Detection of changing conductance indicating sequential interactions between nucleic acid bases

Abstract

Methods are provided that include causing a relative movement of a first nucleic acid through an opening formed at least in part by an electrolyzed second nucleic acid. Such methods include detecting a change in conductance along the first nucleic acid or a change in conductance between the first nucleic acid and an electrode proximate to the first nucleic acid during the relative movement, the change in conductance indicating a continuous interaction between the nucleobase of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid. The change in conductance comprises a conductance fingerprint for different nucleobases in the first nucleic acid. In certain embodiments, the method includes determining the identity of one or more nucleotides based on the change in conductance, and optionally determining the sequence of the first nucleic acid. Also provided are computer readable media and systems that find use in implementing the methods of the present disclosure, for example.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 263,803, filed November 9, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Introduction Nucleic acid sequencing is the process of determining the order of nucleotides in a nucleic acid sequence (e.g., DNA, RNA), and has become a fundamental process in fields such as medical diagnostics, forensic biology, and biological research. Available techniques for sequencing include, for example, the Sanger method. This procedure involves the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. In addition, next-generation sequencing platforms (sometimes referred to as "second-generation" sequencing) use different techniques for sequencing, such as pyrosequencing, sequencing-by-synthesis, sequencing-by-ligation, or nanopore-based sequencing. However, new sequencing methodologies are desirable to address issues related to cost, sequencing quality, sequencing time, efficiency, and ease of use.

[0003] Recently, sequencing methods have been proposed that exploit the electrochemical properties of nucleic acids, such as DNA. In particular, an approach has been contemplated in which DNA is electrically driven through a nanopore. As the DNA translocates through the nanopore, it may be detected by measuring the ionic blockade or transverse current. However, nanopore sequencing methods that use the electrochemical properties of nucleic acids have not yet been widely adopted due to issues related to feasibility, accuracy, and cost. Therefore, alternative electrochemical sequencing approaches are desirable. Summary of the Invention

[0004] Methods are provided that include causing a relative movement of a first nucleic acid through an opening formed at least in part by an electrolyzed second nucleic acid. Such methods include detecting a change in conductance along the first nucleic acid or a change in conductance between the first nucleic acid and an electrode proximate to the first nucleic acid during the relative movement, the change in conductance indicating a continuous interaction between the nucleobase of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid. In certain embodiments, the change in conductance includes a conductance fingerprint for different nucleobases in the first nucleic acid. According to some embodiments, the method includes determining the identity of one or more nucleotides of the first nucleic acid based on the change in conductance. In certain embodiments, the method includes determining the nucleotide sequence of the first nucleic acid based on the change in conductance. Also provided are computer readable media and systems that find use in practicing the methods of the present disclosure, for example.

[0005] The disclosed method, system, and computer readable medium are based in part on the inventors' recognition that different nucleotides with different nucleobases, such as adenine (A), thymine (T), guanine (G), cytosine (C), uracil (U), or non-natural variants thereof, and their various sequences, each with different chemical compositions and structures, can be associated with specific signatures of transverse tunneling (perpendicular to the DNA or RNA axis). In support of this, DNA base pairs have been shown to behave as biological Aviram-Ratner electrical rectifiers (see, for example, Agapito et al. Nanotechnology, 23(13), 135202) due to the spatial separation and weak binding between the nucleobases, and because the current flowing across these base pairs varies based on the specific properties of the particular nucleotide. In other words, DNA base pairs can function as unidirectional conductors of electric current, and the interaction between given nucleobases is characterized by a specific "conductance fingerprint". Such is shown in Figures 1A-1D (adopted from Agapito et al., supra). Top and side views of the CG (Figure 1A) and TA (Figure 1C) junctions are shown, along with the densities of states projected onto the purine and pyrimidine components of the CG (Figure 1B) and TA (Figure 1D) junctions.

[0006] The inventors conclude that DNA sequencing may be performed by assessing conductance for the presence of a conductance fingerprint when a nucleic acid to be sequenced migrates relative to a second nucleic acid. [Brief description of the drawings]

[0007] [Figure 1] It shows the "conductance fingerprint" associated with a particular interaction between given nucleobases. [Diagram 2] 1 presents a schematic diagram of a method according to an embodiment of the present disclosure, comprising relative movement of a first nucleic acid through a plurality of openings formed by a plurality of electrolyzed nucleic acids. [Diagram 3]1 presents a schematic diagram of a method according to an embodiment of the present disclosure, comprising relative movement of a first nucleic acid through a plurality of openings formed by a plurality of electrolyzed nucleic acids. [Figure 4] 1 presents a schematic diagram of a method according to an embodiment of the present disclosure, comprising relative movement of a first nucleic acid through a plurality of openings formed by a plurality of electrolyzed nucleic acids. [Diagram 5] Schematic diagrams of various electrolyzed nucleic acid configurations that find use in practicing the methods of the present disclosure are presented. [Figure 6] A schematic diagram of an embodiment is presented in which a changing conductance between a first nucleic acid and an electrode proximate to the first nucleic acid is detected, the changing conductance indicating a continuous interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed nucleic acid in the form of a bridge structure. [Figure 7] A schematic diagram of an embodiment is presented in which a changing conductance between a first nucleic acid and an electrode proximate to the first nucleic acid is detected, the changing conductance indicating a sequential interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed nucleic acid. [Figure 8] 1 shows an embodiment of the invention in which changing conductance between a first nucleic acid and an electrode proximate to the first nucleic acid may be detected. "A" = ammeter. [Figure 9] 1 illustrates a chip architecture for use in the subject method according to certain embodiments. [Figure 10] 1 presents a micrograph showing an insulating layer between the right and left electrodes and the gate electrode according to a specific embodiment. [Figure 11] 1 presents a micrograph taken via helium ion microscope of a tip for use in the subject method. [Figure 12] A prototype of the chip constructed as shown in Figures 9A-9B is shown. [Figure 13] We present micrographs of the tip-electrodes obtained via atomic force microscopy (AFM). [Figure 14]9A-9B show the topography and conductivity aspects of a chip prototype constructed as shown in FIG. [Figure 15] We present an AFM image showing single-stranded DNA immobilized on a gold substrate via a thio bond. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Before describing the methods, systems, and computer-readable media of the present disclosure in more detail, it should be understood that the methods, systems, and computer-readable media are not limited to particular embodiments described, as such may, of course, vary, and the scope of the methods, systems, and computer-readable media will be limited only by the appended claims, and therefore it should be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0009] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is to be understood that each intermediate value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, and any other stated value or intermediate value within that stated range, is encompassed within the present methods, systems, and computer-readable media. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the present methods, systems, and computer-readable media, subject to any specific excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the present methods, systems, and computer-readable media.

