herein. 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. A first solution on a first side, a second solution on a second side, or a nanopore disclosed herein, or any combination thereof, may be configured to translocate an analyte using an electro-osmotic flow. 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 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.

[0606] In some aspects, the present disclosure provides methods comprising providing a nanopore system described herein. The system may comprise a fluidic chamber. The system may comprise a membrane. The membrane may comprise an engineered biological nanopore described herein. The membrane may separate the fluid chamber. The fluid chamber may be separated into a first side (e.g., a cis side) and/or a second side (e.g., a trans side). The engineered biological nanopore may be an engineered biological nanopore described herein. For example, the engineered biological nanopore may comprise a channel. The channel may comprise a first region. The channel may comprise a second region. The second region may have a constriction region. The first region of the engineered biological nanopore may be modified. The first region may be modified to be more net negative than a respective region of a wild-type biological nanopore. The second region of the engineered biological nanopore may be modified. The second region may be modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore. The second region may be modified to be more net neutral than a respective region of a wild-type biological nanopore. The second region may be modified to be more net negative than a respective region of a wild-type biological nanopore. The first region may be modified to be more net negative than a respective region of a wild-type biological nanopore and (ii) the second region may be modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore. The first region of the channel may be adjacent to the second region of the channel. The method may comprise contacting the engineered biological nanopore with a biopolymer. The biopolymer may be any biopolymer described herein.

[0607] In some aspects, the present disclosure provides methods comprising providing a nanopore system described herein. The system may comprise a fluidic chamber. The system may comprise a membrane. The membrane may comprise an engineered biological nanopore described herein. The membrane may separate the fluid chamber. The fluid chamber may be separated into a first side (e.g., a cis side) and/or a second side (e.g., a trans side). The engineered biological nanopore may be an engineered biological nanopore described herein. For example, the engineered biological nanopore may comprise a channel. The channel may comprise a first region. The channel may comprise a second region. The second region may have a constriction region. The first region of the channel may be adjacent to the second region of the channel. The first region of the channel may have a negative charge. The second region of the channel may have a neutral charge. The first region of the channel may be adjacent to the second region of the channel. The method may comprise contacting the engineered biological nanopore with a biopolymer. The biopolymer may be any biopolymer described herein.

[0608] In some aspects, the present disclosure provides methods comprising providing a nanopore system described herein. The system may comprise a fluidic chamber. The system may comprise a membrane. The membrane may comprise an engineered biological nanopore described herein. The membrane may separate the fluid chamber. The fluid chamber may be separated into a first side (e.g., a cis side) and/or a second side (e.g., a trans side). The engineered biological nanopore may be an engineered biological nanopore described herein. For example, the engineered biological nanopore may comprise a channel. The channel may comprise a first region. The channel may comprise a second region. The second region may have a constriction region. The first region of the channel may be adjacent to the second region of the channel. The first region of the channel may have a negative charge. The second region of the channel may have a net charge. The net charge of the second region of the channel may be at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore (e.g., a constriction region of a wild-type biological nanopore). The method may comprise contacting the engineered biological nanopore with a biopolymer. The biopolymer may be any biopolymer described herein.

[0609] In another embodiment the translocase is coupled to the nanopore. In the system of the present disclosure the translocase may not be coupled to the top of the nanopore to optimally feed the analyte into the nanopore. Instead the strong cis-to-trans EOF of the present disclosure enables the portion of an analyte extruded from the translocase to be captured into the nanopore and translocated, which will in turn pull the translocase atop the pore, whereupon it will continue to control the movement of the extruded analyte. In this embodiment the analyte may not have stall or capture motifs due to the proximity of the extruded analyte to the nanopore entrance. [0610] 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. [0611] Provided herein comprises a method for improving the sensing properties of a nanopore disclosed herein. The method comprises increasing a net negative charge at (1) one or more first portions and/or one or more third portions, and/or (2) one or more second portions disclosed here.

[0612] In some aspects, the present disclosure provides a method comprising (a) providing a mixture. The mixture may contain or be suspected of containing an analyte. The analyte may comprise polypeptide and/or protein. The analyte may be any analyte described herein. The method may comprise using an engineered biological nanopore. The engineered biological nanopore may be any nanopore described herein. The engineered biological nanopore may be used to determine a sequence. The engineered biological nanopore may be used to generate a measure of a concentration or relative amount of an analyte in a mixture at an accuracy of at least 80%. The engineered biological nanopore may be used to determine a sequence and generate a measure of a concentration or relative amount of an analyte in a mixture at an accuracy of at least 80%.

[0613] In some cases, the mixture may contain or be suspected of containing an additional analyte. The additional analyte may comprise an additional protein, polypeptide, peptide, or any combination thereof. The additional analyte may be the same as the analyte. The additional analyte may be different from the analyte. In some cases, a nanopore (e.g., an engineered biological nanopore described herein) may be used to generate one or more measures (e.g., a concentration and/or relative amount). The measure may be a concentration and/or relative amount of an additional analyte. The measure (e.g., a concentration and/or relative amount) may be made with a level of accuracy. For example, the measure (e.g., a concentration and/or relative amount) of the additional analyte in a mixture may be generated at an accuracy of at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater than about 99%. For example, the measure (e.g., a concentration and/or relative amount) of the additional analyte in a mixture may be generated at an accuracy of at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, or less than about 60%. The measure (e.g., a concentration and/or relative amount) of the additional analyte in a mixture may be generated at an accuracy of 100%.

[0614] In some cases, a sequence of the analyte may be determined and/or a measure (e.g., a concentration and/or relative amount) of the analyte may be generated. The sequence or measure may be determined at a level of accuracy. For example, a sequence of the analyte may be determined and/or a measure (e.g., a concentration and/or relative amount) of the analyte may be generated at an accuracy of at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater than about 99%. For example, a sequence of the analyte may be determined and/or a measure (e.g., a concentration and/or relative amount) of the analyte may be generated at an accuracy of at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, or less than about 60%. The sequence of the analyte may be determined and/or a measure (e.g., a concentration and/or relative amount) of the analyte may be generated at an accuracy of 100%.

