HK1235090A1 - Target sequence detection by nanopore sensing of synthetic probes - Google Patents

Description

Target sequence detection by nanopore probing of synthetic probes

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional patent application No.62/056,378, filed on 26/9/2014, the disclosure of which is incorporated herein by reference.

Technical Field

The present disclosure relates to methods and compositions for target sequence detection using a nanopore device.

Background

The detection, localization, and copy number determination of specific sequence regions within nucleic acid strands, referred to herein as "target sequence detection," have applications in biomedical science and technology, medicine, agriculture, and forensic medicine, among other fields. The detection of genes and their modifications, sequences, positions or numbers is important for the development of molecular diagnostics in medicine. DNA microarrays, PCR, Southern blotting, and FISH (fluorescence in situ hybridization) are all methods that can be used to perform or facilitate the detection of target sequences. These methods are slow and laborious and have limited accuracy and resolution. Newer methods such as real-time PCR and Next Generation Sequencing (NGS) techniques have increased throughput, but still do not have sufficient resolution for many applications.

Solid state nanopores have been demonstrated to detect molecules by applying a voltage across the pore and measuring the resistance of the molecule as it passes through the nanopore. The overall effectiveness of any given nanopore device depends on its ability to accurately and reliably measure resistance and distinguish between different types of molecules passing through. Experiments disclosed in the literature demonstrate the detection of DNA and RNA strands that pass through the pore and synthetic molecules that hybridize to specific sequences thereon. However, they have not been able to be used to create a high throughput and reliable nanopore device for detecting probes on specific DNA or RNA sequences. The probes that have been developed to date are not sufficient for reliable sequence detection. Therefore, what is needed are probe sets and probe complexes for detection in nanopores that enable sequence-specific binding.

Disclosure of Invention

Provided herein is a method of detecting a polynucleotide comprising a target sequence in a sample, the method comprising: contacting the sample with a probe that specifically binds to the polynucleotide comprising the target sequence under conditions that promote binding of the probe to the target sequence to form a polynucleotide-probe complex; loading the sample into a first chamber of a nanopore device, wherein the nanopore device comprises at least one nanopore and at least the first and second chambers, wherein the first and second chambers are in electrical and fluid communication through the at least one nanopore, and wherein the nanopore device further comprises an independently controlled voltage across each of the at least one nanopore and a sensor associated with each of the at least one nanopore, wherein the sensor is configured to identify objects passing through the at least one nanopore, and wherein the polynucleotide-probe complex translocated through the at least one nanopore provides a detectable signal associated with the polynucleotide-probe complex; and determining the presence or absence of said polynucleotide-probe complex in said sample by observing said detectable signal, thereby detecting said polynucleotide comprising said target sequence. In one embodiment, the method further comprises generating a voltage potential across the at least one nanopore, wherein the voltage potential generates a force on the polynucleotide-probe complex to pull the polynucleotide-probe complex through the at least one nanopore, causing the polynucleotide-probe complex to translocate through the at least one nanopore to generate the detectable signal.

In some embodiments, the polynucleotide is DNA or RNA. In one embodiment, the detectable signal is an electrical signal. In one embodiment, the detectable signal is an optical signal. In one embodiment, the probe comprises a molecule selected from the group consisting of: a protein, peptide, nucleic acid, TALEN, CRISPR, peptide nucleic acid, or chemical compound. In one embodiment, the probe comprises a molecule selected from the group consisting of: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), Peptide Nucleic Acid (PNA), DNA/RNA hybrids, polypeptides or any chemically derivatized polymer.

In one embodiment, the probe comprises a PNA molecule bound to a second molecule configured to facilitate detection of the probe bound to the polynucleotide during translocation through the at least one nanopore. In a further embodiment, the second molecule is PEG. In a further embodiment, the PEG has a molecular weight of at least 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa or 10 kDa.

In one embodiment, the method of detecting a polynucleotide comprising a target sequence in a sample further comprises administering to the sample a condition suspected of altering a binding interaction between the probe and the target sequence. In a further embodiment, the conditions are selected from: removing the probe from the sample, adding an agent that competes with the probe for binding to the target sequence, and changing initial pH, salt, or temperature conditions.

In one embodiment, the polynucleotide comprises a chemical modification configured to alter binding of the polynucleotide to the probe. In a further embodiment, the chemical modification is selected from biotinylation, acetylation, methylation, small ubiquitin-like modification (sumoylation), glycosylation, phosphorylation and oxidation.

In one embodiment, the probe comprises a chemical modification coupled to the probe via a cleavable bond. In one embodiment, the probe interacts with the target sequence of the polynucleotide by covalent, hydrogen, ionic, metallic, van der waals, hydrophobic, or planar stacking interactions. In one embodiment, the method of detecting a polynucleotide comprising a target sequence in a sample further comprises contacting the sample with one or more detectable labels capable of binding to the probe or to the polynucleotide-probe complex. In one embodiment, the polynucleotide comprises at least two target sequences.

In one embodiment, the nanopore is about 1nm to about 100nm in diameter and 1nm to about 100nm in length, and wherein each chamber includes an electrode. In one embodiment, the nanopore device comprises at least two nanopores configured to simultaneously control the motion of the polynucleotide in both nanopores. In one embodiment, the method of detecting a polynucleotide comprising a target sequence in a sample further comprises reversing the independently controlled voltages after initial detection by the polynucleotide-probe complex of the detectable signal, such that movement of the polynucleotide through the nanopore is reversed after the probe-bound portion passes through the nanopore, thereby again determining the presence or absence of the polynucleotide-probe complex.

In one embodiment, the nanopore device comprises two nanopores, and wherein the polynucleotide is located within both of the two nanopores simultaneously. In a further embodiment, the method of detecting a polynucleotide comprising a target sequence in a sample comprises adjusting the magnitude and/or direction of a voltage in each of the two nanopores such that opposing forces are generated by the nanopores to control the rate of translocation of the polynucleotide through the nanopores.

Also provided herein is a method of detecting a polynucleotide or polynucleotide sequence in a sample comprising: contacting the sample with a first probe and a second probe, wherein the first probe specifically binds to a first target sequence of the polynucleotide under conditions that promote binding of the first probe to the target sequence, wherein the second probe specifically binds to a second target sequence of the polynucleotide under conditions that promote binding of the second probe to the target sequence; contacting the sample with a third molecule configured to simultaneously bind to the first and second probes when the first and second probes are sufficiently proximate to each other under conditions promoting binding of the third molecule to the first probe and the second probe, thereby forming a fusion complex comprising the polynucleotide, the first probe, the second probe, and the third molecule; loading the sample into a first chamber of a nanopore device, wherein the nanopore device comprises at least one nanopore and at least the first and second chambers, wherein the first and second chambers are in electrical and fluid communication through the at least one nanopore, and wherein the nanopore device further comprises a controlled voltage potential across each of the at least one nanopore and a sensor associated with each of the at least one nanopore, wherein the sensor is configured to identify objects passing through the at least one nanopore, and wherein the fusion complex displaced by the at least one nanopore provides a detectable signal associated with the fusion complex; and determining the presence or absence of said fusion complex in said sample by observing said detectable signal.

In one embodiment, the polynucleotide is DNA or RNA. In one embodiment, the detectable signal is an electrical signal. In one embodiment, the detectable signal is an optical signal. In one embodiment, the sufficient proximity is less than 3, 4, 5,6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides. In one embodiment, the third molecule comprises PEG or an antibody.

In one embodiment, said third molecule and said first and second probes are bound to ssDNA, and wherein said ssDNA to which said third molecule is attached comprises a region complementary to a region of ssDNA attached to said first probe and complementary to a region of ssDNA attached to said second probe. In one embodiment, the method of detecting a polynucleotide or polynucleotide sequence in a sample further comprises contacting the sample with one or more detectable labels capable of binding to the third molecule or to the fusion complex.

Also provided herein are kits comprising a first probe, a second probe, and a third molecule, wherein the first probe is configured to bind to a first target sequence on a target polynucleotide, wherein the second probe is configured to bind to a second target sequence on the target polynucleotide, and wherein the third molecule is configured to bind to the first probe and the second probe when the first and second probes bind to the polynucleotides at the first and second target sequences, thereby positioning the first and second probes in sufficient proximity to allow the third molecule to bind to the first and second probes simultaneously.

In one embodiment, the first probe and the second probe are selected from the group consisting of: a protein, peptide, nucleic acid, TALEN, CRISPR, peptide nucleic acid, or chemical compound. In one embodiment, the third molecule comprises PEG or an antibody. In one embodiment, the third molecule comprises a modification that alters the binding affinity to the probe.

Also provided herein are nanopore devices comprising at least two chambers and a nanopore, wherein the device comprises a modified PNA probe bound to a polynucleotide within the nanopore.

Also provided herein is a dual-pore, dual-amplifier device for detecting a charged polymer through two pores, the device comprising an upper chamber, a middle chamber, and a lower chamber, a first pore connecting the upper chamber and the middle chamber, and a second pore connecting the middle chamber and the lower chamber, wherein the device comprises a modified PNA probe bound to a polynucleotide within the first or second pore.

In one embodiment, the device is configured to control the movement of the charged polymer through both the first aperture and the second aperture simultaneously. In one embodiment, the modified PNA probe is conjugated to at least one PEG molecule. In one embodiment, the apparatus further comprises a power supply configured to provide a first voltage between the upper chamber and the middle chamber and a second voltage between the middle chamber and the lower chamber, each voltage being independently adjustable, wherein the middle chamber is connected to a common ground with respect to the two voltages, wherein the apparatus provides dual amplifier electronics configured for independent voltage control and current measurement of each aperture, wherein the two voltages may be different in magnitude, wherein the first and second apertures are configured such that the charged polymer can move across both apertures simultaneously in either direction and in a controlled manner.

Drawings

The accompanying drawings, which are provided as embodiments of the invention, are illustrated by way of example only and not by way of limitation.

Fig. 1 illustrates the detection of a target molecule bound to a modified probe in a nanopore pair as one embodiment of the method of the present disclosure.

FIG. 2 shows the effect of probe binding to a target molecule on the electrical signal generated when the complex translocates through the nanopore.

Fig. 3A and 3B each show embodiments of two probes bound to a polynucleotide at respective target sequences and a third bridging molecule (e.g., an antibody) that facilitates detection of the probes in the nanopore when the two probes are bound to the scaffold.

FIG. 4 shows two probes bound to a polynucleotide at respective target sequences, wherein a third bridging molecule is PEG and is attached to the probes by complementary ssDNA linkers to enable probe detection when the two probes are bound to the scaffold in sufficient proximity.

FIG. 5 shows two probes that bind to a polynucleotide in sufficient proximity at the corresponding target sequences to allow detection of an optical signal generated by their proximity, e.g., byResonance Energy Transfer (FRET).

Fig. 6A is a schematic diagram of a system that combines a nanopore device with an epifluorescence microscope to enable detection of fluorophore-modified binding agents. Figure 6B is a graphical representation of what is seen by the detector as the fluorophore passes through the two nanopore devices in plane. Fig. 6C shows the change in current magnitude and corresponding fluorescence signal as the scaffold passes through the nanopore.

Figure 7 shows the binding of probes with cleavable groups (e.g., fluorophores) to aid detection.

FIG. 8 illustrates the multiplexing capability of the present technology by including probes of different sizes, each binding to a unique target sequence in a target bearing molecule. In this illustration, a double-stranded DNA is a polynucleotide having a target sequence and a plurality of different DNA binding probes that bind to the target sequence desired to be detected.