[0010] In this specification, certain ranges are presented with the term "about" preceding numerical values. In this specification, the term "about" is used to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present method, system, and computer readable medium belongs. Although any methods, systems, and computer readable media similar or equivalent to those described herein may also be used in the practice or testing of the present method, system, and computer readable medium, representative exemplary methods, systems, and computer readable media are described herein.

[0012] All publications and patents cited herein are incorporated by reference herein to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference herein to disclose and describe in connection with the materials and / or methods for which the publications are cited. The citation of any publication is for its disclosure prior to the filing date, and should not be construed as an admission that the methods, systems, and computer-readable media are not entitled to antedate such publications, which dates may be different from the actual publication dates, which may need to be independently confirmed.

[0013] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely" and "only" in connection with the recitation of claim elements, or for the use of a "negative" limitation.

[0014] For clarity, it is understood that certain features of the method, system, and computer-readable medium described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the method, system, and computer-readable medium described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments are specifically encompassed by the present disclosure, and to the extent such combinations include operable processes and / or compositions, each and every combination is disclosed herein as if it were individually and explicitly disclosed. In addition, all subcombinations listed in the embodiments describing such variables are also specifically encompassed by the method, system, and computer-readable medium, and each and every such subcombination is disclosed herein as if it were individually and explicitly disclosed herein.

[0015] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual elements and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the method. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0016] definition The term "nucleotide" is intended to include moieties that contain not only naturally occurring purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term "nucleotide" includes moieties that contain haptens or fluorescent labels and may contain other sugars as well as traditional ribose and deoxyribose sugars. Modified nucleosides or nucleotides also include modifications in the sugar moiety, for example, where one or more of the hydroxyl groups have been replaced with halogen atoms or aliphatic groups, or have been functionalized as ethers, amines, and the like.

[0017] As used herein, an "oligonucleotide" is a single-stranded multimer of nucleotides, from 2 to 500 nucleotides, e.g., from 2 to 200 nucleotides. Oligonucleotides may be synthetic or enzymatically produced, and in some embodiments are 5 to 50 nucleotides in length (e.g., 9 to 50 nucleotides in length). Oligonucleotides may contain ribonucleotide monomers (i.e., oligoribonucleotides or "RNA oligonucleotides") or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides or "DNA oligonucleotides"). Oligonucleotides may be, for example, 5 to 9, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 200, up to 500 or more nucleotides in length.

[0018] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 bases, greater than 100,000 bases, greater than about 1,000,000 bases, up to about 10 10It describes a polymer of more than one base, which may be produced enzymatically or synthetically (e.g., PNA, as described in U.S. Pat. No. 5,948,902 and references cited therein), that can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to that of two naturally occurring nucleic acids, for example, participating in Watson-Crick base pairing interactions. Naturally occurring nucleotides include guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively). Whereas DNA and RNA have deoxyribose and ribose sugar backbones, respectively, the backbone of PNA is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA, the various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. Locked nucleic acids (LNA), often referred to as inaccessible RNA, are modified RNA nucleotides. The ribose moiety of LNA nucleotides is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. The bridge "locks" the ribose in the 3'-endo(North) conformation that is often found in A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides whenever desired. The term "unstructured nucleic acid" or "UNA" refers to a nucleic acid that contains non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain G' and C' residues, which correspond to non-naturally occurring forms, i.e., analogs of G and C that base pair with each other with reduced stability but retain the ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acids are described in US2005 / 0233340, which is incorporated herein by reference for its disclosure of UNAs.

[0019] method As summarized above, aspects of the present disclosure include methods that include detecting a changing conductance along a first nucleic acid or between the first nucleic acid and an electrode proximate to the first nucleic acid, which may be measured and utilized to sequence the first nucleic acid. As will be understood upon review of the present disclosure, the present methods constitute improvements over existing sequencing techniques, for example, because the present methods may be performed much more quickly, with increased accuracy and portability, and at reduced cost (e.g., essentially no consumables) compared to existing approaches for nucleic acid sequencing.