[0615] 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 is to be added to the first side (e.g., cis side) and translocated through the nanopore to the 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 cases, the nanopore system is 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 is captured in the nanopore with on top of the nanopore the translocase controlling the translocation. The nanopore system can have a cis to trans EOF resulting from a net ionic current flow cis-lo-trans over total ionic current flow of greater than 0.2 or less than -0.2, greater than 0.3 or less than -0.3, greater than 0.35 or less than -0.35. Without wishing to be bound by theory, under an applied positive potential, a nanopore system described herein can have a cis to trans electro-osmotic force resulting from a cis to trans net ionic current flow. The cis to trans electro-osmotic force may translocate a portion of an analyte (e.g., at least a portion of a biopolymer) through a nanopore described herein.

[0616] In some cases, a first side (e.g., cis) to second side (e.g., 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, greater than about 0. 1, greater than about 0.2, greater than about 0.3, greater than about 0.4, greater than about 0.5, greater than about 0.6, greater than about 0.7, greater than about 0.8, greater than about 0.9, greater than about 0.95, or greater than about 0.99. In some embodiments, a first side (e.g., cis) to second side (e.g., trans) EOF results from a net ionic current flow trans to cis over a total ionic current flow, also referred to as a relative net current flow cis to trans, of less than about 0.0, less than about - 0. 1, less than about -0.2, less than about -0.3, less than about -0.4, less than about -0.5, less than about -0.6, less than about -0.7, less than about -0.8, less than about -0.9, less than about -0.95, or less than about -0.99. [0617] In some cases, the absolute relative net electro-osmotic current over applied voltage, is greater than about 0.01, greater than about 0.02, greater than about 0.03, greater than about 0.04, greater than about 0.05, greater than about 0.06, greater than about 0.07, greater than about 0.08, greater than about 0.09, greater than about 0. 10, greater than about 0. 15, greater than about 0.2, greater than about 0.3, greater than about 0.4, greater than about 0.5, greater than about 0.6, greater than about 0.7, greater than about 0.8, greater than about 0.9, or greater than about 1 picoampere per millivolt (pA/mV). In some cases, the absolute relative net electro-osmotic current over applied voltage, is less than about 0.01, less than about 0.02, less than about 0.03, less than about 0.04, less than about 0.05, less than about 0.06, less than about 0.07, less than about 0.08, less than about 0.09, less than about 0. 10, less than about 0. 15, less than about 0.2, less than about 0.3, less than about 0.4, less than about 0.5, less than about 0.6, less than about 0.7, less than about 0.8, less than about 0.9, or less than about 1 pA/mV.

[0618] The methods provided herein may comprise contacting an analyte (e.g., a biopolymer) with a nanopore. An analyte may contact a nanopore at any location of the nanopore. An analyte may contact a nanopore at a first opening (e.g., cis opening) or a second opening (e.g., a trans opening). An analyte may contact a nanopore within the pore (e.g., channel) of the nanopore. The analyte may contact and/or interact with amino acid residues within the channel. The analyte may contact a constriction region of the nanopore. [0619] A method provided herein comprises measuring a signal generated by the translocation of the analyte (e.g., the biopolymer) to the pore (e.g., the engineered biological nanopore) and reside in the pore. 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 and/or conductivity can also be measured. In some cases, current rectification can be measured. In some cases, fluorescence probes for reporting ionic flux or field effect transistor systems can be used to measure properties of a translocation and/or capture event. In some cases, changes in the system’s ionic concentrations can be measured without an applied electric potential. Instead, the changes may be measured by a chemical gradient of ions and/or analytes can provide the driving force for translocation of analytes to a pore and create measurable signals. In some cases, the applied potential can be a chemical potential or applied electric potential. A system described herein can comprise electrodes, spectroscopy tools, microscopes, etc. to measure the signals.

[0620] An applied electric potential can be maintained at a constant or fluctuating voltage for a fixed period (milliseconds, seconds, minutes, hours). In some cases, 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, sinusoidal, etc.). Waveforms of different amplitudes, frequencies, and shapes can be used to translocate analytes, which can produce different signals from the same analytes.

[0621] In some cases, the absolute relative net electro-osmotic flow over applied voltage (IrelV), can be at least about 0.01 pA/mV, at least about 0.02 pA/mV, at least about 0.03 pA/mV, at least about 0.04 pA/mV, at least about 0.05 pA/mV, at least about 0.06 pA/mV, at least about 0.07 pA/mV, at least about 0.08 pA/mV, at least about 0.09 pA/mV, at least about 0. 10 pA/mV, at least about 0. 15 pA/mV, at least about 0.2 pA/mV, at least about 0.3 pA/mV, at least about 0.4 pA/mV, at least about 0.5 pA/mV, at least about 0.6 pA/mV, at least about 0.7 pA/mV, at least about 0.8 pA/mV, at least about 0.9 pA/mV, at least about 1 pA/mV, or greater than about 1 pA/mV. In some cases, the absolute relative net electro-osmotic flow over applied voltage (IrelV), can be at most about 1 pA/mV, 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 0.15, at most about 0. 10, at most about 0.09, at most about 0.08, at most about 0.07, at most about 0.06, at most about 0.05, at most about 0.04, at most about 0.03, at most about 0.02, at most about 0.01, or less than about 0. 1 pA/mV.