Figure 9A shows PNA ligands that have been modified to increase the ligand charge and thus facilitate detection through the nanopore. FIG. 9B shows an example where double stranded DNA is used as the target-carrying polymer and a plurality of different DNA binding probes are bound to the target sequence desired to be detected.

Figure 10 shows a plurality of different sequence-specific probes that bind to DNA as it traverses a nanopore to allow for multiplexed detection.

Figures 11A-C show nanopores and representative current signatures and populations from translocation of molecules through the nanopores. In fig. 11A, a solid state pore and voltage path are shown. Fig. 11B shows the current retardation and residence time of the molecule through the nanopore. Fig. 11C shows different molecular populations through the nanopore based on residence time and average current magnitude.

FIG. 12A shows an example of the use of PNA probes bound to biotin, complexed with larger neutravidin to allow detection of sequences on the DNA backbone. FIG. 12B shows the binding sites of PNA probes on a DNA backbone.

FIG. 13 shows the displacement of unbound DNA, free neutravidin and composite PNA-biotin bound to DNA and to neutravidin. The current signature (current on the y-axis, time on the x-axis) obtained when the molecule migrates through the nanopore under the applied voltage is also shown for each complex.

The DNA/PNA/neutravidin complex results in a detectable translocation current profile above other background event types (e.g., unbound DNA alone and neutravidin alone) and thus can be event labeled as a detectable PNA probe bound to DNA (i.e., DNA/PNA/neutravidin complex).

Figure 14A shows a scatter plot of events characterized by duration and mean conductance shift due to translocation through the nanopore in three populations, DNA alone (x), neutravidin alone (squares), and DNA complexed with a biotin probe linked to neutravidin (circles). Fig. 14B shows a histogram of the residence time probabilities associated with each of the three populations. FIG. 14C shows gel migration analysis (gel shift assay) of DNA only (lane 2), samples containing DNA, PNA with 3 biotin sites binding to neutravidin and neutravidin (lane 3), samples containing DNA, PNA with 7 biotin sites binding to neutravidin and neutravidin (lane 3), samples containing DNA, PNA with 16 biotin sites binding to neutravidin and neutravidin (lane 3), and samples containing DNA, PNA with 36 biotin sites binding to neutravidin and neutravidin (lane 3).

Fig. 15 shows a diagram of probe binding sites on a DNA backbone, where the probe is a VspR protein.

Figure 16A shows a graphical representation of the passage of unbound DNA molecules through a nanopore and representative current characteristics associated with the passage of a single molecule through the nanopore. Fig. 16B shows a graphical representation of VspR-bound DNA molecules passing through a nanopore and representative current characteristics associated with their passage through the nanopore.

Fig. 17 shows another ten representative current decay events consistent with VspR-binding scaffold passing through the pore.

FIG. 18A shows PNA-PEG probes bound to target sequences on dsDNA molecules. Fig. 18B shows the results of gel migration analysis using the following samples: DNA only (lane 1), DNA/PNA (lane 2), DNA/PNA-PEG (10kDa) (lane 3) and DNA/PNA-PEG (20kDa) (lane 4). Fig. 18C shows the results of gel migration analysis using the following samples: DNA labeling (lane 1), random DNA sequences incubated with PNA probes (lane 2), DNA with a single mismatch at the target sequence incubated with the corresponding PNA probe (lane 3), and DNA with the target sequence mixed with the corresponding PNA probe specific to the target sequence (lane 4).

Fig. 19A shows a representative current signature event for a molecule depicted under each current signature as it migrates through a nanopore under an applied voltage. Fig. 19B shows the results in three populations: scattergrams of events characterized by duration and mean conductance shift due to translocation through the nanopore in DNA/bisPNA (squares), DNA/bisPNA-PEG5kDa (circles), and DNA/bisPNA-PEG 10kDa (diamonds). Fig. 19C shows a histogram of the mean conductance offset probabilities associated with each of the three populations. Fig. 19D shows a histogram of event duration probabilities associated with each of the three populations.

FIG. 20A shows representative event signatures associated with displacement of PNA-PEG probes bound to DNA molecules. Figure 20B shows a plot of mean conductance shift versus duration for each recorded event in nanopores from samples containing bacterial DNA and PNA-PEG probes. Fig. 20C and 20D show the corresponding histograms characterizing these detected events by the mean conductance offset and duration of each event, respectively. Fig. 20E shows the results of the gel migration analysis, which shows: 100bp ladder (lane 1), 300bp DNA with wild type cftr sequence incubated with PNA-PEG probe (lane 2) and 300bp DNA with cftr Δ F508 sequence incubated with PNA-PEG probe (lane 3).

FIG. 21A shows the results of gel migration analysis, lane 1 contains light Streptococcus (S.mitis) bacterial DNA without bound bisPNA-PEG and lane 2 contains light Streptococcus DNA with bound site-specific bisPNA-PEG. Fig. 21B shows a scatter plot of mean conductance shift (dG) vs. time duration on the vertical axis for all recorded events in two consecutive experiments. The first sample included bacterial DNA with PEG-modified PNA probe (DNA/bisPNA-PEG). The second sample comprises bacterial DNA alone.

Some or all of the figures are schematic illustrations of examples; thus, they do not necessarily depict the actual relative sizes or positions of the elements presented. The drawings are presented for purposes of illustrating one or more embodiments and are to be distinctly understood as not limiting the scope or meaning of the following claims.

Detailed Description

Throughout this application, the text refers to various embodiments of the nutrients, compositions and methods of the present invention. The various embodiments described are intended to provide a number of illustrative examples and should not be construed as descriptions of alternative categories. Rather, it should be noted that the descriptions of the various embodiments provided herein may overlap in scope. The embodiments discussed herein are merely illustrative and are not intended to limit the scope of the invention.

Also throughout this disclosure, various publications, patents and published patent specifications are cited by explicit citations. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into this disclosure to more fully describe the state of the art to which this invention pertains.

As used in the specification and in the claims, the singular form of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "an electrode" includes a plurality of electrodes, including mixtures thereof.

As used herein, the term "comprising" means that the devices and methods comprise the recited ingredients or steps, but do not exclude other ingredients or steps. "consisting essentially of … …" when used to define devices and methods is meant to exclude other ingredients or steps of any substantial significance to the combination. "consisting of … …" is meant to exclude other ingredients or steps. Embodiments defined by each of these transitional terms are within the scope of the present invention.

All numerical expressions, such as distance, magnitude, temperature, time, voltage, and concentration, including ranges, are approximations that are intended to encompass ordinary experimental variations in the measurement of parameters and that are intended to be within the scope of the described embodiments. It is to be understood, although not always explicitly stated that all numerical expressions are preceded by the term "about". It is also to be understood that, although not always explicitly stated, the ingredients described herein are exemplary only and that equivalents of such ingredients are known in the art.

As used herein, the term "nanopore" (or simply "pore") refers to a single nanoscale opening in a membrane separating two volumes. The pores may be protein channels, for example, inserted into lipid bilayer membranes, or may be engineered through a thin solid matrix (such as silicon nitride or silicon dioxide or graphene or a combination of these or other materials) by drilling or etching or using a voltage pulse method. Geometrically, the pores have dimensions of no less than 0.1nm in diameter and no more than 1 micron in diameter; the length of the pores is controlled by the film thickness, which may be sub-nanometer thickness or a thickness of up to 1 micron or more. For films with a thickness greater than a few hundred nanometers, nanopores may be referred to as "nanochannels.

As used herein, the term "nanoporosimetry" refers to a device that combines one or more nanopores (in parallel or series) with a circuit for detecting a single molecular event. In particular, a nanopore meter uses a sensitive voltage clamp amplifier to apply a specified voltage across one or more pores while simultaneously measuring the ionic current through the pores. When a single charged molecule, such as double-stranded dna (dsdna), is captured and driven through the pore by electrophoresis, the measured current changes, which are indicative of the capture event (i.e., translocation of the molecule through the nanopore or capture of the molecule in the nanopore) and the amount of change (magnitude of current) and duration of the event, are used to characterize the molecule captured in the nanopore. After many events are recorded during the experiment, the distribution of events is analyzed according to their change (i.e., their current signature) to characterize the corresponding molecules. In this way, nanopores provide a simple, label-free, pure-electron, single-molecule biomolecule detection method.

As used herein, the term "event" refers to the translocation of a detectable molecule or molecular complex through a nanopore and its associated measurement. This may be defined by its current, duration, and/or other detectable characteristics of the molecules in the nanopore. Multiple events with similar characteristics indicate a population of molecules or complexes that are the same or have similar characteristics (e.g., volume, charge).

Molecular detection

The present disclosure provides methods and systems for molecular detection and quantification. In addition, the methods and systems may also be configured to measure the affinity of the probe for binding to a target molecule. Furthermore, such detection, quantification and measurement can be performed in a multiplexed manner, thereby greatly improving its efficiency.

FIG. 1 provides an illustration of one embodiment of the disclosed method and system. More specifically, the system comprises a target-carrying molecule (102) comprising the motif of interest 101, which it is desired to detect or quantify. The probe (103) is capable of binding to a specific binding motif 101 on the target-carrying molecule 102. Additional molecules may be added to aid in the detection of the probe (107) on the target-carrying polynucleotide, if present.

Thus, if present in solution in its entirety, probe 103 binds to the target motif through its specific recognition of target motif 101. This binding results in the formation of a complex comprising the probe and the target sequence.

The formed complex (101/103 or 101/103/107) can be detected by a device (104) comprising two apertures (105 and 106) that divide the interior space of the device into 3 volumes and a sensor adjacent to the apertures configured to identify objects passing through the apertures. This embodiment is a dual nanopore device having two nanopores in series. In some embodiments, the nanopore device includes electronic components that route a controlled voltage across the nanopore (which in some embodiments can be independently controlled and clamped) and circuitry for measuring the current across the nanopore. The voltage can be adjusted to move the polynucleotide from one volume to another across the pore in a controlled manner. The polynucleic acids are charged or modified to contain a charge, and an applied trans-pore potential or voltage difference assists and controls the movement of the charged scaffold by applying electrostatic forces on the charged molecules exposed to the electric field. Although fig. 1 shows a dual nanopore device, the principles described above may also be applied to other embodiments of the invention using a single nanopore device. Unless indicated below, the references to pore or nanoporous devices are intended to encompass single pore, dual pore or porous devices within the spirit of the invention.

When a sample comprising the formed complex is loaded into the nanopore, the nanopore may be configured to pass a target-carrying molecule through the pore. When the target motif is within or adjacent to the pore, the binding state of the target motif can be detected by the sensor.

As used herein, the "binding state" of a target motif refers to whether the binding motif is occupied by a probe. Essentially, the bound state is bound or unbound. Either (i) the target motif is free and not bound to the probe (see 201 and 204 in FIG. 2), or (ii) the target motif is bound to the probe (see 202 and 205 in FIG. 2). In addition, probes of different sizes or with different probe binding sites can be used to give additional current profiles (see, e.g., 203 and 206 in fig. 2) to enable detection of more than one target sequence in one target-carrying molecule.

Detection of the binding state of the target motif can be accomplished by various methods. In one aspect, by virtue of the different sizes of the target motif in each state (i.e., occupied or unoccupied), the different sizes result in different currents across the pore as the target motif passes through the pore. In this respect, no separate sensor is required for detection, as the electrodes (which are connected to a power supply and can detect current) can be used for the detection function. Thus, both electrodes may be used as "sensors".

In some aspects, a reagent (e.g., 107 in fig. 1) is added to the complex to increase detection. This agent is capable of binding to the probe or polynucleotide/probe complex. In one aspect, the reagent includes a charge (negative or positive) to facilitate detection. In another aspect, the reagent is increased in size to facilitate detection. In another aspect, the reagent comprises a detectable label such as a fluorophore.