[0020] According to some embodiments, the method includes causing relative movement of a first nucleic acid (e.g., a nucleic acid to be sequenced) through an opening at least partially formed by the electrolyzed second nucleic acid. "Causing relative movement" means that at least one of the first nucleic acid and the electrolyzed second nucleic acid changes position relative to the other. In some versions of the subject method, the first nucleic acid moves while the second nucleic acid remains stationary. In other embodiments, the second nucleic acid moves while the first nucleic acid remains stationary. In still other embodiments, both the first and second nucleic acids move. When the method includes causing movement of a first nucleic acid relative to a second nucleic acid, the movement may be accomplished using any convenient approach. For example, the method may include pulling the first nucleic acid through an opening at least partially formed by the second nucleic acid. "Pulling" means exerting a force on the nucleic acid sufficient to cause the nucleic acid to change position. The required force may be present in any convenient form. For example, in some cases, the first nucleic acid is attached to an elongated structure that can be used to pull (i.e., physically pull) the nucleic acid. The force may be driven using any convenient approach, including, but not limited to, chemical propulsion, magnetic propulsion, ultrasonically driven propulsion, light driven propulsion, electrically driven propulsion, and combinations thereof. Nanoscale methods of propulsion are described, for example, in Wang et al. Chemical reviews, 115(16), 8704-8735, which is incorporated herein by reference in its entirety. The rate of relative movement may vary. In some examples, the rate of relative movement is in the range of 1 to 1000 nucleotides / second.

[0021] In certain versions, the elongated structures are nanowires. The nanowires may have any convenient diameter, such as a range of diameters from 0.5 nm to 500 nm, including, for example, 1 nm to 200 nm, and 5 nm to 100 nm. The nanowires may comprise any convenient material. Exemplary nanowire materials include, but are not limited to, carbon, germanium, silicon, gold, copper, yttrium barium copper oxide (YBCO), indium phosphide, gallium nitride, nickel, platinum, combinations thereof, and the like.

[0022] In additional embodiments, the elongated structure is a nanotube. Nanotubes are generally tubular structures that include carbon (e.g., fullerenes, graphene). Various techniques have been developed to create carbon nanotubes. By way of example, methods for forming carbon nanotubes are described in U.S. Patent Nos. 5,753,088 and 5,482,601, the disclosures of which are incorporated herein by reference. Non-limiting techniques for the creation of nanotubes include laser vaporization, electric arc, and gas phase techniques.

[0023] In still further embodiments, the extension structure is a biopolymer. In some instances, the biopolymer is a protein. In other instances, the biopolymer is a nucleic acid. In other words, the biopolymer extension structure may be used in the same manner as the nanotubes or nanowires described above to tension a first nucleic acid. The biopolymer extension structure may have any convenient amino acid or nucleotide structure, as desired.

[0024] When the method involves pulling a first nucleic acid through an extension structure, the extension structure may be attached to the first nucleic acid via any convenient approach. In some cases, the first nucleic acid may be attached (e.g., covalently attached) to an end of the extension structure. In alternative cases, an adaptor may be associated with one end (e.g., the 5' or 3' end) of the first nucleic acid. Any convenient adaptor may be used. In alternative versions, the extension structure may have a nucleic acid sequence complementary to the adaptor associated with the first nucleic acid such that they may hybridize when the two molecules are placed in close proximity to each other, thereby attaching the extension structure to the first nucleic acid. In examples where the extension structure is a nucleic acid, alternative nucleobases at the ends (e.g., the 5' or 3' ends) of the extension structure may be complementary to the adaptor associated with the first nucleic acid such that they may hybridize when the two molecules are placed in close proximity to each other.

[0025] In other cases, the force that causes the movement of the first nucleic acid relative to the second nucleic acid is an electromagnetic force. In such cases, the method includes applying a voltage across an opening formed at least in part by the second nucleic acid such that the first nucleic acid moves relative to the second nucleic acid. In some cases, the rate at which the nucleic acid is pulled can be adjusted by adjusting the applied voltage. Voltages for use in the subject methods may vary and in some instances may range from 25 mV to 500 mV, including 50 mV to 400 mV, including 75 mV to 300 mV, and 100 mV to 200 mV. In additional cases, the force that causes the movement of the first nucleic acid relative to the second nucleic acid is a magnetic force. In some such cases, the first nucleic acid includes magnetic particles (e.g., magnetic beads) attached thereto. The type of magnetic particles used may vary and may include, for example, iron nanoparticles, nickel nanoparticles, cobalt nanoparticles, and the like. In embodiments, applying a magnetic field to the magnetic particles attached to the first nucleic acid is sufficient to provide a pulling force to the first nucleic acid.

[0026] The second nucleic acid can be any convenient electrolyzed nucleic acid configured in such a manner as to at least partially form an opening. In embodiments, the second nucleic acid includes at least a nucleobase that base pairs with adenine, a nucleobase that base pairs with thymine or uracil, a nucleobase that base pairs with guanine, and a nucleobase that base pairs with cytosine. In certain cases, the second nucleic acid includes one or more abasic nucleotides. The second nucleic acid may be a single-stranded nucleic acid in some cases. As discussed herein, an "electrolyzed" nucleic acid refers to a nucleic acid having an electric current passing through it. Without being bound by theory, it is believed that the DNA backbone can support multiple charge transfer mechanisms resulting from small activation gaps induced by water and counterions. In some embodiments, the second nucleic acid is modified to modulate the electrical conductivity. For example, the electrical conductivity of the nucleic acid can be modified with dopants such as conductive metal nanoparticles. Metal nanoparticles of interest include, but are not limited to, gold, lead sulfide, lead selenide, germanium, and silver. In some embodiments, the conductivity of the nucleic acid can be modified with dopants such as conductive carbons, such as carbon nanotubes, carbon nanorods, carbon black, graphene sheets, graphene nanoribbons, and carbon nanofibers. In additional cases, the conductivity of the second nucleic acid is modified by molecular doping with intercalators, such as anthraquinone, ferrocene, norbornadiene, methylene blue, ethidium, coralyne, and cryptolepin. Nucleic acid intercalation involves the insertion of one intercalation moiety (monointercalator), two intercalation moieties (bisintercalator), or multiple intercalation moieties into the nucleic acid structure. Intercalation changes the conductivity / resistance of the complex, which affects the overall conductive characteristics of the nucleic acid chain. In one embodiment, the dopant can be reduced or oxidized by an applied current.