[0622] In some cases, the absolute relative net electro-osmotic flow over applied voltage (IrelV), can be from about 0.01 pA/mV to about 1 pA/mV. In some cases, the absolute relative net electro-osmotic flow over applied voltage (IrelV), can be from about 0.01 pA/mV to about 0.02 pA/mV, about 0.01 pA/mV to about 0.04 pA/mV, about 0.01 pA/mV to about 0.06 pA/mV, about 0.01 pA/mV to about 0.08 pA/mV, about 0.01 pA/mV to about 0.1 pA/mV, about 0.01 pA/mV to about 0.15 pA/mV, about 0.01 pA/mV to about 0.2 pA/mV, about 0.01 pA/mV to about 0.4 pA/mV, about 0.01 pA/mV to about 0.6 pA/mV, about 0.01 pA/mV to about 0.8 pA/mV, about 0.01 pA/mV to about 1 pA/mV, about 0.02 pA/mV to about 0.04 pA/mV, about 0.02 pA/mV to about 0.06 pA/mV, about 0.02 pA/mV to about 0.08 pA/mV, about 0.02 pA/mV to about 0.1 pA/mV, about 0.02 pA/mV to about 0. 15 pA/mV, about 0.02 pA/mV to about 0.2 pA/mV, about 0.02 pA/mV to about 0.4 pA/mV, about 0.02 pA/mV to about 0.6 pA/mV, about 0.02 pA/mV to about 0.8 pA/mV, about 0.02 pA/mV to about 1 pA/mV, about 0.04 pA/mV to about 0.06 pA/mV, about 0.04 pA/mV to about 0.08 pA/mV, about 0.04 pA/mV to about 0. 1 pA/mV, about 0.04 pA/mV to about 0. 15 pA/mV, about 0.04 pA/mV to about 0.2 pA/mV, about 0.04 pA/mV to about 0.4 pA/mV, about 0.04 pA/mV to about 0.6 pA/mV, about 0.04 pA/mV to about 0.8 pA/mV, about 0.04 pA/mV to about 1 pA/mV, about 0.06 pA/mV to about 0.08 pA/mV, about 0.06 pA/mV to about 0. 1 pA/mV, about 0.06 pA/mV to about 0. 15 pA/mV, about 0.06 pA/mV to about 0.2 pA/mV, about 0.06 pA/mV to about 0.4 pA/mV, about 0.06 pA/mV to about 0.6 pA/mV, about 0.06 pA/mV to about 0.8 pA/mV, about 0.06 pA/mV to about 1 pA/mV, about 0.08 pA/mV to about 0. 1 pA/mV, about 0.08 pA/mV to about 0.15 pA/mV, about 0.08 pA/mV to about 0.2 pA/mV, about 0.08 pA/mV to about 0.4 pA/mV, about 0.08 pA/mV to about 0.6 pA/mV, about 0.08 pA/mV to about 0.8 pA/mV, about 0.08 pA/mV to about 1 pA/mV, about 0. 1 pA/mV to about 0. 15 pA/mV, about 0. 1 pA/mV to about 0.2 pA/mV, about 0. 1 pA/mV to about 0.4 pA/mV, about 0.1 pA/mV to about 0.6 pA/mV, about 0.1 pA/mV to about 0.8 pA/mV, about 0.1 pA/mV to about 1 pA/mV, about 0.15 pA/mV to about 0.2 pA/mV, about 0.15 pA/mV to about 0.4 pA/mV, about 0.15 pA/mV to about 0.6 pA/mV, about 0.15 pA/mV to about 0.8 pA/mV, about 0. 15 pA/mV to about 1 pA/mV, about 0.2 pA/mV to about 0.4 pA/mV, about 0.2 pA/mV to about 0.6 pA/mV, about 0.2 pA/mV to about 0.8 pA/mV, about 0.2 pA/mV to about 1 pA/mV, about 0.4 pA/mV to about 0.6 pA/mV, about 0.4 pA/mV to about 0.8 pA/mV, about 0.4 pA/mV to about 1 pA/mV, about 0.6 pA/mV to about 0.8 pA/mV, about 0.6 pA/mV to about 1 pA/mV, or about 0.8 pA/mV to about 1 pA/mV.

[0623] In some cases, the absolute relative net electro-osmotic flow over applied voltage (IrelV), can be about 0.01 pA/mV, about 0.02 pA/mV, about 0.03 pA/mV, about 0.04 pA/mV, about 0.05 pA/mV, about 0.06 pA/mV, about 0.07 pA/mV, about 0.08 pA/mV, about 0.09 pA/mV, about 0. 10 pA/mV, about 0.15 pA/mV, about 0.2 pA/mV, about 0.3 pA/mV, about 0.4 pA/mV, about 0.5 pA/mV, about 0.6 pA/mV, about 0.7 pA/mV, about 0.8 pA/mV, about 0.9 pA/mV, or about 1 pA/mV.

[0624] In some cases, electrodes of a sensor system described herein can provide an applied voltage. The applied voltage may generate the electro-osmotic force (EOF) which may assist in translocating the analyte to the pore. In some cases, the applied voltage may be a negative voltage on a first side of a fluid chamber of the system. In some cases, the applied voltage may be a positive voltage on a first side of a fluid chamber of the system. In some cases, the applied voltage may be a negative voltage on a second side of a fluid chamber of the system. In some cases, the applied voltage may be a positive voltage on a second side of a fluid chamber of the system. In some cases, the applied voltage across the membrane can be at least about 1 mV, at least about 5 mV, at least about 10 mV, at least about 20 mV, at least about 30 mV, at least about 40 mV, at least about 50 mV, at least about 60 mV, at least about 70 mV, at least about 80 mV, at least about 90 mV, at least about 100 mV, at least about 150 mV, at least about 200 mV, at least about 250 mV, at least about 300 mV, at least about 350 mV, at least about 400 mV, at least about 450 mV, at least about 500 mV, at least about 600 mV, at least about 700 mV, at least about 800 mV, at least about 900 mV, at least about 1000 mV, or greater than about 1000 mV in magnitude. In some cases, the applied voltage across the membrane can be at least about 1000 mV, at most about 900 mV, at most about 800 mV, at most about 700 mV, at most about 600 mV, at most about 500 mV, at most about 450 mV, at most about 400 mV, at most about 350 mV, at most about 300 mV, at most about 250 mV, at most about 200 mV, at most about 150 mV, at most about 100 mV, at most about 90 mV, at most about 80 mV, at most about 70 mV, at most about 60 mV, at most about 50 mV, at most about 40 mV, at most about 30 mV, at most about 20 mV, at most about 10 mV, at most about 5 mV, at most about 1 mV, or less than about 1 mV in magnitude.