In this case, recognition of the bound state (ii) indicates that the target sequence in the target-carrying molecule is complexed with the probe. In other words, the target sequence is detected.

In another embodiment, the bound molecules are spaced apart to individually detect the bound molecules by a change in impedance, wherein each bound molecule gives an impedance value that is not masked by adjacent bound molecules.

In one embodiment, the bound probes are separated by a distance of at least 1nm (i.e., about 3bp for a nucleic acid-based polynucleotide). In another embodiment, the bound probes are separated by a distance of at least 10nm (i.e., about 33bp for a nucleic acid-based polynucleotide). In another embodiment, the bound probes are separated by a distance of at least 100nm (i.e., about 333bp for a nucleic acid-based polynucleotide). In another embodiment, the bound probes are separated by a distance of at least 500nm (i.e., about 1666bp for nucleic acid-based polynucleotides).

In some aspects, the method further comprises having two separate probes that can bind a third molecule if they are close enough to each other once bound to the polynucleotide. This binding of the third molecule provides a different translocation current characteristic, thus providing evidence of the close proximity of two independent probes.

The mechanism of determining whether probes bind closely allows us to use short probes that can discriminate single base pair mismatches and thus detect alleles with single nucleotide polymorphisms (or single nucleotide mutations) and longer probes that act as markers to determine unique sites in the genome. In one embodiment, we use two probes in combination to determine whether a particular sequence is associated with a particular target gene. In another embodiment, this method is used to determine structural rearrangements by using probes as markers for regions in the genome. In another embodiment, we used two probes in combination to determine whether a particular sequence was chemically (epigenetically, e.g., methylated, hydroxymethylated) modified and associated with a particular target gene.

In one embodiment, the third molecule is an antibody (301) that binds only probes (305 and 306) (if at least two probes bind to polynucleotide (304) and are significantly close to each other (0.01 nm-50 nm)) (fig. 3A). In another embodiment, half of the epitopes of antibody (301) are attached to each probe molecule (302 and 303) (by covalent attachment, ionic, H-bond, or other means) resulting in antibody binding to both epitopes depending on the location in close proximity, indicating that the probes are sufficiently close to each other (fig. 3B). In one embodiment, each probe is a PNA molecule, and each PNA molecule comprises or is attached to a fragment (covalently linked, ionic, H-bonded, or otherwise) of a binding epitope of an antibody. In this embodiment, a portion of the epitope must be sufficiently close for the antibody to bind to form a complex with the polynucleotide.

In another embodiment, the PEG molecule (310) is used as a third molecule to bind two probes (302 and 303) in close proximity and to the polynucleotide (304). In some embodiments, the PEG is modified to provide sufficient volume, charge, or other characteristics (when present) that allow for unique characteristics (fig. 4). In some embodiments, PEG is modified to increase binding affinity to probes in close proximity. Such binding modifications on PEG can be, for example, single-stranded dna (ssDNA) molecules at each end of PEG that are complementary to free ssDNA attached to each probe. In one embodiment, the energy barrier required for PEG binding to the probe is met only when two ssDNA oligomers bind to their complementary sequences attached to the probe (fig. 4). Thus, probes with the distance spanned by PEG are detected in the nanopore by detection of the PEG/probe/polynucleotide complex, while those at greater intervals are not. As recognized by those skilled in the art, ssDNA may be replaced by synthetic nucleic acid analogs such as PNA or by RNA.

In some aspects, two independent probes are modified to allow detection in their close proximity to bind to a polynucleotide. In one embodiment, modification of the probe comprises altering the ionic charge of the probe to alter the current signature when the probe is bound in close proximity to the polynucleotide and passed through the nanopore, as distinguished from the current signature of a single probe/polynucleotide complex when passed through the nanopore without being in close proximity to a second probe. In one embodiment, the addition of a positive charge to both probes (e.g., by labeling the probes with 2-hydroxyethyl thiosulfonate (MTSET)) provides different translocation current characteristics when the two probes are sufficiently close in space along the polynucleotide and the effect of the charge is additive, as opposed to they binding to the polynucleotide at a more distant interval.

In some aspects, the method further comprises using a probe that is long enough to be able to bind only one unique sequence in the target population, but also has the ability to not bind to the target site in the presence of only a single base pair mismatch. This is possible when using PNA probes. As shown (Strand-Invasion of Extended, Mixed-Sequence B-DNA by γ PNAs, G.He, D.Ly et al, J Am Chem Soc.2009September 2; 131(34): 12088-12090. doi:10.1021/ja900228J), a 20bp γ PNA probe was able to bind perfectly matched target sequences efficiently, but binding was abolished when the target and probe sequences differed by only one base. When considering the human genome, which contains 31 megabases, a 20 base pair sequence is likely to occur randomly 0.003 times. Thus, probes designed to bind 20 base pairs to the specific sequence under study are less likely to bind to undesired locations and provide false positives. The examples contained therein (FIGS. 19c and 21) show that PNA and PNA-PEG probes selectively bind only complementary sequences.

In some aspects, the method further comprises having two separate probes comprising an element that emits a detectable signal when the two probes are linked in sufficient proximity to the polynucleotide. In one embodiment, each probe is labeled with a fluorophore (see, e.g., fig. 5(315, 316)). The emission spectrum is detected by a detector (317) when the probes are sufficiently close to produce a detectable signal. In one embodiment, the two probes are labeled with different colored fluorophores. When the probes are in close proximity, the colors are imaged together (or blended to provide a new color) which can be detected with an external sensor such as a camera or microscope and demonstrate that the two probes are in spatial proximity. In a related embodiment, detection of the FRET (or BRET) type is used to determine the proximity of two probes, such that one fluorescently labeled probe affects the energy emission spectrum of the other probe in close proximity.

In some embodiments, the detectable label is a fluorophore. To detect fluorophores, a nanopore device fabricated in-plane with a coverslip can be combined with an epifluorescence microscope to enable dual current magnitudes and fluorescence signal detection. Figure 6A shows how such a device can be used to detect added fluorophore labels. The nanopore device is placed under the objective of an epifluorescence microscope. As the nanopore measurement progresses, the microscope continues to image the nanopore region. The nanopore region is illuminated by a broadband excitation light source that is filtered so as to allow only wavelengths corresponding to the excitation spectrum of the fluorophore to pass. The dichroic filter selectively allows transmission of wavelengths corresponding to the emission spectrum of the fluorophore while emitting all other wavelengths simultaneously. As the fluorophore-modified binding molecule passes through the nanopore, the fluorophore absorbs the excitation spectrum and re-emits an emission spectrum. An emission filter in front of the detector ensures that only the wavelengths corresponding to the emission spectrum of the fluorophore are detected. Thus, the detector only has a signal when the fluorophore passes through the nanopore. Figure 6B shows a top view of the nanopore device viewed through a microscope during fluorophore emission. Figure 6C demonstrates how detection of a fluorophore can be used in conjunction with the signal from the nanopore. Two signals are used to enhance the reliability of biomolecule detection.

In some aspects, the method further comprises using probes having features that allow for detection of attachment by the sensor, but which are linked to the probes using cleavable linkers. Thus, probe sets that can be distinguished from each other in the nanopore bind to the target carrying polynucleotide. Once the probe set is detected in the nanopore, the feature is cut away and a new probe set is added that also has a cleavable detection feature (fig. 7). The addition/cleavage/washing cycle may continue until all sequence information is extracted from the captured target molecules. Examples of molecules that aid in probe detection are described above. Examples of Cleavable linkers are reducing agent cleaved linkers (disulfide linkers cleaved by TCEP), acid cleaved linkers (hydrazone/hydrazide bonds), amino acid sequences cleaved by proteases, nucleic acid linkers cleaved by endonucleases (site-specific restriction enzymes), alkali cleaved linkers or photocleaved linkers [ Leriche, Geoffray, Louise Chisholm and Alain wagner.

Target motif

For nucleic acids and polypeptides to which the target sequence detection method applies, the target binding motif can be a nucleotide or peptide sequence that is recognized by the probe molecule. The target motif may be chemically modified (e.g., methylated) or occupied by other molecules (e.g., activators or repressors), and depending on the nature of the probe, the state of the target motif may be elucidated. In some aspects, the target sequence comprises a chemical modification for binding the probe to the polynucleotide. In some aspects, the chemical modification is selected from the group consisting of acetylation, methylation, small ubiquitin-like modified proteolysis, glycosylation, phosphorylation, and oxidation.

Probe molecule

In the present technology, a probe molecule is detected or quantified by its binding to a polynucleotide carrying a target.

A probe as used herein is understood to be capable of specifically binding to a site on a polynucleotide, wherein the site is characterized by sequence or structure. Examples of probe molecules include PNA (protein nucleic acid), bis-PNA, γ -PNA, PNA-conjugates that increase the size or charge of the PNA. Other examples of probe molecules are from the group of natural or recombinant proteins, fusion proteins, DNA binding domains of proteins, peptides, nucleic acids, oligonucleotides, TALENs, CRISPR, PNA (protein nucleic acid), bis-PNA, γ -PNA, PNA-conjugates (e.g. oligomer-labeled) or any other PNA-derived polymer that increases size, charge, fluorescence or functionality, and chemical compounds.

In some aspects, the probe comprises γ -PNA. Gamma-PNAs have simple modifications in peptide-like backbones, especially at the gamma-position of the N- (2-aminoethyl) glycine backbone, thus generating chiral centers (Rapireddy S. et al, 2007.J. Am. chem. Soc.,129: 15596-152; He G et al, 2009, J. Am. chem. Soc.,131: 12088-90; Chema V et al, 2008, Chemiochem 9: 2388-91; Dragulescu-Andrasi, A. et al, 2006, J.Am. chem. Soc.,128: 10258-10267). Unlike bis-PNA, γ -PNA can bind dsDNA without sequence restriction, leaving one of the two DNA strands available for further hybridization.

In some aspects, the probe functions to hybridize to a polynucleotide having a target sequence through complementary base pairing to form a stable complex. The PNA molecule may additionally be combined with additional molecules to form a complex having a cross-sectional area large enough to produce a detectable change or contrast in the magnitude of the signal above background. The background is the average or mean signal magnitude corresponding to a cross-section of the non-probe-bound polynucleotide.

The stability of the binding of polynucleotide target sequences to PNA molecules is important for their detection by nanopore devices. Binding stability must be maintained throughout translocation of the polynucleotide carrying the target through the nanopore. If the stability is weak or unstable, the probe can be separated from the target polynucleotide and will not be detected when the polynucleotide carrying the target passes through the nanopore.

In certain embodiments, an example of a probe is a PNA-conjugate in which the PNA moiety specifically recognizes a nucleotide sequence and the binding moiety increases the size/shape/charge difference between different PNA-conjugates.

As shown in fig. 8, ligands A, B, C and D each specifically bind to a site on a DNA molecule, and these ligands can be recognized and distinguished from each other by their width, length, size, and/or charge. If their corresponding sites are designated A, B, C and D, respectively, recognition of the ligand results in revealing those DNA sequences, A-B-C-D, in terms of the composition and order of the sites.

Different reactive moieties may be incorporated into the ligand to provide a chemical treatment (chemical handle) to which the tag can be coupled. Examples of reactive moieties include, but are not limited to, primary amines, carboxylic acids, ketones, amides, aldehydes, boronic acids, hydrazones, thiols, maleimides, alcohols, and hydroxyl groups, and biotin.