[0027] In embodiments, the second nucleic acid is attached to a surface. The second nucleic acid may be stably associated with the surface in any suitable manner. By "stably associated" it is meant that the second nucleic acid does not readily dissociate from the surface. In certain cases, the electrolyzed second nucleic acid includes first and second discontinuous regions attached to the surface. By "discontinuous" regions it is meant that the first and second regions are not the same region (i.e., they do not overlap). The discontinuous regions of the second nucleic acid may be attached to the same or different surfaces. Each surface may comprise any convenient material. In certain cases, the surface is a metal surface (e.g., a gold surface). In some embodiments, the second nucleic acid is stably associated with the surface via thiol binding chemistry. For example, in embodiments where the surface is a gold surface, a thiol may react directly with the gold surface to form an Au-S bond via a redox reaction. In additional embodiments, stably associating the second nucleic acid with the surface comprises dip pen nanolithography. In such embodiments, an atomic force microscope may be used to imprint a thiolate onto the surface. Dip pen nanolithography is described, for example, in U.S. Patent Nos. 8,261,662 and 9,403,180, the disclosures of which are incorporated herein by reference in their entirety.In other embodiments, the second nucleic acid is stably associated with a surface via biotin-streptavidin interaction.In alternative cases, the second nucleic acid is in contact with multiple surfaces.In other words, one end of the second nucleic acid may be in contact with a first surface, and the other end of the second nucleic acid may be in contact with a second surface that is different from the first surface.

[0028] In an optional case, the method includes spatially targeting the attachment of the second nucleic acid by coating the surface with a biocompatible layer. The biocompatible layer may be various and may include, for example, a polymer matrix, a gel, or a self-assembled monolayer (SAM). In certain embodiments, an alkanethiol SAM is generated on one or more of the surfaces. In such a case, the surface (e.g., a gold surface) is incubated with a thiol-functionalized blocking molecule. The thiol-functionalized blocking molecule includes, but is not limited to, 1-mercapto-11-undecanol, 1-mercapto-6-hexanol, hexadecanethiol, combinations thereof, and the like. SAM formation is described, for example, in Szymonik et al. Nanotechnology, 27(39), 395301, which is incorporated herein by reference in its entirety. The subject method may additionally include applying a negative voltage to the surface in solution, thereby breaking the thiol bond and releasing the blocking molecule. Suitable methods for such electrochemical desorption can be found, for example, in Widrig et al. Journal of electroanalytical chemistry and interfacial electrochemistry, 310(1-2), 335-359, which is incorporated herein by reference in its entirety. After release of the blocking molecule, the method according to certain embodiments includes incubating a mixture of the 5'- or 3'-thiol-functionalized nucleic acid of interest (e.g., oligonucleotide) with the thiol-containing blocking molecule to form a new electrode-bound monolayer interspersed with the nucleic acid of interest. Methods that may be employed for use in surface functionalization are described, for example, in Walti et al. Langmuir, 19(4), 981-984, which is incorporated herein by reference in its entirety.

[0029] In some cases, the surface to which the second nucleic acid is bound is an electrode. In other words, to pass a current through the electrolyzed second nucleic acid, the 5' and / or 3' ends of the nucleic acid may be in contact with at least one electrode in some cases. In alternative versions, one end of the second nucleic acid is stably associated with an electrode while the other end is stably associated with a non-electrode surface. In other cases, both ends of the second nucleic acid are stably associated with different electrodes. The electrode may comprise any convenient material. In some embodiments, the electrode is a metal electrode. Materials for use in metal electrodes include, but are not limited to, platinum, gold, titanium nitride, silver, and graphite. In certain embodiments, the electrode is a gold electrode.

[0030] In select embodiments, a surface (e.g., an electrode surface) is functionalized with oligonucleotides that include sequences complementary to the first and second discontinuous regions, and the first and second discontinuous regions are attached to the surface via hybridization to the oligonucleotides. In other words, embodiments of the subject method include stably associating a second nucleic acid with the surface by anchoring the oligonucleotide to one or more of the surfaces (e.g., via thiol coupling chemistry, etc.). The oligonucleotides may be any suitable short (e.g., 5-20 nucleotides) single-stranded DNA or RNA molecules. In certain cases, each oligonucleotide has a sequence that is complementary to the sequence of the second nucleic acid, i.e., such that the two molecules hybridize. Because the oligonucleotides are anchored to the surface (e.g., an electrode surface), the second nucleic acid hybridized to the oligonucleotides can be stably associated with at least one surface.

[0031] As discussed above, the second nucleic acid forms at least a portion of the opening. By "at least a portion" of the opening, it is meant that the second nucleic acid may constitute one component of the opening, or the entire opening. The second nucleic acid may be arranged in any convenient configuration that promotes sequential interactions between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid. In some examples, the method includes arranging the second nucleic acid in a "bridge structure" configuration. As described herein, a bridge structure refers to a configuration of the second nucleic acid in which the nucleic acid is stably associated with a surface (e.g., an electrode) in such a manner that a bridge-like shape is formed. In certain examples, the electrolyzed second nucleic acid includes first and second discontinuous regions attached to the surface or surfaces such that the electrolyzed second nucleic acid forms a bridge structure. In an embodiment of the method, the movement of the first nucleic acid includes pulling the first nucleic acid through the opening of the bridge structure (e.g., via any of the methods discussed above).