[0625] In some cases, the applied voltage across the membrane can be from about 1 mV to about 100 mV in magnitude. In some cases, the applied voltage across the membrane can be from about 1 mV to about 5 mV, about 1 mV to about 10 mV, about 1 mV to about 20 mV, about 1 mV to about 30 mV, about 1 mV to about 40 mV, about 1 mV to about 50 mV, about 1 mV to about 60 mV, about 1 mV to about 70 mV, about 1 mV to about 80 mV, about 1 mV to about 90 mV, about 1 mV to about 100 mV, about 5 mV to about 10 mV, about 5 mV to about 20 mV, about 5 mV to about 30 mV, about 5 mV to about 40 mV, about 5 mV to about 50 mV, about 5 mV to about 60 mV, about 5 mV to about 70 mV, about 5 mV to about 80 mV, about 5 mV to about 90 mV, about 5 mV to about 100 mV, about 10 mV to about 20 mV, about 10 mV to about 30 mV, about 10 mV to about 40 mV, about 10 mV to about 50 mV, about 10 mV to about 60 mV, about 10 mV to about 70 mV, about 10 mV to about 80 mV, about 10 mV to about 90 mV, about 10 mV to about 100 mV, about 20 mV to about 30 mV, about 20 mV to about 40 mV, about 20 mV to about 50 mV, about 20 mV to about 60 mV, about 20 mV to about 70 mV, about 20 mV to about 80 mV, about 20 mV to about 90 mV, about 20 mV to about 100 mV, about 30 mV to about 40 mV, about 30 mV to about 50 mV, about 30 mV to about 60 mV, about 30 mV to about 70 mV, about 30 mV to about 80 mV, about 30 mV to about 90 mV, about 30 mV to about 100 mV, about 40 mV to about 50 mV, about 40 mV to about 60 mV, about 40 mV to about 70 mV, about 40 mV to about 80 mV, about 40 mV to about 90 mV, about 40 mV to about 100 mV, about 50 mV to about 60 mV, about 50 mV to about 70 mV, about 50 mV to about 80 mV, about 50 mV to about 90 mV, about 50 mV to about 100 mV, about 60 mV to about 70 mV, about 60 mV to about 80 mV, about 60 mV to about 90 mV, about 60 mV to about 100 mV, about 70 mV to about 80 mV, about 70 mV to about 90 mV, about 70 mV to about 100 mV, about 80 mV to about 90 mV, about 80 mV to about 100 mV, or about 90 mV to about 100 mV in magnitude.

[0626] In some cases, the applied voltage across the membrane can be from about 100 mV to about 1,000 mV in magnitude. In some cases, the applied voltage across the membrane can be from about 100 mV to about 150 mV, about 100 mV to about 200 mV, about 100 mV to about 250 mV, about 100 mV to about 300 mV, about 100 mV to about 400 mV, about 100 mV to about 500 mV, about 100 mV to about 600 mV, about 100 mV to about 700 mV, about 100 mV to about 800 mV, about 100 mV to about 900 mV, about 100 mV to about 1,000 mV, about 150 mV to about 200 mV, about 150 mV to about 250 mV, about 150 mV to about 300 mV, about 150 mV to about 400 mV, about 150 mV to about 500 mV, about 150 mV to about 600 mV, about 150 mV to about 700 mV, about 150 mV to about 800 mV, about 150 mV to about 900 mV, about 150 mV to about 1,000 mV, about 200 mV to about 250 mV, about 200 mV to about 300 mV, about 200 mV to about 400 mV, about 200 mV to about 500 mV, about 200 mV to about 600 mV, about 200 mV to about 700 mV, about 200 mV to about 800 mV, about 200 mV to about 900 mV, about 200 mV to about 1,000 mV, about 250 mV to about 300 mV, about 250 mV to about 400 mV, about 250 mV to about 500 mV, about 250 mV to about 600 mV, about 250 mV to about 700 mV, about 250 mV to about 800 mV, about 250 mV to about 900 mV, about 250 mV to about 1,000 mV, about 300 mV to about 400 mV, about 300 mV to about 500 mV, about 300 mV to about 600 mV, about 300 mV to about 700 mV, about 300 mV to about 800 mV, about 300 mV to about 900 mV, about 300 mV to about 1,000 mV, about 400 mV to about 500 mV, about 400 mV to about 600 mV, about 400 mV to about 700 mV, about 400 mV to about 800 mV, about 400 mV to about 900 mV, about 400 mV to about 1,000 mV, about 500 mV to about 600 mV, about 500 mV to about 700 mV, about 500 mV to about 800 mV, about 500 mV to about 900 mV, about 500 mV to about 1,000 mV, about 600 mV to about 700 mV, about 600 mV to about 800 mV, about 600 mV to about 900 mV, about 600 mV to about 1,000 mV, about 700 mV to about 800 mV, about 700 mV to about 900 mV, about 700 mV to about 1,000 mV, about 800 mV to about 900 mV, about 800 mV to about 1,000 mV, or about 900 mV to about 1,000 mV in magnitude.

Kits

[0627] In some aspects, the present disclosure provides kits of analysis of one or more characteristics of one or more analytes. Kits can comprise one or more elements disclosed herein in relation to any of the various aspects, in any combination thereof. In some cases, the kit can comprise a pore described herein (e.g., an engineered biological nanopore). In some cases, the kit can comprise a nanopore system. In some cases, the nanopore system can comprise a fluidic chamber with a membrane. In some cases, the membrane can split the fluidic chamber into a first side (e.g., cis side) and a second side (e.g., trans side). In some cases, the fluidic chamber can further comprise a nanopore (e.g., an engineered biological nanopore). In some cases, the nanopore is embedded into the membrane. In some cases, the kit can further comprise a first solution. In some cases, the first solution can be added to the first side (e.g., cis side) of the fluidic chamber. In some cases, the kit can comprise a second solution. In some cases, the second solution can be added to the second side (e.g., trans side) of the fluidic chamber. In some cases, the first solution and the second solution can be the same solution. In some cases, the first solution and the second solution can be different solutions. In some cases, the kit can further comprise a sample analyte for testing. In some cases, the sample analyte can be used to set up the system of the present disclosure. In some cases, the kit can comprise an informational manual describing an instruction of using the kit.

[0628] In another aspect, the present disclosure provides a device comprising an array of the system. In some cases, the system can comprise any one of the systems disclosed herein.

[0629] In another aspect, the present disclosure provides a method of characterizing at least one structural feature of the polymer analyte. In some cases, the method can comprise any of the methods disclosed herein.