Figure 9A shows PNA ligands modified to increase ligand charge and thus facilitate detection through a nanopore. Specifically, this ligand, which binds to the target DNA through complementary base pairing between bases on the PNA molecule and bases in the target DNA and Hoogsteen base pairing, has cysteine residues incorporated into the backbone, which provides a free thiol chemical treatment for labeling. Here, cysteine is tagged to the peptide 2-aminoethylmethane thiosulfonate (MTSEA) via a maleimide linker, which provides a means to detect whether a ligand binds to its target sequence, as the tag/peptide results in an increase in the ligand charge. This greater charge results in a greater change in current through the pores compared to unlabeled PNA.

In some aspects, to increase the contrast of the change between the ligand-binding polynucleotide and other background molecules present in the sample, the pseudopeptide backbone can be modified to alter the overall size of the ligand (e.g., PNA) to increase contrast. See, for example, fig. 9B, which shows PNAs with incorporated cysteine residues (301) modified with SMCC linkers (302) to enable coupling to peptides (303) through their N-terminal amides. In addition to increasing the charge by labeling the ligand (e.g., as in FIG. 9A), the selection of more charged amino acids rather than non-polar amino acids can also be used to increase the charge of the PNA. In addition, small particles, molecules, proteins, peptides or polymers (e.g., PEG) can be coupled to the pseudopeptide backbone to increase the volume or cross-sectional area of the ligand and the polynucleotide complex carrying the target. The increased volume serves to increase the signal magnitude contrast so that any difference signal caused by the increased volume can be easily detected. Examples of small particles, molecules, proteins or peptides that can be coupled to the pseudopeptide backbone include, but are not limited to, alpha-helix forming peptides, nanoscale gold particles or rods (e.g., 3nm), quantum dots, polyethylene glycol (PEG). Methods of molecular coupling are well known to those skilled in the art, for example, in U.S. Pat. Nos. 5,180,816,6,423,685,6,706,252,6,884,780, and 7,022,673, which are incorporated herein by reference in their entirety.

The above embodiments describe PEG labeling by cysteine residues, but other residues may also be used. For example, lysine residues are easily interchanged with cysteine residues to achieve attachment chemistry using NHS-esters and free amines. Moreover, PEG can be easily interchanged between bifunctional linkers and PNAs with other volume-increasing components such as Dendrons, beads or rods, or with Dendron coupled directly. One skilled in the art will recognize the flexibility of this system in that amino acids can be changed and the attachment chemistry can be changed for that particular amino acid, such as a serine reactive isocyanate. Some examples of linkage chemistries that can be used in this reaction are listed in the following table.

Table 1: chemical action of ligation

Reactive group	Target functional group
		Arylazide compounds	Nonselective or primary amines
Carbodiimides	Amine/carboxyl
		Hydrazides	Carbohydrate compound
Hydroxymethyl phosphine	Amines as pesticides
		Imido ester	Amines as pesticides
Isocyanates	Hydroxy radical
		Carbonyl radical	Hydrazine
Maleimide	Mercapto group
		NHS-esters	Amines as pesticides
PFP-esters	Amines as pesticides
		Psoralen	Thymidine
Pyridine disulfide	Mercapto group
		Vinyl sulfone	Mercaptoamine, hydroxy

Figures 3A, 3B, 4, 5, 9A, 9B, and 18A show PNA probes modified to increase probe size, comprise an epitope, comprise a ssDNA oligomer, comprise a fluorophore, an added charge, or an added size to facilitate detection or detection of two probes in close proximity.

Different reactive moieties may be incorporated into the probe to provide a chemical treatment to which the label may be coupled. Examples of reactive moieties include, but are not limited to, primary amines, carboxylic acids, ketones, amides, aldehydes, boronic acids, hydrazones, thiols, maleimides, alcohols, and hydroxyl groups, and biotin.

A common method for incorporating chemical treatments is to include specific amino acids into the backbone of the probe. Examples include, but are not limited to, cysteine (to provide thiolates), lysine (to provide free amines), threonine (to provide hydroxyl groups), glutamic acid, and aspartic acid (to provide carboxylic acids).

Different types of labels can be added using reactive moieties. These include the following notations:

1. the size of the probe is increased, e.g. biotin/streptavidin, peptides, nucleic acids.

2. Altering the charge of the probe, such as a charged peptide (6xHIS) or protein (e.g., charthodotoxin), or a small molecule or peptide (e.g., MTSET).

3. Changing or increasing the fluorescence of the probe, e.g. common fluorophores, FITC, rhodamine, Cy3, Cy 5.

4. An epitope or interaction site for binding a third molecule, e.g., a peptide for binding an antibody, is provided.

Multiplexing

In some embodiments, instead of including the same kind of probes as described above, a collection of different probes is added, each binding a unique site or target motif.

With this arrangement, a plurality of different probes can be used to detect a plurality of different target sites within the same target-carrying polynucleotide. Fig. 10 illustrates this approach. Here, double-stranded DNA 1002 contains multiple different target motifs, two copies 1003, two copies 1004, and one copy 1005.

By using probes 1006, 1007, and 1008, each providing a unique current signature (e.g., by differing in size), the present technology can detect different target motifs within the same molecule, thereby providing a means to multiplex target motif detection. In addition, by counting how many of each unique probe is bound, the number (or copy number) of each target can be determined. By adjusting the conditions that affect binding, the system can obtain more detailed binding dynamics information.

Similarly, multiplexing can be accomplished by employing mix-and-match with a collection of probes having different properties and any of a variety of combinations, the only requirement being that probes that bind to different sequences be distinguishable from each other. For example, the assay may use probes distinguishable by size and additional probes distinguishable by size (fig. 9A, 9B and 19).

Additional methods of multiplexing include designing probes that bind to polynucleotides at known sequences at fixed positions relative to each other to interrogate a sample comprising a collection of nucleic acids from different species. As an example of this method, if we test a water source for three different bacteria with known sequences, we can locate two probes spaced 1000 base pairs apart for species a, two probes spaced 3000bp base pairs apart for species B and two probes spaced 5000 base pairs apart for species C. Species a and B are present but species C does not reach detectable levels if probes separated by 1000 base pairs and 3000 base pairs are detected. This same approach with designed spacing can also be used for multiplexing of the detection of known or mutated sequences in a particular target sample.

Nanopore device

The provided nanopore device includes an aperture that forms an opening in a structure that divides an interior space of the device into two volumes, and is configured to identify an object passing through the aperture, e.g., with a sensor (e.g., by detecting a change in a parameter indicative of the object). A nanopore device for use in the methods described herein is also disclosed in PCT publication WO/2013/012881, which is incorporated by reference in its entirety.

The pores in the nanoporous devices are nano-sized or micro-sized. In one aspect, each pore is sized to allow passage of small or large molecules or microorganisms. In one aspect, each pore is at least about 1 nanometer in diameter. Optionally, each pore diameter is at least about 2 nanometers, 3 nanometers, 4 nanometers, 5 nanometers, 6 nanometers, 7 nanometers, 8 nanometers, 9 nanometers, 10 nanometers, 12 nanometers, 13 nanometers, 14 nanometers, 16 nanometers, 17 nanometers, 18 nanometers, 19 nanometers, 20 nanometers, 25 nanometers, 30 nanometers, 35 nanometers, 40 nanometers, 45 nanometers, 50 nanometers, 60 nanometers, 70 nanometers, 80 nanometers, 90 nanometers, or 100 nanometers.

In one aspect, the pores are no more than about 100 nanometers in diameter. Alternatively, the pore diameter is no more than about 95 nanometers, 90 nanometers, 85 nanometers, 80 nanometers, 75 nanometers, 70 nanometers, 65 nanometers, 60 nanometers, 55 nanometers, 50 nanometers, 45 nanometers, 40 nanometers, 35 nanometers, 30 nanometers, 25 nanometers, 20 nanometers, 15 nanometers, or 10 nanometers.

In some aspects, each pore has a diameter of at least about 100 nanometers, 200 nanometers, 500 nanometers, 1000 nanometers, 2000 nanometers, 3000 nanometers, 5000 nanometers, 10000 nanometers, 20000 nanometers, or 30000 nanometers. In one aspect, the pore diameter is no more than about 100000 nanometers. Alternatively, the pore diameter is no more than about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm.

In one aspect, the pore diameter is between about 1 nanometer and about 100 nanometers, or alternatively, between about 2 nanometers and about 80 nanometers, or between about 3 nanometers and about 70 nanometers, or between 4 nanometers and about 60 nanometers, or between about 5 nanometers and about 50 nanometers, or between about 10 nanometers and about 40 nanometers, or between about 15 nanometers and about 30 nanometers.

In some aspects, the pores in the nanopore device are of a larger scale for detection of large microorganisms or cells. In one aspect, the size of each pore allows large cells or microorganisms to pass through. In one aspect, each pore is at least about 100 nanometers in diameter. Optionally, each pore diameter is at least about 200 nanometers, 300 nanometers, 400 nanometers, 500 nanometers, 600 nanometers, 700 nanometers, 800 nanometers, 900 nanometers, 1000 nanometers, 1100 nanometers, 1200 nanometers, 1300 nanometers, 1400 nanometers, 1500 nanometers, 1600 nanometers, 1700 nanometers, 1800 nanometers, 1900 nanometers, 2000 nanometers, 2500 nanometers, 3000 nanometers, 3500 nanometers, 4000 nanometers, 4500 nanometers, or 5000 nanometers.

In one aspect, the pore diameter is no more than about 100000 nanometers. Optionally, the pore diameter is no more than about 90000 nanometers, 80000 nanometers, 70000 nanometers, 60000 nanometers, 50000 nanometers, 40000 nanometers, 30000 nanometers, 20000 nanometers, 10000 nanometers, 9000 nanometers, 8000 nanometers, 7000 nanometers, 6000 nanometers, 5000 nanometers, 4000 nanometers, 3000 nanometers, 2000 nanometers, or 1000 nanometers.

In one aspect, the pores have a diameter between about 100 nanometers and about 10000 nanometers, or alternatively between about 200 nanometers and about 9000 nanometers, or between about 300 nanometers and about 8000 nanometers, or between about 400 nanometers and about 7000 nanometers, or between about 500 nanometers and about 6000 nanometers, or between about 1000 nanometers and about 5000 nanometers, or between about 1500 nanometers and about 3000 nanometers.

In some aspects, the nanopore device further comprises means for moving the polymer scaffold across the aperture and/or means for identifying an object passing through the aperture. Further details are provided below, which are described in the context of a dual aperture device.

A dual-pore device can be more easily configured to provide good control of the speed and direction of movement of the polymer scaffold across the pores compared to a single-pore nanopore device.

In certain embodiments, the nanopore device comprises a plurality of chambers, each chamber communicating with an adjacent chamber through at least one aperture. Of these apertures, two apertures (i.e., a first aperture and a second aperture) are positioned such that at least a portion of the polymer scaffold is allowed to move out of the first aperture and into the second aperture. In addition, the device includes a sensor capable of identifying the polymer scaffold during movement. In one aspect, identification entails identifying individual polymer scaffold components. In another aspect, identification requires identification of fusion molecules and/or target analytes bound to the polymer scaffold. When a single sensor is employed, the single sensor may comprise two electrodes placed across the aperture to measure the ionic current across the aperture. In another embodiment, a single sensor comprises components other than electrodes.

In one aspect, the device comprises three chambers connected by two apertures. Devices having more than three chambers can be readily designed to include one or more additional chambers on either side of a three-chamber device or between any two of the three chambers. Likewise, more than two apertures connecting the chambers may be included in the device.

In one aspect, there may be two or more apertures between two adjacent chambers to allow multiple polymer scaffolds to move from one chamber to the next simultaneously. Such a porous design may improve the throughput of polymer scaffold assays in the device.