[0032] In some cases, the electrolyzed second nucleic acid is one of a plurality of electrolyzed nucleic acids. Any suitable number of electrolyzed nucleic acids may be used. In some cases, the number of electrolyzed nucleic acids ranges from 1 to 20, including, for example, 1 to 15, and 1 to 10. In some embodiments, the method of the present invention involves the use of two or more electrolyzed nucleic acids. In yet other embodiments, the method of the present invention involves the use of three or more electrolyzed nucleic acids. Each of the plurality of electrolyzed nucleic acids may adopt the same or different configurations. In some embodiments, each of the plurality of electrolyzed nucleic acids is arranged in a bridge structure configuration.

[0033] As discussed above, the method of the present invention includes detecting the conductance that changes along the first nucleic acid, which indicates the sequential interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid. As discussed above in the summary section with respect to Agapito et al. and FIG. 1, the interaction between given nucleobases of a nucleic acid is characterized by a specific conductance fingerprint. In other words, DNA base pairs behave as biological Aviram-Ratner electric rectifiers because of the spatial separation and weak bonds between the nucleobases, and because the current flowing across these base pairs varies based on the specific nature of the bonds. Thus, the method includes identifying these conductance fingerprints as the first nucleic acid passes through an opening at least partially formed by the second nucleic acid, thereby identifying features of the first nucleic acid, including but not limited to the sequence of the first nucleic acid. Thus, the method includes applying a current to the second nucleic acid (e.g., via electrodes) in a manner sufficient to pass the current through the backbone of the second nucleic acid.

[0034] Detecting the change in conductance "along" the first nucleic acid means that the current travels across the electrolyzed second nucleic acid (e.g., bridge structure) to the DNA / RNA / molecule to be sequenced (i.e., the first nucleic acid). Thus, in these repetitions, electrons travel along the first nucleic acid to complete the circuit. The interaction between the base to be sequenced and the second nucleic acid base leads to a change in conductance fingerprint that allows the identification of the sequenced base. The measurement of the electrons traveling along the first nucleic acid allows the evaluation of said conductance fingerprint.

[0035] Conductance along the first nucleic acid may be measured via any convenient approach. In some embodiments, an electrometer is used to provide a bias current and analyze the resulting current. Commercially available electrometers that may be suitable for use in the subject methods include, for example, Keithley® instruments. In some embodiments, detecting the changing conductance includes an impedance-based approach. In certain versions, the method includes identifying the nucleic acid base based on its characteristic energy level. Characteristic energy levels of interest include, but are not limited to, the highest occupied molecular orbital (HOMO) energy. Measurement techniques that may be employed for use in the subject methods can be found, for example, in Pedersen et al. Nanotechnology, 28(1), 015502, and Ohshiro et al. 2012 12th IEEE International Conference on Nanotechnology (IEEE-NANO) (pp. 1-2). IEEE, which are incorporated herein by reference in their entireties.

[0036] FIG. 2 presents a schematic diagram of a method according to an embodiment of the present disclosure, which includes relative movement of a first nucleic acid through a plurality of openings formed by a plurality of electrolyzed nucleic acids. In this example, the electrolyzed second nucleic acids 202 each include first and second discontinuous regions attached to a surface such that the electrolyzed nucleic acids 202 form a bridge structure. During the relative movement, a varying conductance is detected along the first nucleic acid 201, which indicates a continuous interaction between the nucleobases of the first nucleic acid 201 and one or more nucleobases of the electrolyzed bridge structure 202. In this example, the relative movement includes pulling the first nucleic acid through the openings formed by the bridge structure 202. An adaptor 203 is hybridized to the first nucleic acid 201, which connects the first nucleic acid 201 to an elongation structure (not shown) to pull the first nucleic acid 201 through the bridge structure 202. In the embodiment of FIG. 2, the first nucleic acid 201 is attached to a magnetic bead 204.

[0037] FIG. 3 presents a schematic diagram of a method according to an embodiment of the present disclosure, including relative movement of a first nucleic acid through a plurality of openings formed by a plurality of electrolyzed nucleic acids. In this example, the electrolyzed second nucleic acids 302 each include first and second discontinuous regions attached to a surface such that the electrolyzed nucleic acids form a bridge structure. During the relative movement, a changing conductance is detected along the first nucleic acid 301, indicative of a continuous interaction between the nucleobases of the first nucleic acid 301 and one or more nucleobases of the electrolyzed bridge structure 302. In this example, the relative movement includes pulling the first nucleic acid through the openings formed by the bridge structures, the pulling being via an elongation structure 305 (e.g., a nanotube, nanowire, or biopolymer (e.g., nucleic acid)) in the direction in which the first nucleic acid 301 is to be pulled. The elongation structure 305 is attached to the first nucleic acid 301 via an adaptor 303.

[0038] In an embodiment, causing the relative movement includes moving the surface relative to the first nucleic acid. In other words, the relative movement is caused by the movement of the surface, and the movement of the first nucleic acid is negligible. In a particular example, the first nucleic acid is immobilized during the relative movement. Any convenient approach may be used to move the surface, such as using a piezoelectric material / actuator. In some embodiments, materials and approaches used in mechanically controllable break junctions and AFM may be used to move the surface.

[0039] 4 provides an illustration of a method according to an embodiment of the present disclosure that includes relative movement of a first nucleic acid through a plurality of openings formed by a plurality of electrolyzed nucleic acids. In this example, the electrolyzed nucleic acids each include first and second discontinuous regions attached to a surface 406 such that the electrolyzed nucleic acids form a bridge structure 402. During the relative movement, a changing conductance is detected along the first nucleic acid 401 indicative of successive interactions between the nucleobases of the first nucleic acid 401 and one or more nucleobases of the electrolyzed bridge structure 402. In this example, the relative movement includes moving the surface 406 (and in turn the bridge structure 402) relative to the first nucleic acid 401.