[0630] In another aspect, the present disclosure provides a method for analysis of an amino acid sequence or amino acid composition of one or more polymer analytes. In some cases, the analysis can be performed at a single molecule level. In some cases, the method can comprise any of the methods disclosed herein.

[0631] In another aspect, the present disclosure provides a system for characterizing at least one structural feature of the polymer analyte. In some cases, the system can comprise any of the systems disclosed herein.

[0632] In another aspect, the present disclosure provides a system for analysis of an amino acid sequence or amino acid composition of one or polymer analytes. In some cases, the analysis can be performed at a single molecule level. In some cases, the system can comprise any one of the systems disclosed herein.

Computer Systems [0633] The present disclosure provides computer systems that are programmed to implement methods of determining one or more characteristics of an analyte. FIG. 7 shows a computer system 601 that is programmed or otherwise configured to determine one or more characteristics of an analyte. The computer system 601 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 601 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.

[0634] The computer system 601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 601 also includes memory or memory location 610 (e.g., randomaccess memory, read-only memory, flash memory), electronic storage unit 615 (e.g., hard disk), communication interface 620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 625, such as cache, other memory, data storage and/or electronic display adapters. The memory 610, storage unit 615, interface 620 and peripheral devices 625 are in communication with the CPU 605 through a communication bus (solid lines), such as a motherboard. The storage unit 615 can be a data storage unit (or data repository) for storing data. The computer system 601 can be operatively coupled to a computer network (“network”) 630 with the aid of the communication interface 620. The network 630 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 630 in some cases is a telecommunication and/or data network. The network 630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 630, in some cases with the aid of the computer system 601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 601 to behave as a client or a server.

[0635] The CPU 605 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 610. The instructions can be directed to the CPU 605, which can subsequently program or otherwise configure the CPU 605 to implement methods of the present disclosure. Examples of operations performed by the CPU 605 can include fetch, decode, execute, and writeback.

[0636] The CPU 605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0637] The storage unit 615 can store files, such as drivers, libraries and saved programs. The storage unit 615 can store user data, e.g., user preferences and user programs. The computer system 601 in some cases can include one or more additional data storage units that are external to the computer system 601, such as located on a remote server that is in communication with the computer system 601 through an intranet or the Internet. [0638] The computer system 601 can communicate with one or more remote computer systems through the network 630. For instance, the computer system 601 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 601 via the network 630.

[0639] 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 601, such as, for example, on the memory 610 or electronic storage unit 615. 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 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605. In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610.

[0640] 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.

[0641] Aspects of the systems and methods provided herein, such as the computer system 601, 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. [0642] 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 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.

[0643] The computer system 601 can include or be in communication with an electronic display 635 that comprises a user interface (UI) 640 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.

[0644] 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 605.

[0645] Another aspect of the present disclosure provides a non-fransitory 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.

[0646] 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.

EXAMPLES

Example 1. Preparation of MspA nanopores.

Cloning

[0647] The gene encoding for MspA WT monomers, with a Strep-tag at the end of the gene, was placed in a pT7-SCl plasmid (Miles et al., 2001b), controlled by a T7 promoter. Mutations to the MspA gene were introduced using USER™ cloning (Bitinaite et al., 2007) The resulting plasmids were transformed into E. coli cells to amplify the DNA and the mutations were checked by sequencing.

Expression and purification

[0648] MspA nanopores were expressed in E. coli BL21 (DE3) cells in 2-YT medium. When the optical density at 600 nm reached 0.8, 0. 1 mM IPTG was added and the cells were grown at 20 °C for 20 h. Cells were harvested by centrifugation (6500 rpm, 15 min, 4 °C), lysed by one freeze-thaw cycle and resuspended in lysis buffer (15 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 1 pg/ml of DNasel, 0.2 mg/ml of lysozyme and 1 mM MgC12. MspA nanopore oligomerisation was induced by the addition of 0.8 % DDM in the cell lysate. The mixture was then incubated for 20 min at 37 °C on a tube shaker. The cells were furthered lysed by sonication on ice (Branson sonifier 450 at 40 % output power for 3 x 40 seconds). After centrifugation (6500 rpm, 30 min, 4°C) the supernatant from 200 ml culture was collected. Afterwards the supernatant was incubated with 200 pl of bed volume Strep-Tactin beads for 1 hour at 4 °C on a tube rotator at 20 RPM, allowing the Strep-tag at the C-terminus of the protein monomers to bind to the Strep-Tactin beads. Protein was eluted with 100 ul Tris buffer (50 Mm Tris-HCl, 150 mM NaCl, 0.02% DDM, pH 8.5) supplemented with 2.5 mM desthiobiotin. Success of purification and oligomerization was checked on 12 % SDS-PAGE gel and MspA oligomers were stored at 4°C.

Example 2. Electrophysiological analysis of MspA nanopores

Preparation of ClpX Translocase

[0649] E. coli ClpX was employed as exemplary translocase to control the movement of the polypeptide through the nanopore. 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 (Olivares et al. 2016, Nature Reviews Microbiology Vol. 14, pg. 33 44). The monomer and covalently linked trimer of N-terminal truncated ClpX variants (residues 61 423) were purified as previously described (Singh, et al. J. Biol. Chem.276.31 (2001): 29420-29429; Martin et al., Nature 437.7062 (2005): 1115-1120) with minor modifications and used for ClpX nanopore experiments. Specifically, the gene encoding for monomer and trimer of ClpX-AN 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 (3-D-l- 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 KC1, 20 mM imidazole, 1 mM dithiothreitol (DTT), 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 pL of Ni-NTA resin (Qiagen) to a 50 mL falcon tube, which was preequilibrated with wash buffer (50 mM HEPES, pH 7.5, 300 mM KC1, 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 pL elution buffer (600 mM imidazole, 1 mM dithiothreitol (DTT), 100 uM EDTA, 200 mM KC1, 25 mM MgC12, 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 KC1, 25 mM MgC12, 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. The sequences of the ClpX can be found in Table 2.