In some aspects, the device further comprises means for moving the polymer scaffold from one chamber to another chamber. In one aspect, the movement results in loading the polymer scaffold simultaneously across both the first aperture and the second aperture. In another aspect, the means further enables the polymeric bone strands to move in the same direction through both apertures.

For example, in a three-chamber two-aperture device ("two-aperture" device), each chamber may contain electrodes for connection to a power source, so that separate voltages may be applied across the various apertures between the chambers.

According to one embodiment of the present invention, there is provided an apparatus comprising an upper chamber, a middle chamber, and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first aperture and the middle chamber is in communication with the lower chamber through a second aperture. Such devices may have any of the dimensions or other features previously disclosed in U.S. publication No.2013-0233709, entitled dual aperture device, which is hereby incorporated by reference in its entirety.

In some embodiments as shown in fig. 7A, the apparatus comprises an upper chamber 705 (chamber a), a middle chamber 704 (chamber B), and a lower chamber 703 (chamber C). The chambers are separated by two separate layers or membranes (702 and 701), each having a separate aperture (711 or 712). In addition, each chamber contains an electrode (721, 722, or 723) for connection to a power supply. The designations of the upper, middle and lower chambers are relative expressions and do not indicate, for example, that the upper chamber is placed above the middle or lower chamber with respect to the ground and vice versa.

Each pore 711 and 712 independently has a size that allows passage of small or large molecules or microorganisms. In one aspect, each pore is at least about 1 nanometer in diameter. Optionally, each pore diameter is at least about 2 nanometers, 3 nanometers, 4 nanometers, 5 nanometers, 6 nanometers, 5 nanometers, 7 nanometers, 8 nanometers, 9 nanometers, 10 nanometers, 11 nanometers, 12 nanometers, 13 nanometers, 14 nanometers, 15 nanometers, 16 nanometers, 17 nanometers, 18 nanometers, 19 nanometers, 20 nanometers, 25 nanometers, 30 nanometers, 35 nanometers, 40 nanometers, 45 nanometers, 50 nanometers, 60 nanometers, 70 nanometers, 80 nanometers, 90 nanometers, or 100 nanometers.

In one aspect, the pores are no more than about 100 nanometers in diameter. Alternatively, the pore diameter is no more than about 95 nanometers, 90 nanometers, 85 nanometers, 80 nanometers, 75 nanometers, 70 nanometers, 65 nanometers, 60 nanometers, 55 nanometers, 50 nanometers, 45 nanometers, 40 nanometers, 35 nanometers, 30 nanometers, 25 nanometers, 20 nanometers, 15 nanometers, or 10 nanometers.

In one aspect, the pore diameter is between about 1 nanometer and about 100 nanometers, or alternatively between about 2 nanometers and about 80 nanometers, or between about 3 nanometers and about 70 nanometers, or between about 4 nanometers and about 60 nanometers, or between about 5 nanometers and about 50 nanometers, or between about 10 nanometers and about 40 nanometers, or between about 15 nanometers and about 30 nanometers.

In other aspects, each pore diameter is at least about 100 nanometers, 200 nanometers, 500 nanometers, 1000 nanometers, 2000 nanometers, 3000 nanometers, 5000 nanometers, 10000 nanometers, 20000 nanometers, or 30000 nanometers. In one aspect, each pore diameter is 50000 nanometers to 100000 nanometers. In one aspect, the pore diameter is no more than about 100000 nanometers. Alternatively, the pore diameter is no more than about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm.

In some aspects, the pores are substantially circular. As used herein, "substantially circular" refers to a shape that is at least about 80 or 90% cylindrical in form. In some embodiments, the aperture shape is square, rectangular, triangular, oval, or hexagonal.

The apertures 711 and 712 independently have respective depths (i.e., the lengths of the apertures extending between two adjacent volumes). In one aspect, each pore is at least about 0.3 nanometers deep. Optionally, each pore depth is at least about 0.6 nanometers, 1 nanometer, 2 nanometers, 3 nanometers, 4 nanometers, 5 nanometers, 6 nanometers, 7 nanometers, 8 nanometers, 9 nanometers, 10 nanometers, 11 nanometers, 12 nanometers, 13 nanometers, 14 nanometers, 15 nanometers, 16 nanometers, 17 nanometers, 18 nanometers, 19 nanometers, 20 nanometers, 25 nanometers, 30 nanometers, 35 nanometers, 40 nanometers, 45 nanometers, 50 nanometers, 60 nanometers, 70 nanometers, 80 nanometers, or 90 nanometers.

In one aspect, each pore depth is no more than about 100 nanometers. Optionally, the depth is no more than about 95 nanometers, 90 nanometers, 85 nanometers, 80 nanometers, 75 nanometers, 70 nanometers, 65 nanometers, 60 nanometers, 55 nanometers, 50 nanometers, 45 nanometers, 40 nanometers, 35 nanometers, 30 nanometers, 25 nanometers, 20 nanometers, 15 nanometers, or 10 nanometers.

In one aspect, the depth of the pores is between about 1 nanometer and about 100 nanometers, or alternatively between about 2 nanometers and about 80 nanometers, or between about 3 nanometers and about 70 nanometers, or between about 4 nanometers and about 60 nanometers, or between about 5 nanometers and about 50 nanometers, or between about 10 nanometers and about 40 nanometers, or between about 15 nanometers and about 30 nanometers.

In some aspects, the nanopores extend through the membrane. For example, the pores may be protein channels inserted into the lipid bilayer membrane, or they may also be engineered by drilling, etching, or otherwise forming pores through a solid matrix (such as silica, silicon nitride, graphene, or layers formed from combinations of these or other materials). In some aspects, the length or depth of the nanopores is large enough to form a channel connecting two otherwise separated volumes. In some such aspects, the depth of each pore is greater than 100 nanometers, 200 nanometers, 300 nanometers, 400 nanometers, 500 nanometers, 600 nanometers, 700 nanometers, 800 nanometers, or 900 nanometers. In some aspects, the depth of each pore is no more than 2000 nanometers or 1000 nanometers.

In one aspect, the pores are spaced apart by a distance between about 10 nanometers and about 1000 nanometers. In some aspects, the distance between pores is greater than 1000 nanometers, 2000 nanometers, 3000 nanometers, 4000 nanometers, 5000 nanometers, 6000 nanometers, 7000 nanometers, 8000 nanometers, or 9000 nanometers. In some aspects, the pore spacing is no more than 30000 nanometers, 20000 nanometers, or 10000 nanometers. In one aspect, the distance is at least about 10 nanometers, or alternatively at least about 20 nanometers, 30 nanometers, 40 nanometers, 50 nanometers, 60 nanometers, 70 nanometers, 80 nanometers, 90 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, or 300 nanometers. In another aspect, the distance is no more than about 1000 nanometers, 900 nanometers, 800 nanometers, 700 nanometers, 600 nanometers, 500 nanometers, 400 nanometers, 300 nanometers, 250 nanometers, 200 nanometers, 150 nanometers, or 100 nanometers.

In yet another aspect, the distance between the pores is between about 20 nanometers and about 800 nanometers, between about 30 nanometers and about 700 nanometers, between about 40 nanometers and about 500 nanometers, or between about 50 nanometers and about 300 nanometers.

The two apertures may be arranged in any position as long as they allow fluid communication between the chambers and have a defined size and spacing. In one aspect, the pores are arranged such that there is no direct blockage therebetween. In yet another aspect, the apertures are substantially coaxial, as shown in fig. 7A.

In one aspect, as shown in fig. 7A, the device is connected to one or more power sources through electrodes 721, 722, and 723 in chambers 703, 704, and 705, respectively. In some aspects, the power supply includes a voltage clamp or patch clamp that can provide a voltage across each aperture and independently measure a current through each aperture. In this aspect, the power supply and electrode configuration may place the middle chamber as a common ground for both power supplies. In one aspect, the one or more power supplies are configured to apply a first voltage V between the upper chamber 705 (chamber a) and the middle chamber 704 (chamber B)₁And between the middle chamber 704 and the lower chamber 703 (chamber C)Applying a second voltage V₂。

In some aspects, the first voltage V₁And a second voltage V₂Are independently adjustable. In one aspect, the middle chamber is regulated to a ground voltage with respect to the two voltages. In one aspect, the middle chamber contains a medium for providing electrical conductance between the respective apertures and the electrodes in the middle chamber. In one aspect, the middle chamber contains a medium for providing electrical resistance between each aperture and an electrode in the middle chamber. Keeping this resistance small enough relative to the nanopore resistance facilitates decoupling of the two voltages and currents across the pore, which facilitates independent adjustment of the voltages.

The adjustment of the voltage may be used to control the movement of charged particles within the chamber. For example, when the two voltages are set to the same polarity, suitably charged particles can be moved sequentially from the upper chamber to the middle chamber and to the lower chamber, or vice versa. In some aspects, when the two voltages are set to opposite polarities, charged particles may move from the upper or lower chamber to the middle chamber and stay there.

The adjustment of the voltage in the device can be used in particular for the control of the movement of large molecules, such as charged polymer scaffolds, which are long enough to span both pores simultaneously. In this regard, the direction and speed of molecular movement can be controlled by the relative magnitude and polarity of the voltage, as described below.

The device may comprise a material suitable for containing a liquid sample, in particular a biological sample, and/or a material suitable for nanofabrication. In one aspect, such materials include dielectric materials such as, but not limited to, silicon nitride, silicon dioxide, graphene, carbon nanotubes, TiO₂、HfO₂、Al₂O₃Or other metal layers, or any combination of these materials. In some aspects, for example, a monolithic graphene film about 0.3 nanometers thick can be used as the pore-bearing film.

Devices that are microfluidic devices and that house a dual pore microfluidic chip facility can be fabricated by a variety of means and methods. For microfluidic chips consisting of two parallel membranes, the two membranes can be drilled simultaneously through a single beam to form two concentric apertures, although it is also possible to use different beams on each side of the membrane in conjunction with any suitable alignment technique. In general terms, the housing ensures sealed separation of the chambers A-C. In one aspect shown in fig. 7B, the housing provides a minimum access resistance between the voltage electrodes 721, 722, and 723 and the nanopores 711 and 712 to ensure that each voltage is applied primarily across the respective pores.

In one aspect, the device comprises a microfluidic chip (labeled "dual core chip") composed of two parallel membranes connected by a spacer. Each membrane contains pores formed by single beam drilling through the center of the membrane. Further, the device preferably has a chip for the chipA housing. The enclosure ensures sealed separation of the chambers a-C and provides a minimum access resistance for the electrodes to ensure that each voltage is applied primarily across the respective aperture.

More specifically, the pore-bearing film can be fabricated with a Transmission Electron Microscopy (TEM) grid having silicon, silicon nitride or silicon dioxide windows of 5-100 nanometers thick. The spacers may be of an insulator (e.g., SU-8, photoresist, PECVD oxide, ALD alumina) or vaporized metallic material (e.g., silver, gold, or platinum) and occupy a small space in the otherwise aqueous portion of chamber B between the membranes for separating the membranes. The holder is placed in an aqueous bath constituted by the largest volume part of chamber B. Chambers a and C may be reached through larger diameter channels (for low access resistance) which results in a membrane seal.

A focused electron or ion beam can be used to drill an aperture through the membrane and align it naturally. The pores can also be engraved (shrunk) to smaller dimensions by applying an appropriate beam focus to each layer. Any single nanopore drilling method may also be used to drill a pore pair in both membranes, taking into account the drilling depth and membrane thickness possible for a given method. Pre-drilling of the micropores to a specified depth and then drilling of the nanopores through the remainder of the membrane is also possible to further optimize the thickness of the membrane.