[0040] In certain versions of the disclosed method, the electrolyzed second nucleic acid at least partially forms a loop structure, and the relative movement of the first nucleic acid is through the opening of the loop structure. For example, the electrolyzed second nucleic acid may include a first end attached to a surface (e.g., an electrode) to form a stem-loop structure. In additional embodiments, the electrolyzed second nucleic acid includes a first end attached to a surface and first and second discontinuous regions hybridized to a third nucleic acid molecule such that the electrolyzed second nucleic acid and the third nucleic acid molecule form a loop structure. The third nucleic acid may be substantially similar to the second nucleic acid described herein. In alternative examples, the third nucleic acid is also electrolyzed. In certain cases, the third nucleic acid has one or more regions that are complementary to regions of the second nucleic acid such that the two nucleic acids may hybridize, thereby forming a loop structure. In alternative cases, the second nucleic acid is attached to a first surface and the third nucleic acid is attached to a second surface. In other cases, only one of the second and third nucleic acids is attached to the surface. The second and / or third nucleic acids may be attached to the substrate via any convenient mechanism, such as those described above. In an alternative embodiment, the first end of the electrolyzed second nucleic acid is attached to the surface via biotin-streptavidin interaction. In an additional embodiment, the first end of the electrolyzed second nucleic acid is attached to the surface via magnetic attraction.

[0041] FIG. 5 presents a schematic diagram of various electrolyzed nucleic acid configurations that find use in the practice of the disclosed method. As shown on the left, the electrolyzed second nucleic acid may include a first end attached to a surface and form a stem-loop structure. As shown in the center, the electrolyzed second nucleic acid may include a first end attached to a surface and first and second discontinuous regions hybridized to a third nucleic acid molecule such that the electrolyzed second nucleic acid and the third nucleic acid molecule form a loop structure. In this example, the third nucleic acid includes an end attached to a second surface. As shown on the right, the electrolyzed second nucleic acid may include a first end attached to a surface and first and second discontinuous regions hybridized to a third nucleic acid molecule such that the electrolyzed second nucleic acid and the third nucleic acid molecule form a loop structure. In this example, the third nucleic acid does not include an end attached to a second surface. The DNA to be sequenced (ie, the first nucleic acid marked by an "X") is inside the loop, and the concept and method of completing the circuit is the same as in Figures 1-4.

[0042] 5, in some embodiments, the structures shown (e.g., the central structure attached to each substrate) are arranged in a manner such that the loop forms a nanopore that is used to sequence the first nucleic acid. For example, the DNA that forms the hairpin may have regions that are conductive and regions that act as isolators.

[0043] In some cases, the method includes detecting a changing conductance between the first nucleic acid and an electrode in proximity to the first nucleic acid. Similar to the embodiment discussed above with respect to Figures 1-5, the changing conductance indicates a continuous interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid, and a current flows along the DNA molecule to be sequenced. However, without being bound by theory, the circuit can also be completed by placing a conductive electrode in proximity to the second nucleic acid and the nucleic acid to be sequenced (i.e., the first nucleic acid). The interaction between the base to be sequenced and the second nucleobase leads to a changing conductance fingerprint that allows identification of the sequenced base. The electrode in proximity to the first nucleic acid may be any convenient electrode, such as those described above.

[0044] Figure 6 presents a schematic diagram of an embodiment in which a changing conductance between a first nucleic acid and an electrode proximate to the first nucleic acid is detected, the changing conductance indicating a successive interaction between the nucleobase of the first nucleic acid and one or more nucleobases of an electrolyzed nucleic acid in the form of a bridge structure. In the example of Figure 6, a relative movement occurs (e.g., via any one of the mechanisms described above) between a first nucleic acid 601 and a plurality (i.e., three) electrolyzed second nucleic acids 602. The current from the plurality of electrolyzed nucleic acids 602 passes through the first nucleic acid 601 and proceeds to an electrode 603 in the direction of the arrow when a base-to-base interaction occurs between the nucleic acids. The conductance may then be evaluated for the fingerprint described above to identify the nucleobase of the first nucleic acid 601.

[0045] In some cases, the electrolyzed second nucleic acid is disposed in the channel, and causing relative movement between the first nucleic acid and the second nucleic acid includes translocating the first nucleic acid into the channel through an opening formed at least in part by the electrolyzed second nucleic acid. Any channel suitable for translocating nucleic acids may be used. The size (e.g., diameter) of the channel may vary. Exemplary diameters range from 0.5 nm to 20 nm. Materials from which the channel may be constructed include, but are not limited to, silicon (e.g., silicon nitride), graphene, and the like, and combinations thereof. In some cases, the channel is a nanopore (e.g., a nanopore to which a potential difference is applied), and the method includes exposing the nucleic acid bases to the nanopore in a sequential manner while monitoring the electrical signal.

[0046] The second nucleic acid may be disposed in the channel in any convenient manner. In some embodiments, the second nucleic acid is attached to the channel at one end at a first point and at the other end at a second point. In some cases, the second point is opposite the first point. The second nucleic acid may be attached to the channel by any convenient technique, including but not limited to the techniques described above (e.g., thiol-based techniques). In certain embodiments, the second nucleic acid is one of a plurality of electrolyzed nucleic acids disposed in the channel. In these embodiments, the electrolyzed nucleic acids may be disposed relative to each other in any convenient manner. In an alternative version, the electrolyzed nucleic acid is disposed in a "cross-hair" configuration. In other words, the electrolyzed nucleic acid is attached to the pore such that the resulting shape of the electrolyzed nucleic acid resembles a cross from a vantage point at the top of the pore. The electrolyzed nucleic acid may be attached to an electrode (e.g., a positive electrode) in the channel that is configured to apply a current therethrough. The cross-hair configuration results in the creation of four quadrants in the channel, one of which the first nucleic acid may pass through. In an embodiment, each quadrant is associated with an electrode (e.g., a negative electrode). The current from the electrolyzed nucleic acid may be conducted through the first nucleic acid passing through a given quadrant before reaching the respective negative electrode. The varying conductance between the first nucleic acid and the electrode proximate to the first nucleic acid may then be used to identify a feature (e.g., sequence information) of the first nucleic acid.