Table 2. Sequences of ClpX

Preparation of Protein Analytes

[0650] Maltose Binding Protein (MBP-1) was used to test protein translocation through the nanopores. The model proteins were provided with a His-affmity tag and further modified via genetic fusions to express full length substrates with C- terminal extensions that enabled binding to ClpX (ssrA recognition motif), stalling of the ClpX (polyglycine stall motif) and EPF capture motifs that enabled nanopore capture (e.g. polycation stretches) (see Table 3).

[0651] The gene encoding for MBP-1 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 with 50 mg/L of kanamycin. The cells were induced at an A600 of ~0.6 by addition of 0.5 mM isopropyl P- D-l -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 pL elution buffer (600 mM imidazole, 1 mM dithiothreitol (DTT), 150 mM KC1, 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 3. Sequence of maltose binding protein (MBP-1)

Planar lipid bilayer electrophysiological recordings system

[0652] For each experiment a single nanopore was inserted in a planar lipid bilayer system as described previously (Maglia et al., 2010, Methods Enzymol, 475, pg. 591-623). Briefly, the electrophysiology chamber consisted of two compartments separated by a 25 pm thick Teflon (Goodfellow Cambridge Ltd) membrane. The Teflon membrane contained an aperture with a diameter of approximately 100-200 pm. Lipid membranes were formed by first applying 5 pl of 5% hexadecane (Sigma Aldrich) in pentane (Sigma Aldrich) to the Teflon membrane, near the aperture. The pentane was left to dry and 400 pl of the appropriate buffered solution was added to each compartment. 20 pl 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 compartment of the chamber. 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). All recordings were carried out at 22°C.

Determination of nanopore system EOF from ion-selectivity and electroosmotic flow

[0653] The ion-permeability parameters P<K+) and P(Giu-) for the ions K+ and Glu- were determined by carrying out ion-selectivity measurements using asymmetric salt concentrations to reveal the relative catiomanion selectivity of each nanopore system. Briefly, reverse voltage from ion-selectivity measurements were performed in the planar lipid bilayer electrophysiological recording system described above. 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 KC1 solution. For reverse voltage measurements, both compartments were first filled with 400 pL 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(Giu-) for K+ and Glu- were determined using a solution-A of 2 M KGlu, 50 mM Tris pH 7.5 and a solution-B of 0.5 M KGlu, 50 mM Tris pH 7.5.

[0654] 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 (Fr), which is the voltage offset required to achieve zero ionic current flow, was estimated by linear regression of the curve between -20 and +20 mV. The pores were measured in triplicates.

[0655] The average reversal voltage was then used in the following equation to determine the relative permeability ratio:

[0656] wherein P<x+j 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 mean activity coefficients of KGlu in 0.5 M and 2.0 M are 0.68 and 0.719 respectively (Bonner et al., 1981, J. Chem. Eng. Data., 26, 2, pg. 147-148). Recordings of protein translocation

[0657] Both compartments of the nanopore system were filled with an electrolyte solution (I M potassium glutamate, 50 mM Tris, 25 mM MgCE . 1 mM EDTA, 10 mM DTT buffered to pH 7.5). MspA nanopores were added to the cis compartment to achieve a single inserted nanopore. After insertion of a single nanopore the open pore current was recorded at -80 mV, unless stated otherwise. Afterwards, ClpX translocase and maltose binding protein (MBP-1) substrate were added in a 2: 1 molar ratio together with 2.5 mM ATP to the cis compartment. The MBP-1 translocation events were recorded at -80 mV applied potential, unless stated otherwise. FIGs. 3A-3H show the ionic current recordings over time (s) for the wild-type MspA nanopore and six MspA mutants. The current output shows an open-pore current (I_o). Once the analyte (e.g., MBP-1) occupied the pore, the current displays peaks for the blockage current. The upward peaks designate capture of the MBP-1 analyte. The results of the recordings are shown in Table 4. P(k/glu) indicates the ion selectivity of potassium (K⁺) over glutamate (Glu‘), and whether the nanopores were able to translocate the test MBP- 1 protein substrate under moderate applied voltage. The results demonstrate that neutralizing the constriction of MspA reduced the cation selectivity and thus reduced the cis-lo-trans EOF. As a result, the 2N (D90N-D91N) double mutation nanopore could not effectively translocate protein substrates. The data in Table 3 and FIGs. 3A-3H further show that a strong EOF could be restored to the 2N nanopore by engineering in additional negative mutations into the nearby positions outside the constriction, enabling protein substrates to be translocated cis-lo-trans . Further, the data in Table 3 and FIGs. 3A-3H show that multiple negative mutations are additive, working together to further enhance the P(+)/P(-) ion selectivity and thus the strength of the EOF. For example, the 2N-I105E and 2N-N108E nanopores have enhanced EOF versus the 2N nanopore, but the combination of both into the 2N-I105E-N108E nanopore further enhances the ion selectivity and EOF effect.

Table 4. Summary of results from current recordings. Example 3. CsgG and CsgG-CsgF nanopores

Preparation of CsgG and CsgG/F nanopores

[0658] Engineered CsgG and CsgG-CsgF nanopores are prepared. Briefly, E. colt cells transformed with genes coding for CsgG and CsgF subunits are resuspended in 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 5 mM MgC12, 0.4 mM AEBSF, 1 qgml-1 leupeptin, 0.5 mgml-1 DNase I and 0.1 mgml-1 lysozyme. The cells are lysed and then incubated for 30 min with 1% n-dodecyl-P-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 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 6 10/30 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 mlmin-l.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 °C 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 run and assuming 1/1 stoichiometry.

[0659] For preparation of only CsgG nanopores, the same preparation as described above is employed with omission of the CsgF genes and the TEV protease digestion step.

[0660] The CsgG protein sequence is the following:

CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVPQSATAM LVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSIIGYESNVKS GGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGE VGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES (SEQ ID NO.: 4).

[0661] Underlined residues are those residues suitable for engineering EOF. Bolded residues are those residues that are most preferable for modification.

[0662] The CsgF protein sequence is the following:

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNSYKDPSYNDDFGIETPSALDNFTQAIQSQILGGLLSNI NTGKPGRMVTNDYIVDIANRDGQLQLNVTDRKTGQTSTIQVSGLQNNSTDF (SEQ ID NO: 5). [0663] Underlined residues are those residues suitable for engineering EOF. Bolded residues are those residues that are most preferable for modification.