In another aspect, biological nanopores inserted into solid-state nanopores to form hybrid pores can be used for either or both pores in a dual-pore approach. Biological pores can increase the sensitivity of ion current measurements and are useful when only single-stranded polynucleotides are captured and controlled (e.g., for sequencing) in a dual pore device.

Charged molecules can move through the pores between the chambers by the voltage present at the pores of the device. The speed and direction of movement can be controlled by the magnitude and polarity of the voltage. Furthermore, since the two voltages can be adjusted independently of each other, the direction and speed of movement of the charged molecules can be finely controlled in each chamber.

One example involves a charged polymer scaffold, such as DNA, that is longer than the combined distance comprising the depth of two pores plus the distance between two pores. For example, a 1000bp dsDNA is about 340 nm in length and is significantly greater than the 40nm distance spanned by two 10nm deep gaps spaced 20 nm apart. In the first step, the polynucleotide is loaded into the upper or lower chamber. Because it is negatively charged under physiological conditions at a pH of about 7.4, the polynucleotide can move across the pore to which the voltage is applied. Thus, in the second step, two voltages of the same polarity and the same or similar magnitude are applied to the pores to move the polynucleotide sequentially across the two pores.

One or both of the voltages may be changed approximately when the polynucleotide reaches the second pore. Since the distance between the two pores is chosen to be shorter than the length of the polynucleotide, when the polynucleotide reaches the second pore, it is also in the first pore. Thus, the sudden change in voltage polarity at the first aperture creates a force that pulls the polynucleotide away from the second aperture, as shown in fig. 7C.

Suppose two pores have the same voltage-force effect, and | V₁|＝|V₂The value V > 0 (or < 0) can be set to | + VV₁L (| (or V))₂) The direction is adjusted by obtaining adjustable movement. In practice, although the voltage-induced forces at the individual pores are not due to V₁＝V₂But the same, calibration experiments may determine the appropriate bias voltage to produce an equal pull force for a given dual aperture chip; and the variation around this bias voltage can then be used for directional control.

If, at this point, the magnitude of the voltage-induced force at the first aperture is less than the magnitude of the voltage-induced force at the second aperture, the polynucleotide continues to move across both apertures toward the second aperture, but at a slower rate. In this regard, it is readily understood that the speed and direction of polynucleotide motion can be controlled by the polarity and magnitude of the two voltages. As will be described further below, this fine control of motion has wide application.

Accordingly, in one aspect, a method for controlling the movement of a charged polymer scaffold through a nanoporous device is provided. The method entails (a) loading a sample comprising a charged polymer scaffold into one of the upper, middle or lower chambers of the device of any of the above embodiments, wherein the device is connected to one or more power supplies for providing a first voltage between the upper and middle chambers and a second voltage between the middle and lower chambers; (b) setting an initial first voltage and an initial second voltage to move the polymer scaffold between the chambers to position the polymer scaffold across both the first and second apertures; and (c) adjusting the first and second voltages such that both voltages generate a force that pulls the charged polymer scaffold away from the middle chamber (voltage competition mode), wherein under controlled conditions, the two voltages are different in magnitude such that the charged polymer scaffold moves across the two pores in either direction and in a controlled manner.

To establish the voltage competition pattern in step (c), the relative forces exerted by the voltages at the respective pores are determined for each of the dual pore devices used, and this can be done using calibration experiments by observing the effect of different voltage values on the movement of the polynucleotide, which can be measured by probing for detectable features in the polynucleotide and known locations (examples of which are set forth in detail in this disclosure). For example, if the forces are equal at each common voltage, the use of the same voltage value (common polarity in the upper and lower chambers relative to the grounded middle chamber) at each aperture produces a zero value net motion in the absence of thermal disturbances (the presence and effect of brownian motion discussed below). If the forces are not equal at the respective common voltages, achieving equal forces includes determining and using a larger voltage at the aperture that produces a weaker force at the common voltage. Calibration of the voltage competition mode can be performed for each dual-pore device and for a particular charged polymer or molecule whose characteristics affect the force at which the particular charged polymer or molecule passes through the respective pores.

In one aspect, a sample containing a charged polymer scaffold is loaded into the upper chamber and an initial first voltage is set to pull the charged polymer scaffold from the upper chamber to the middle chamber and an initial second voltage is set to pull the polymer scaffold from the middle chamber to the lower chamber. Likewise, the sample may be initially loaded into the lower chamber and the charged polymer scaffold may be drawn into the middle and upper chambers.

In another aspect, a sample containing a charged polymer scaffold is loaded into the middle chamber; the initial first voltage is set to draw the charged polymer stent from the middle chamber to the upper chamber; and the initial second voltage is set to draw the charged polymer stent from the middle chamber to the lower chamber.

In one aspect, the first voltage and the second voltage adjusted in step (c) are up to about 10 times to about 10000 times the difference/difference between the two voltages in magnitude. For example, the two voltages may be 90 millivolts and 100 millivolts, respectively. The magnitude of the two voltages (about 100 millivolts) is about 10 times the difference/difference between them (10 millivolts). In some aspects, the magnitude of the voltage is up to at least about 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 400 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times, or 9000 times the difference/difference between the two voltages. In some aspects, the voltage magnitude is no more than about 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2000 times, 1000 times, 500 times, 400 times, 300 times, 200 times, or 100 times the difference/difference between the two voltages.

In one aspect, the real-time or on-line adjustment of the first voltage and the second voltage in step (c) is performed by active control or feedback control using dedicated hardware and software at clock frequencies up to several hundred megahertz. The automatic control of the first voltage or the second voltage or both is based on feedback of the first or second or both ion current measurements.

Sensor with a sensor element

In certain embodiments, the nanopore device of the present invention comprises one or more sensors to accomplish identification of the binding state of the target motif.

The sensor used in the device may be any sensor suitable for recognizing molecules or particles, such as charged polymers. For example, the sensor may be configured to identify the charged polymer by measuring a current, a voltage, a pH, an optical characteristic, or a residence time associated with the charged polymer or one or more individual components of the charged polymer. In some embodiments, the sensor includes a pair of electrodes placed on opposite sides of the pore to measure the cross-pore ionic current as a molecule or particle, particularly a charged polymer (e.g., polynucleotide), moves through the pore.

In certain embodiments, the sensor measures an optical characteristic of the polymer or a component (or unit) of the polymer. One example of such a measurement includes identifying the unique absorption band of a particular cell by infrared (or ultraviolet) spectroscopy.

When using dwell time measurements, the size of a cell is associated with a particular cell based on the length of time that the particular cell spends through the sensing device.

In some embodiments, the sensor is functionalized with reagents that form different non-covalent bonds with each probe. In this regard, the gap may be larger and still allow for effective measurements. For example, a gap of 5nm can be used to detect probe/target complexes that are approximately measured at 5 nm. Channel sensing with functionalized sensors is referred to as "recognition tunneling". Probes that bind to the target motif are readily identified using a Scanning Tunneling Microscope (STM) with a recognition tunneling effect.

Thus, the methods of the present technology can provide charged polynucleotide (e.g., DNA) transmissibility control for one or more recognition tunnel sites, each recognition tunnel site located in one or two nanopore channels or between pores, and voltage control can ensure that each probe/target complex resides at each site for a sufficient period of time for stable recognition.

The sensors in the devices and methods of the present disclosure may comprise gold, platinum, graphene, or carbon, or other suitable materials. In a particular aspect, the sensor includes a component made of graphene. Graphene can act as a conductor and insulator, so tunneling currents through graphene and across the nanopore can sequence displaced DNA.

In some embodiments, the tunnel gap has a width of about 1 nanometer to about 20 nanometers. In one aspect, the width of the gap is at least about 1 nanometer, or alternatively at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,6,7, 8, 9, 10, 12, or 15 nanometers. In another aspect, the width of the gap is no greater than about 20 nanometers, or alternatively no greater than about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nanometers. In some aspects, this width is between about 1 nanometer and about 15 nanometers, between about 1 nanometer and about 10 nanometers, between about 2 nanometers and about 10 nanometers, between about 2.5 nanometers and about 10 nanometers, or between about 2.5 nanometers and about 5 nanometers.

In some embodiments, the sensor is an electrical sensor. In some embodiments, the sensor detects the fluorescent detection means when the probe has a label that produces a unique fluorescent signature. A radiation source at the outlet may be used to detect this feature.

It should be understood that while the invention has been described in conjunction with the above embodiments, the foregoing description and the following examples are intended to illustrate, but not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Examples

Example 1 DNA alone in solid-State nanopore experiments

Nanopore instruments use a sensitive voltage-clamp amplifier to apply a voltage V across the pore while measuring the ionic current I through the open pore₀. Measured from I when a single charged molecule such as double stranded DNA (dsDNA) is captured and driven through the pore by electrophoresis₀To I_BCurrent offset and offset Δ I ═ I₀-I_BAnd duration t_DFor characterizing events. Analysis at Δ I vs. t after recording many events during the experiment_DThe events on the curve are distributed to characterize the corresponding molecules in the population on the curve. In this way, the nanopore provides a simple, label-free, pure-electron single-molecule approach for biomolecule detection.

The single nanopore fabricated in a silicon nitride (SiN) substrate is a 40nm diameter pore in a 100nm thick SiN film (fig. 11A), which is shown as an example of a solid state nanopore. In FIG. 11B, a representative current trace shows a blocking event caused by 5.6kb dsDNA passing through an 11nm diameter nanopore in 10nm thick SiN in a single row (unfolded) fashion at 200mV in a buffer containing 1M KCl. The current is reduced when the DNA passes through the pore at KCl concentrations at or above 0.3M, and the current is increased when the DNA passes through the pore at KCl concentrations below 0.3M. Average open channel current is I₀9.6nA, and average event amplitude I_B9.1nA, and average event duration t_D0.064 ms. The amplitude shift of the dsDNA molecule through nanopore translocation is Δ I ═ I₀-I_B0.5 nA. In fig. 11C, a scatter plot shows | Δ I | vs.t for all 1301 events recorded within 16 minutes_D。

Example 2 Capture of molecules comprising PNA and Biotin for target sequence detection

We have demonstrated an approach that allows for the detection of binding of compounds in solution by engineering polymer scaffolds. We provide PNA probes that have been modified to contain a biotin moiety that binds neutravidin. Neutravidin increases volume and thus makes PNAs detectable in large (e.g., 15-30nm diameter) nanopores. Specifically, we engineered a 5.6kb dsDNA scaffold to bind 12-mer peptide-nucleic acid (PNA) probe molecules, with each PNA probe having 3 biotinylation sites, each for binding to neutravidin (fig. 12A). We engineered the dsDNA scaffold to have 25 different sites (binding motifs) to which our PNA probes bind (fig. 12B). We provided solutions containing polymer scaffold only, free neutravidin or probe/DNA complexes (fig. 13). The resulting current event stamps from each population (fig. 13) showed that the DNA/PNA/neutravidin complexes resulted in a detectably shifted current stamp above other background event types (e.g., unbound DNA alone, neutravidin alone, PNA/neutravidin alone) and thus could be recognized in a nanopore device. In the remainder of this example, we demonstrate that the DNA/PNA/neutravidin complex can be detected with high confidence using a nanopore.