[0047] When practicing the subject method, any suitable nanopore device / apparatus for translocating a first nucleic acid therethrough and detecting / monitoring the change in conductance during translocation may be used. For example, a suitable nanopore device may include a chamber containing an aqueous solution and a membrane separating the chamber into two sections, the membrane including a nanopore formed therein. Electrical measurements may be performed using a single channel recording instrument such as those described in, for example, Lieberman et al. (2010) J.Am.Chem.Soc. 132(50):17961-72, Stoddart et al. (2009) PNAS 106(19):7702-7, U.S. Patent No. 9,481,908, and U.S. Patent Application Publication No. 2014 / 0051068, the disclosures of which are incorporated herein by reference in their entirety for all purposes. Alternatively, electrical measurements may be made using a multi-channel system, such as that described in U.S. Patent Application Publication No. 2015 / 346149, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0048] In nanopore-based analyses (e.g., sequencing), the nanopore functions as a biosensor, providing the only pathway by which the ionic solution on the cis side of the membrane contacts the ionic solution on the trans side. A constant voltage bias (positive on the trans side) generates an ionic current through the nanopore, driving the polynucleotide in the cis chamber through the pore to the trans chamber. A processive enzyme (e.g., helicase, polymerase, nuclease, etc.) may bind to the polynucleotide such that its stepwise translocation controls the nucleotide, ratcheting nucleobase by nucleobase through the small nanopore.

[0049] Suitable conditions for nanopore-based analysis (e.g., protein pores, solid-state pores, etc.) are known in the art. Typically, a voltage is applied across the membrane and pore. The voltage used may be between +2V and -2V, e.g., between -400mV and +400mV. The voltage used may be within a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. The voltage may be within a range of 100mV to 240mV, e.g., between 120mV and 220mV.

[0050] The method is typically carried out in the presence of a suitable charge carrier, such as a metal salt, e.g. an alkali metal salt, a halide salt, e.g. a chloride salt, e.g. an alkali metal chloride salt. The charge carrier may include an ionic liquid or an organic salt, e.g. tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or l-ethyl-3-methylimidazolium chloride. Typically, the salt is present in an aqueous solution in the chamber. For example, potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) may be used. The salt concentration may be saturated. The salt concentration may be 3M or less, typically 0.1-2.5M, 0.3-1.9M, 0.5-1.8M, 0.7-1.7M, 0.9-1.6M, or 1M-1.4M. The salt concentration may be 150mM to 1M. The method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio, allowing one to identify currents indicative of the presence of a nucleotide against a background of normal current fluctuations.

[0051] In some embodiments, the rate at which the first nucleic acid is exposed to the nanopore is controlled using a processive enzyme. Non-limiting examples of processive enzymes that may be used include polymerases (e.g., phi29 or other suitable polymerases) and helicases, such as Hel308 helicase, RecD helicase, Tral helicase, Tral subgroup helicase, XPD helicase, and the like. The processive enzyme may, for example, bind to the nucleic acid, and the resulting complex may then be drawn to the nanopore, for example, by a potential difference applied across the nanopore. In other embodiments, the processive enzyme may be located at the nanopore (e.g., attached to or adjacent to the nanopore), such that the processive enzyme, for example, binds to the nucleic acid upon reaching the nanopore.

[0052] The nanopore may be present in a solid state film, a biological membrane, etc. In some embodiments, the nanopore is a solid state nanopore. In other embodiments, the nanopore is a biological nanopore. The biological nanopore may be, for example, an alpha-hemolysin-based nanopore, a Mycobacterium smegmatis porin A (MspA)-based nanopore, etc.

[0053] 7A-7B present an embodiment of the invention in which an electrolyzed second nucleic acid is disposed within a channel. FIG. 7A presents a top view of the channel 705, while FIG. 7B presents a side view of the channel 705. As shown in FIG. 7A, the channel (i.e., nanopore) 705 includes a negative electrode 703 and a positive electrode 704. The electrolyzed nucleic acid 702 is attached to the channel 705 via the electrodes 704 and is disposed in a cross-hair configuration resulting in dividing the interior of the channel 705 into four quadrants. The nucleic acid to be sequenced (i.e., the first amino acid) can pass through any one of the four quadrants. The molecule to be sequenced can be pulled through the pore and through a bridge structure via electrophoresis or via the flow of fluid / buffer through the pore. Illustrated is a base "A" that allows for the circuit to be completed. FIG. 7B presents an embodiment of the channel 705 in which there are multiple sets of electrolyzed nucleic acids 702. FIG. 7B does not show the electrodes shown in FIG. 7A, although their presence is suggested.

[0054] In an embodiment, the subject method is performed on an integrated device configured to carry out the steps of the invention. The integrated device may be configured to analyze (e.g., sequence) one or more nucleic acids (i.e., a first nucleic acid) using one or more electrolyzed nucleic acids (i.e., a second nucleic acid) described herein. The integrated device may include components necessary to electrolyze the second nucleic acid, such as a power source, electrodes, conduits, and switches. The integrated device may be configured to detect changing conductance along the first nucleic acid or to detect changing conductance between the first nucleic acid and an electrode proximate to the first nucleic acid, as desired. In an alternative embodiment, the integrated device is a chip. The chip may be constructed from any convenient material. Exemplary materials include silicon (e.g., silicon dioxide).