CsgG and CsgG/F nanopores with enhanced EOF

[0664] The amino-acid positions in Tables 5 and 6 are determined to be the regions for engineering enhanced EOF in CsgG and CsgG/F respectively. For CsgG nanopores (FIG. 4), EOF enhancement is achieved by increasing negative charges in and near the constriction. For CsgG/F nanopores (FIG. 5), optimal EOF enhancement is achieved by increasing negative charges in and near the constriction (502) of the CsgG component (501) and/or the narrowest lumen facing regions (504) of the CsgF component (503). The highest EOF enhancement is achieved by increasing the charge in the residues with the narrowest C(alpha)-C(alpha) diameter. Further enhancement of EOF is achieved by combining multiple sets of mutations to increase net charge.

Table 5. Residue mutations in CsgG for creating CsgG or CsgG/F nanopores with enhanced ion selectivity and EOF. n Table 5, * indicates residues suitable for engineering EOF enhancement and ** indicates residue mutations for optimal EOF enhancement. Table 6. Residue mutations in CsgF for creating CsgG/F nanopores with enhanced ion selectivity and EOF. Table 6, * indicates residues suitable for engineering EOF enhancement and ** indicates residue mutations for optimal EOF enhancement.

[0665] In Table 7, example mutations of CsgG nanopores is shown. For each D mutation, a similar EOF is achieved with an E mutation at the same position.

Table 7. Examples of CsgG nanopores with enhanced EOF. n Table 7, the number of * indicates qualitatively increased EOF. For example, a CsgG nanopore labeller **** would indicate a stronger EOF than a CsgG nanopore labelled **.

[0666] Any of the engineered CsgG nanopores in Table 7 can be combined with further mutations at any of the listed positions in Table 8 to further enhance EOF.

Table 8. Additional examples of CsgG nanopores with enhanced EOF. n Table 8, the number of * indicates qualitatively increased EOF. For example, a CsgG nanopore labeller ***** _wouy j_nc j_ca[_{e a} stronger EOF than a CsgG nanopore labelled ****.

Planar lipid bilayer electrophysiological recordings system

[0667] Experiments with single CsgG or single CsgG/F nanopores are performed as described in the Example 2 in a planar lipid bilayer system under the same conditions.

Measurement of ion-selectivity and Recordings of protein translocation

[0668] Measurements of ion-selectivity on single CsgG or single CsgG/F nanopores are performed as described in the Example 2 in a planar lipid bilayer system under the same conditions.

[0669] Measurements of translocase controlled protein translocation through CsgG and CsgG/F nanopores are carried out according to the system described in the previous example with the same protein substrate, under the same conditions. 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 in Example 2) 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 MgCE. After pre-incubation, the ClpX:MBP-l complex is added to the cis -compartment.

[0670] Electrical recordings are acquired over a range of voltages from -60 mV to -200 mV. ClpX: MBP- 1 complex translocation events through the CsgG and CsgG/F nanopores with enhanced EOF 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.

[0671] 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. 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.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method comprising:

(a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and/or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel; and

(b) contacting the engineered biological nanopore with a biopolymer.

2 The method of claim 1, wherein the first region is more net negative than the second region.

3 The method of claim 1 or 2, wherein one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids.

4 The method of any one of claims 1-3, wherein one or more amino acids in the first region is mutated to one or more negative amino acids.

5 The method of any one of claims 1-4, wherein when one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the first region is mutated to one or more negative amino acids.

6 The method of claim 5, wherein the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

7 The method of claim 6, wherein the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region.

8 The method of claim 6 or 7, wherein a first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from a second ring of charge comprising the mutated at least one amino acid in the second region.

9 The method of any one of claims 1-8, wherein the engineered biological nanopore comprises one or more monomers.

10 The method of any one of claims 1-9, wherein a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region.

11 The method of claim 10, wherein a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion.

12. The method of claim 10 or 11, wherein the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

13. The method of any one of claims 10-12, wherein the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge.

14. The method of claim 13, wherein the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion.

15. The method of any one of claims claim 1-14, wherein the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore.

16. The method of claim 15, wherein the first region modified to be more net negative and the second region modified to be more net neutral or more net negative generate the EOF.

17. The method of any one of claims 1-16, wherein the engineered biological nanopore has a cationselectivity P(+)/P(-) of at least about 1.5.

18. The method of any one of claims 1-17, wherein the second region of the channel comprises a first entrance and a second entrance.

19. The method of claim 18, wherein the first region of the channel is adjacent to the first entrance of the second region of the channel.

20. The method of claim 18, wherein the first region of the channel is adjacent to the second entrance of the second region of the channel.

21. The method of any one of claims 1-20, wherein one or more amino acid mutations in the second region of the engineered biological nanopore increase a cation selectivity p(K+/Cl-) relative to the wild-type biological nanopore.

22. The method of any one of claims 1-21, wherein one or more amino acid mutations in the first region of the engineered biological nanopore increase a cation selectivity p(K+/Cl-) relative to the wild-type biological nanopore.

23. The method of any one of claims 1-22, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 5 nm.

24. The method of any one of claims 1-23, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 2 nm.

25. The method of any one of claims 1-24, wherein a net charge of the first region is at least about 50% more negative as compared to another region adjacent to the respective region of the wild-type biological nanopore.

26. The method of any one of claims 1-25, wherein the second region is more net neutral as compared to the respective region of the wild-type biological nanopore.

1. The method of any one of claims 1-26, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

28. The method of any one of claims 1-27, wherein the constriction region has a neutral charge.

29. The method of any one of claims 1-28, wherein the second region comprises one or more amino acid mutations that increase a neutral charge in the residues that contribute to a narrowest region of the engineered biological nanopore comprising a diameter of at most 1.0 nm.

30. The method of any one of claims 1-29, wherein the engineered biological nanopore comprises a cation selectivity P(+)/P(-) of at least about 1.8.

31. A method comprising:

(a) providing a nanopore system, wherein the nanopore system comprises (1) a fluidic chamber and (2) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm; and

(b) contacting the engineered biological nanopore with a biopolymer.