Probes that bind to specific DNA sequences are protein nucleic acid molecules (PNA) that bind to a unique sequence (GAAAGTGAAAGT, uSeq1) that is repeated 25 times throughout the scaffold. The PNAs used in the experiments have the sequence GAA AGT, wherein it means that biotin is incorporated into the PNA backbone at the gamma position by coupling with lysine amino acids, and thus, each PNA has three biotin molecules and potentially binds 3 neutravidin molecules (PNABio). To bind PNA, a 60nM scaffold was heated to 95 ℃ for 2 minutes, cooled to 60 ℃ and incubated with a 10X excess of PNA for possible PNA-binding sites on the scaffold in 15mM NaCl for 1hr and then cooled to 4 ℃. Excess PNA was dialyzed against 10mM Tris pH8.0 (20k MWCO, ThermoScientific) for 2 hr. This DNA/PNA complex was then labeled with a 10-fold excess of neutravidin (Pierce/Thermo Scientific) bound to the scaffold for the possible biotin sites (assuming 60% PNA reduction during dialysis). Reactions were electrophoresed as described above to assess purity, concentration, and potential aggregation.

FIGS. 14A-B show a comparison of Δ I vs. t from three independent experiments_DDistributed data: dna alone (D), neutravidin (N) alone, and D/P/N reagent (DPN). The largest | Δ I | event in D/P/N experiments was attributed to the D/P/N complex (fig. 13), providing a simple criterion for tagging events based on their binding state (i.e., unbound, scaffold with PNA and bound neutravidin). In particular, we can mark an event as corresponding to a D/P/N complex if for that event, | Δ I>4 nA. For the data set in FIG. 14A, 9.3% (390) of the events in the D/P/N experiment had | Δ I>4nA, and only 0.46% of D and 0.16% of N events in the control exceeded 4 nA. In a separate experiment (data not shown) using 7nM diameter pores at 1M KCl and 200mV applied, no events (0%) exceeded 4nA in the control with PNA alone and neutravidin at a concentration of 0.4 nM. Applying our mathematical criterion, the random variable Q ═ { fraction of tagged events } has a binomial distribution, and using this and other statistical modeling tools, we can calculate a 99% confidence interval for the dataset of 9.29 ± 1.15%. Because of 9.29 percent>0.46% (maximum false positive%) fits well with 99% confidence interval of Q, we have positive test results, and within 8 minutes of data collection. In fact the same 99% confidence interval was obtained for this dataset with only the first 60 seconds of data. Gel migration (fig. 14C) shows that scaffold DNA migration is retarded in a neutravidin-dependent manner; this guides us toA 10x concentration was used in this preliminary experiment because it appeared that all DNA was labeled and a nearly homogeneous population was produced.

Example 3 binding of Vspr protein to DNA scaffold and nanopore detection

The VspR protein Is a90 kDa protein from Vibrio cholerae (V.cholerae) which binds directly to dsDNA with high micromolar affinity in a sequence-specific manner ("VpsR, a number of the Response Regulators of the two-Component regulation Systems Is for Expression of biosyntheses and EPSETr-Associated phenols in Vibrio cholerae O1 El". J. J.A. 90kDa protein from Vibrio cholerae (V.cholerae.) binds directly to dsDNA with high micromolar affinity (see references: Yildiz, Fitnat H., Nadia A. Dolganov. and Gary K. Schoolnik.), "VpsR, a number of the components of the gene regulation Systems and EPSETr-Associated viruses in Vibrio cholerae O1 Tor." journal of bacteria 183, No. 2001 (1716-). 1726). In this target sequence detection embodiment using nanopore technology, VspR functions as a probe molecule with a site-specific DNA binding domain. In this experiment, we show that detection of VspR on a DNA scaffold serves as a model for detecting specific sequences present in DNA using proteins. The DNA scaffold contained 10 VspR-specific binding sites (fig. 15). To maintain the affinity of VspR for dsDNA binding, we used 0.1MKCl, which is the salt concentration where migration of VspR-bound scaffold through the nanopore enhances current flow through the nanopore (fig. 16). We provided a solution containing VspR protein at a concentration of 18nM in recording buffer (recording buffer) and 180nM during the labelling process (binding step). This results in an excess of VspR protein to the 18x binding site on DNA. The experiment was run at pH8.0 (pI of VspR protein is 5.8). Considering Kd and DNA concentration, only 0.1-1% of DNA should be completely occupied by VspR, a larger percentage is partially occupied, and some unknown remaining percentage of DNA is completely unbound. Free VspR protein was also present in the solution during the nanopore experiment.

Two representative events are shown in fig. 16A and 16B. In experiments using VspR, the VspR concentration was 18nM (1.6mg/L), 10nM binding site. The scaffold concentration was 1nM, resulting in capture every 6.6 seconds. The pore size was 15nm diameter and length. The voltage was-100 mV, and note that a negative voltage produced a negative current, so the upward shift corresponded to a decay event, as shown for VspR-bound DNA events (fig. 16B), while the downward shift produced a positive shift, as shown for unbound DNA scaffold events (fig. 16A). This is consistent with the ideal signal pattern and conditions in fig. 2, where the DNA event (fig. 16A) has a shorter duration and opposite polarity compared to the fusion molecule-bound DNA event (fig. 16B). Thus, a key observation derived from this figure is that events of VspR-binding have opposite signal polarity compared to unbound DNA events and are therefore readily detectable indicators of the presence of a particular DNA sequence. Fig. 17 shows another 10 representative current decay events consistent with VspR-bound scaffold passing through the pores. There were 90 such events within 10 minutes of recording, corresponding to 1 VspR-binding event every 6.6 seconds. Events were decays of 50-150pA in amplitude and 0.2-2 milliseconds in duration. As noted, the downward events in fig. 16-17 correspond to current boost events and the upward events correspond to current decay events, and this offset direction is maintained even when the baseline is zeroed for display purposes.

Example 4 sequence-specific Probe Synthesis and target sequence binding

In this example, we show the generation of PNA probes for binding to target sequences of interest, where features are added to the PNA probes to allow for higher detection sensitivity in the nanopore.

We generated a bisPNA probe containing 3 cysteine residues. The bisPNA probe comprises a sequence of PNA capable of binding to a DNA sequence comprising the target sequence of CTTTCCC at the target sequence position of the target DNA molecule. The bisPNA probe was also labeled with maleimido-PEG-Me at 3 cysteine residues on the bisPNA probe to enhance detection of the probe linked to the target DNA molecule in the nanopore. PNA-PEG probes were generated by incubating a 100-fold excess of linker (methyl-PEG (10kDa) -maleimide) with bisPNA (Lys-Lys-Cys-PEG3-JTTTJJJ-PEG-Cys-PEG-CCCTTTC-PEG-Cys-Lys-Lys) under reducing conditions. The maleimide moiety of the linker reacts with the free thiol group in the PNA at pH 7.4, thus producing a pegylated PNA. The addition of lysine increased the affinity of the reagent for its specific homologous DNA sequence, allowing it to remain bound under high salt conditions (1M LiCl). The resulting PNA-PEG probe bound to its target sequence on dsDNA molecule is shown in FIG. 18A.

To confirm the binding of the DNA-PEG probe to its target sequence on the DNA molecule, we incubated different forms of PEG probe with DNA and used the resulting solution for gel migration analysis. For this analysis, we run 4 samples as shown in fig. 18B. Lane 1 is DNA only, lane 2 is DNA + PNA, lane 3 is DNA + PNA-PEG (10kDa) and lane 4 is DNA + PNA-PEG (20 kDa). The upward shift in lanes 2-4 is consistent with the bisPNA species binding to DNA. The circled species is DNA/PNA-PEG and the framed species in lanes 3 and 4, which are present as residual PNA (no PEG) in the labeling experiment, are DNA/PNA. The results of the gel migration analysis showed that complexes of DNA and PNA probes containing the target sequence with homologous DNA sequences complementary to the target sequence were formed independently of the attachment of PEG and PNA. Thus, we demonstrate here successful complex formation of sequence-specific probes that can be detected in the nanopore.

We then performed an analysis to demonstrate the specificity of the PNA probe for its target DNA sequence. Here, we incubated the PNA probe (without PEG) with samples containing DNA without target sequence (lane 2), DNA with target sequence containing a single base mismatch with the PNA probe (lane 3) and DNA with the complete target sequence (lane 4) and analyzed each sample using gel migration analysis, the results of which are shown in fig. 18C. Lane 1 is a DNA marker. As shown by our results, DNA with exact target sequence match (lane 4) binds PNA, while DNA with target sequence comprising single base mismatch sequence (lane 3) and DNA without target sequence (random sequence replacing target sequence) (lane 2) shows no PNA binding. Thus, gel migration analysis shows that PNAs specifically bind to DNA comprising their target sequence, but not to DNA with even a single mismatch in the target sequence.

Example 5 target sequence detection in nanopores Using modified sequence-specific probes

In this example, we show the detection of DNA molecules comprising target sequences bound to our PEG-modified sequence-specific PNA probes.

Here, we provide three different PNA probes to have different volumes based on PEG attachment and PEG length. Three types of probes were used: 1) PNA without PEG, 2) PNA conjugated with 5kDa PEG and 3) PNA conjugated with 10kDa PEG. Each probe was mixed with DNA containing the target sequence and run in a nanopore to observe the detection of DNA bound to the PNA probe. The concentration of each complex in the sample was 2nM in 1M LiCl buffer. The sample was run in a nanopore device at an applied voltage of 100 mV. The results are shown in FIGS. 19A-19E.

Representative single events observed are shown in fig. 19A for DNA bound to each type of probe. The event signature from the DNA/bisPNA event is shown on the left. The event profile from the DNA/bisPNA-PEG complex with a maximum of 3 PEGs bound to each PNA and a PEG size of 5kDa is shown in the middle. The event signature from the DNA/bisPNA-PEG complex with a maximum of 3 PEGs bound to each PNA and a PEG size of 10kDa is shown on the right. The respective event signature is measured by current blocking through the nanopore during translocation of the identified complex. In fig. 19A, a molecular diagram shows scaled linear PEG and DNA for visual comparison. As the probe size (volume) increases, the event signature changes.

We analyzed the event population and generated scatter plots of the mean conductance shift (dG) vs. duration for all events in each dataset. We generated a scatter plot of event average conductance (average current bias removed by voltage) versus event duration (width) from our experiments as shown in fig. 19B. The figure shows that DNA/PNA, DNA/PNA-PEG (5kDa) and DNA/PNA-PEG (10kDa) give overlapping populations that differ based on their event duration and mean conductance. We generated histograms to show the difference in the average conductance shift (dG) observed for each event between the different complexes (fig. 19C). We also generated histograms to show the differences in duration of events observed between different complexes (fig. 19D).

Example 6-detection of mutated cftr Gene target sequences in nanopores to detect human cystic fibrosis

We have demonstrated the specificity of binding of our modified PNA probes to target sequences and the ability to detect target sequences using probes in a nanopore device. Here, we observe the use of modified PNA probes in a nanopore device to detect disease-causing mutations (in particular cystic fibrosis) in a sample from a patient.

We generated (following the method described in example 4) a modified PNA probe (PNA-PEG probe) comprising a PNA molecule which specifically binds to a target DNA sequence comprising the cftr gene with a mutation therein (af 508) causing cystic fibrosis. The PEG bound to the PNA probe was 5 kDa. DNA containing cystic fibrosis disease mutations was incubated with pegylated PNA specific for the mutation. The sample was then placed in a nanopore device with a 26nm pore and the translocation event through the nanopore was recorded and analyzed.