32. The method of claim 31, wherein the first region is more net negative than the second region.

33. The method of claim 31 or 32, wherein one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids

34. The method of any one of claims 31-33, wherein one or more amino acids in the first region is mutated to one or more negative amino acids.

35. The method of claim 34, wherein when one or more amino acids in the second region is modified to (1) one or more neutral amino acids or (2) one or more negative amino acids, then one or more amino acids in the adjacent region is mutated to one or more negative amino acids

36. The method of claim 35, wherein the first region comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

37. The method of claim 36, wherein the mutated at least one amino acid in the first region is at most 10 nm away from a mutated at least one amino acid in the second region.

38. The method of claim 36 or 37, wherein the first ring of charge comprising the mutated at least one amino acid in the first region is at most 10 nm away from the second ring of charge comprising the mutated at least one amino acid in the second region.

39. The method of any one of claims 31-38, wherein the engineered biological nanopore comprises one or more monomers.

40. The method of any one of claims 31-39, wherein a monomer of the engineered biological nanopore comprises a first portion corresponding to the first region and a second portion corresponding to the second region.

41. The method of claim 40, wherein a monomer of the engineered biological nanopore comprises a net charge in the first portion that is more negative as compared to a net charge in the second portion.

42. The method of claim 40 or 41, wherein the first portion comprises at least one amino acid that is mutated to exhibit an increased net negative charge.

43. The method of any one of claims 40-42, wherein the second portion comprises at least one amino acid that is mutated to exhibit an increased net neutral charge or an increased net negative charge.

44. The method of claim 43, wherein the at least one mutated amino acid in the first portion is at most 10 nm away from the at least one mutated amino acid in the second portion.

45. The method of any one of claims 31-44, wherein the engineered biological nanopore generates an electro-osmotic force (EOF) greater than an EOF of the wild-type biological nanopore.

46. The method of claim 45, wherein the first region of the channel and the second region of the channel generates the EOF.

47. The method of claim 45 or 46, wherein the EOF acts in an opposite direction to an electrophoretic force in the nanopore system.

48. The method of any one of claims 31-47, wherein the engineered biological nanopore has a cationselectivity P(+)/P(-) of at least about 1.5.

49. The method of any one of claims 31-48, wherein the second region of the channel comprises a first entrance and a second entrance.

50. The method of claim 49, wherein the first region of the channel is adjacent to the first entrance of the second region of the channel.

51. The method of claim 49, wherein the first region of the channel is adj acent to the second entrance of the second region of the channel.

52. The method of any one of claims 31-51, wherein one or more amino acid mutations in the second region of the engineered biological nanopore increase a cation selectivity p(K+/Cl-) relative to the wild-type biological nanopore.

53. The method of any one of claims 31-52, wherein one or more amino acid mutations in the first region of the engineered biological nanopore increase a cation selectivity p(K+/Cl-) relative to the wild-type biological nanopore.

54. The method of any one of claims 31-53, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 5 nm.

55. The method of any one of claims 31-54, wherein the first region comprises one or more amino acid mutations that increase a negative charge in the residues that contribute to a region of the engineered biological nanopore comprising a diameter of at most 2 nm.

56. The method of any one of claims 31-55, wherein a net charge of the first region is at least about 50% more negative as compared to the respective region of the wild-type biological nanopore.

57. The method of any one of claims 31-56, wherein the second region is more neutral as compared to the respective region of the wild-type biological nanopore.

58. The method of any one of claims 31-57, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to the respective region of the wild-type biological nanopore.

59. The method of any one of claims 31-58, wherein the constriction region has a neutral charge.

60. The method of any one of claims 31-59, wherein the second region comprises one or more amino acid mutations that increase a neutral charge in the residues that contribute to a narrowest region of the engineered biological nanopore comprising a diameter of at most 1.0 nm.

61. The method of any one of claims 31-60, wherein the engineered biological nanopore comprises a cation selectivity P(+)/P(-) of at least about 1.8.

62. A system comprising:

(a) a fluidic chamber; and

(b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein (i) the first region is modified to be more net negative than a respective region of a wild-type biological nanopore and/or (ii) the second region is modified to be more net neutral or more net negative than a respective region of the wild-type biological nanopore, wherein the first region of the channel is adjacent to the second region of the channel, wherein the engineered biological nanopore is configured to contact a biopolymer.

63. A system comprising:

(a) a fluidic chamber; and

(b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region of the channel has a negative charge, wherein a net charge of the second region of the channel is at least about 50% more neutral as compared to a respective region of a wild-type biological nanopore, wherein the engineered biological nanopore is configured to a biopolymer.

64. A system comprising:

(a) a fluidic chamber; and

(b) a membrane comprising an engineered biological nanopore, wherein the membrane separates the fluidic chamber into (1) a first side and (2) a second side, wherein the engineered biological nanopore comprises a channel, wherein the channel comprises a first region and a second region, wherein the second region has a constriction region, wherein the first region of the channel is adjacent to the second region of the channel, wherein the first region is modified to be more net negative as compared to a respective region of a wild-type biological nanopore, wherein a first ring of charge in the first region and a second ring of charge in the second region comprises a distance of at most about 3 nm, wherein the second region comprises a width of at most about 2.5 nm, wherein the engineered biological nanopore is configured to contact a biopolymer.

65. A method comprising (a) providing a mixture containing or suspected of containing an analyte comprising polypeptide or protein, and (b) using an engineered biological nanopore to (1) determine a sequence or (2) generate a measure of a concentration or relative amount of said analyte in said mixture at an accuracy of at least 80%.

66. The method of claim 65, wherein said mixture contains or is suspected of containing an additional analyte comprising an additional polypeptide or protein.

67. The method of claim 65 or 66, further comprising using said engineered biological nanopore to generate a measure of a concentration or relative amount of said additional analyte in said mixture at an accuracy of greater than 80%.

68. The method of any one of claims 65-67, wherein (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 90%.

69. The method of any one of claims 65-68, wherein (1) said sequence is determined or (2) said measure of said concentration or relative amount of said analyte is generated at an accuracy of at least 95%.