The translocation event characteristic associated with translocation of PNA-PEG probes bound to DNA molecules was observed in samples with DNA containing the mutation causing cystic fibrosis (. DELTA.F508). Representative event signatures are shown in fig. 20A. Experiments using samples with only DNA or only DNA/PNA (i.e. no PEG-PNA) did not give a clear translocation event above background, demonstrating the ability of the pores to accurately recognize PNA-PEG probes bound to DNA, and the enhancement of detection was obtained by the modified probes provided herein. For the set of recorded events from samples with the target mutant gene and PNA-PEG probe, events were characterized by mean conductance shift and duration and analyzed. Fig. 20B shows the average conductance shift v. duration plot for each recorded event. Fig. 20C and 20D show corresponding histograms to characterize events detected by mean conductance shift and duration of each event, respectively. The data analyzed matched the expected data for translocation of the DNA/PNA-PEG (5kDa) complex through the nanopore, indicating successful binding and recognition of cftr mutant target sequences in the nanopore device.

We also run gel migration analysis (fig. 20E) on samples containing our PNA-PEG (5kDa) probe specific for Δ F508cftr gene mutations with samples containing 300bp DNA with the wild type cftr sequence (lane 2) and with samples containing 300bp DNA with Δ F508cftr gene mutations (lane 3). This data shows that our PNA-PEG probes specifically bind only to the Δ F508 target sequence, but not to the wild-type sequence.

Thus, we have successfully detected DNA comprising a single base cftr gene mutation (Δ F508) and demonstrated here the use of our system to detect specific sequences of polynucleic acids in a sample, including for the diagnosis or treatment of indications in human patients.

Example 7-infectious bacteria detection in nanopores Using PNA-PEG Probe

In this example, we observe the use of our modified probe to detect the presence of bacterial DNA in a sample using a nanopore device.

We synthesized probes with PNA molecules capable of specifically binding to the DNA of streptococcus mitis (s. The bisPNA comprises a sequence complementary to a sequence specific for a Streptococcus mitis bacterial species.

In this assay, PNA probes are conjugated to 10kDa PEG to allow detection in the nanopore upon binding to bacterial DNA. We mixed PNA probes with bacterial DNA and performed gel migration analysis on the samples to observe binding. FIG. 21A shows the results of gel migration analysis, lane 1 containing bacterial DNA without PNA probes, and lane 2 containing bacterial DNA with PNA probes. Our observations show that our PNA/PEG (10kDa) probe binds to light streptococcal bacterial DNA.

We next prepared two samples for detection in the nanopore. The first sample included bacterial DNA with a PEG-modified PNA probe (DNA/bisPNA-PEG). The second sample included only bacterial DNA. We run these samples through the nanopore device in two consecutive experiments and analyze the resulting events. Figure 21B shows a scatter plot of mean conductance shift (dG) vs. duration on the horizontal axis for all recorded events in two consecutive experiments. Events characterized by labeled sample 1 (squares) and unlabeled sample 2 (circles) are shown.

The tagged molecules are always above the background threshold (broken line), while the untagged molecules are below the line and are consistent with the background population. Populations of molecules from multiple background experiments (DNA/PNA without PEG, filtered serum, etc.) were used to establish thresholds (lines) for labeling tagged events. The background events are not shown here. To accurately detect bacterial DNA in a sample, the DNA must be tagged with a highly site-specific probe.

Our results show that the PNA/PEG-bound population of light streptococcus bacterial DNA can be resolved from background events, whereas DNA alone and DNA/PNA alone cannot. Thus, our modified PNA-PEG sequence-specific probes allow to reliably detect the presence or absence of streptococcus mitis DNA in a sample.

Other embodiments

It is understood that the words which have been used are words of description rather than limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

Although the present invention has been described in considerable detail and with particular reference to several described embodiments, it is not intended to limit the invention to any such specific example or embodiment or any particular embodiment, but rather should be construed with reference to the appended claims so as to provide the broadest possible interpretation of such claims based on the prior art and, therefore, to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Claims (39)

1. A method of detecting a polynucleotide comprising a target sequence in a sample, the method comprising:

contacting the sample with a probe that specifically binds to the polynucleotide comprising the target sequence under conditions that promote binding of the probe to the target sequence to form a polynucleotide-probe complex;

loading the sample into a first chamber of a nanopore device, wherein the nanopore device comprises at least one nanopore and at least the first and second chambers, wherein the first and second chambers are in electrical and fluid communication through the at least one nanopore, and wherein the nanopore device further comprises an independently controlled voltage across each of the at least one nanopore and a sensor associated with each of the at least one nanopore, wherein the sensor is configured to identify objects passing through the at least one nanopore, and wherein the polynucleotide-probe complex translocated through the at least one nanopore provides a detectable signal associated with the polynucleotide-probe complex; and

determining the presence or absence of said polynucleotide-probe complex in said sample by observing said detectable signal, thereby detecting said polynucleotide comprising said target sequence.

2. The method of claim 1, wherein the polynucleotide is DNA or RNA.

3. The method of claim 1, wherein the detectable signal is an electrical signal.

4. The method of claim 1, wherein the detectable signal is an optical signal.

5. The method of claim 1, wherein the probe comprises a molecule selected from the group consisting of: a protein, peptide, nucleic acid, TALEN, CRISPR, peptide nucleic acid, or chemical compound.

6. The method of claim 1, wherein the probe comprises a molecule selected from the group consisting of: deoxyribonucleic acid (DNA), ribonucleic acid (RNA), Peptide Nucleic Acid (PNA), DNA/RNA hybrids, polypeptides or any chemically derivatized polymer.

7. The method of claim 1, wherein the probe comprises a PNA molecule bound to a second molecule configured to facilitate detection of the probe bound to the polynucleotide during translocation through the at least one nanopore.

8. The method of claim 7, wherein the second molecule is PEG.

9. The method of claim 8, wherein the PEG has a molecular weight of at least 1kDa, 2kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, or 10 kDa.

10. The method of claim 1, further comprising applying to the sample a condition suspected of altering a binding interaction between the probe and the target sequence.

11. The method of claim 10, wherein the conditions are selected from the group consisting of: removing the probe from the sample, adding an agent that competes with the probe for binding to the target sequence, and changing initial pH, salt, or temperature conditions.

12. The method of claim 1, wherein the polynucleotide comprises a chemical modification configured to alter binding of the polynucleotide to the probe.

13. The method of claim 12, wherein the chemical modification is selected from biotinylation, acetylation, methylation, small ubiquitin-like modification of protein albinism, glycosylation, phosphorylation and oxidation.

14. The method of claim 1, wherein the probe comprises a chemical modification coupled to the probe via a cleavable bond.

15. The method of claim 1, wherein the probe interacts with the target sequence of the polynucleotide by covalent bonds, hydrogen bonds, ionic bonds, metallic bonds, van der waals forces, hydrophobic interactions, or planar stacking interactions.

16. The method of claim 1, further comprising contacting the sample with one or more detectable labels capable of binding to the probe or the polynucleotide-probe complex.

17. The method of claim 1, wherein the polynucleotide comprises at least two target sequences.

18. The method of claim 1, wherein the nanopores have a diameter of about 1nm to about 100nm and a length of 1nm to about 100nm, and wherein each of the chambers comprises an electrode.

19. The method of claim 1, wherein the nanopore device comprises at least two nanopores configured to simultaneously control movement of the polynucleotide in both nanopores.

20. The method of claim 1, further comprising reversing the independently controlled voltages after initial detection of the polynucleotide-probe complex by the detectable signal, such that movement of the polynucleotide through the nanopore is reversed after the probe-bound portion passes through the nanopore, thereby re-determining the presence or absence of the polynucleotide-probe complex.

21. The method of claim 1, wherein the nanopore device comprises two nanopores, and wherein the polynucleotide is located within both nanopores simultaneously.

22. The method of claim 21, further comprising adjusting the voltage magnitude and/or direction of each of the two nanopores such that opposing forces are generated through the nanopores to control the rate of translocation of the polynucleotide through the nanopores.

23. A method of detecting a polynucleotide or polynucleotide sequence in a sample comprising:

a) contacting the sample with a first probe and a second probe, wherein the first probe specifically binds to a first target sequence of the polynucleotide under conditions that promote binding of the first probe to the first target sequence, wherein the second probe specifically binds to a second target sequence of the polynucleotide under conditions that promote binding of the second probe to the second target sequence;

b) contacting the sample with a third molecule configured to simultaneously bind to the first and second probes when the first and second probes are sufficiently close to each other under conditions promoting binding of the third molecule to the first probe and the second probe, thereby forming a fusion complex comprising the polynucleotide, the first probe, the second probe, and the third molecule;

c) loading the sample into a first chamber of a nanopore device, wherein the nanopore device comprises at least one nanopore and at least the first and second chambers, wherein the first and second chambers are in electrical and fluid communication through the at least one nanopore, and wherein the nanopore device further comprises a controlled voltage potential across each of the at least one nanopore and a sensor associated with each of the at least one nanopore, wherein the sensor is configured to identify objects passing through the at least one nanopore, and wherein the fusion complex displaced by the at least one nanopore provides a detectable signal associated with the fusion complex; and

d) determining the presence or absence of said fusion complex in said sample by observing said detectable signal.

24. The method of claim 23, wherein the polynucleotide is DNA or RNA.

25. The method of claim 23, wherein the detectable signal is an electrical signal.

26. The method of claim 23, wherein the detectable signal is an optical signal.

27. The method of claim 23, wherein the sufficient proximity is less than 3, 4, 5,6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides.

28. The method of claim 23, wherein the third molecule comprises PEG or an antibody.

29. The method of claim 23, wherein said third molecule and said first and second probes bind to ssDNA, and wherein said ssDNA to which said third molecule is attached comprises a region complementary to a region of ssDNA attached to said first probe and is complementary to a region of ssDNA attached to said second probe.

30. The method of claim 23, further comprising contacting the sample with one or more detectable labels capable of binding to the third molecule or to the fusion complex.

31. A kit comprising a first probe, a second probe, and a third molecule, wherein the first probe is configured to bind to a first target sequence on a target polynucleotide, wherein the second probe is configured to bind to a second target sequence on the target polynucleotide, and wherein the third molecule is configured to bind to the first probe and the second probe when the first and second probes bind to the polynucleotides at the first and second target sequences, thereby positioning the first and second probes in sufficient proximity to allow the third molecule to bind to the first and second probes simultaneously.

32. The kit of claim 31, wherein the first probe and the second probe are selected from the group consisting of: a protein, peptide, nucleic acid, TALEN, CRISPR, peptide nucleic acid, or chemical compound.

33. The kit of claim 31, wherein the third molecule comprises PEG or an antibody.

34. The kit of claim 31, wherein the third molecule comprises a modification that alters the binding affinity to the probe.

35. A nanopore device comprising at least two chambers and a nanopore, wherein the device comprises a modified PNA probe bound to a polynucleotide within the nanopore.

36. A dual-pore, dual-amplifier device for detecting a charged polymer through two pores, the device comprising an upper chamber, a middle chamber and a lower chamber, a first pore connecting the upper chamber and the middle chamber and a second pore connecting the middle chamber and the lower chamber, wherein the device comprises a modified PNA probe bound to a polynucleotide within the first or second pore.

37. The device of claim 36, wherein the device is configured to control movement of the charged polymer through both the first aperture and the second aperture simultaneously.

38. The device of claim 36, wherein the modified PNA probe is conjugated to at least one PEG molecule.

39. The apparatus of claim 36, wherein the apparatus further comprises a power supply configured to provide a first voltage between the upper chamber and the middle chamber and a second voltage between the middle chamber and the lower chamber, each voltage being independently adjustable, wherein the middle chamber is connected to a common ground with respect to the two voltages, wherein the apparatus provides dual amplifier electronics configured for independent voltage control and current measurement for each aperture, wherein the two voltages may be different in magnitude, wherein the first and second apertures are configured such that the charged polymer can move across both apertures simultaneously in either direction and in a controlled manner.