[0055] FIG. 8 illustrates an embodiment of the invention in which the change in conductance between a first nucleic acid and an electrode proximate to the first nucleic acid may be detected. As shown in FIG. 8, oligonucleotides are attached to the left electrode 804l and the right electrode 804r (e.g., via thiol chemistry, as described above). A second nucleic acid 802 hybridizes to the oligonucleotide 805, thereby forming an opening through which the nucleic acid to be sequenced may pass. A current resulting from a power source 806 (e.g., a battery) may be applied to one or both of electrodes 804l (left electrode) and 804r (right electrode). For example, current may or may not be applied to electrode 804l depending on whether switch 807a is released. When the second nucleic acid 802 is electrolyzed, the current may jump from the second nucleic acid 802 to electrode 803g. When the nucleic acid to be sequenced passes through the opening formed by the nucleic acid 802, base-to-base interactions between the two nucleic acids lead to the presence of a conductance fingerprint that may be detected by the device. The circuit may be completed depending on whether switch 807b is engaged or released.

[0056] 9A-9B show the architecture of a chip for use in the subject methods. As shown in FIG. 9A, the chip includes a plurality of left and right electrodes (numbered 0-7) (represented by "l" and "r", respectively) corresponding to electrodes 804l and 804r shown in FIG. 8. In addition, the chip includes a "gate" electrode corresponding to electrode 803g in FIG. 8. As shown in FIG. 9B, each of the left and right electrodes is separated by a small gap (e.g., a length in the range of 20 nm to 60 nm) across which a second nucleic acid is positioned (e.g., as shown in FIG. 8). Underneath the left and right electrodes are gate electrodes insulated from the left and right electrodes.

[0057] According to some embodiments, the method of the present disclosure is computer-implemented. "Computer-implemented" means that at least one step of the method is implemented using one or more processors and one or more non-transitory computer-readable media. The computer-implemented method of the present disclosure may further include one or more steps that are not computer-implemented, such as obtaining a sample from a subject, isolating nucleic acid for sequencing, performing contacting and / or combining steps according to the method of the present disclosure, etc.

[0058] The nucleic acid to be sequenced by the disclosed method may be deoxyribonucleic acid (DNA). The DNA of interest includes, but is not limited to, genomic DNA or fragments thereof, complementary DNA (or "cDNA" synthesized from any RNA or DNA of interest) or fragments thereof, recombinant DNA (e.g., plasmid DNA) or fragments thereof, etc. The nucleic acid to be sequenced may be more than about 2 bases, more than about 10 bases, more than about 100 bases, more than about 500 bases, more than 1000 bases, more than 10,000 bases, more than 100,000 bases, more than about 1,000,000 bases, up to about 10 bases, composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides. 10 They may be more than one base and may be produced enzymatically or synthetically (e.g., PNAs as described in U.S. Pat. No. 5,948,902 and the references cited therein) that are capable of hybridizing to naturally occurring nucleic acids in a sequence-specific manner similar to that of two naturally occurring nucleic acids, e.g., participating in Watson-Crick base pairing interactions.

[0059] The nucleic acid to be sequenced by the methods of the present disclosure may be ribonucleic acid (RNA). The RNA may be any type of RNA (or subtype thereof), including, but not limited to, messenger RNA (mRNA), microRNA (miRNA), small interfering RNA (siRNA), trans-acting small interfering RNA (ta-siRNA), naturally occurring small interfering RNA (nat-siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), non-coding RNA (ncRNA), transfer messenger RNA (tmRNA), precursor messenger RNA (pre-mRNA), small Cajal body specific RNA (scaRNA), piwi-interacting RNA (piRNA), endoribonuclease-prepared siRNA (esiRNA), small temporal RNA (stRNA), signal recognition RNA, telomeric RNA, ribozyme, or any combination of these RNA types or subtypes thereof.

[0060] In certain embodiments, the portion of the nucleic acid to be sequenced comprises "non-natural nucleosides" or "non-natural nucleotides," which refer to nucleosides or nucleotides that contain modified nucleobases and / or other chemical modifications, such as modified sugars. In some cases, the non-natural nucleotides / nucleosides have a unique conductance fingerprint that may be recognized by the subject methods. According to some embodiments, the molecular discs are characterized by a higher melting temperature (T) of synthetic strand-nucleic acid hybrids compared to nucleic acid-nucleic acid hybrids. m) includes moieties that contain non-natural nucleobases and / or non-natural nucleotides that modify the Non-limiting examples include modified pyrimidines, such as methyl-dC or propynyl-dU; modified purines, such as G-clamps; 2-amino-2'-deoxyadenosine-5'-triphosphate (2-amino-dATP), 5-methyl-2'-deoxycytidine-5'-triphosphate (5-Me-dCTP), 5-propynyl-2'-deoxycytidine-5'-triphosphate (5-Pr-dCTP), 5-propynyl-2'-deoxyuridine-5'-triphosphate (5-Pr-dUTP), halogenated deoxy-uridines (XdU), such as 5-chloro-2'-deoxyuridine-5'-triphosphate (5-Cl-dUTP), 5-bromo-2'-deoxyuridine-5'-triphosphate (5-Br-dUTP), or any combination thereof.

[0061] The nucleic acid sequenced by the methods of the present disclosure may be nucleic acid from one or more immune cells. Immune cells of interest include, but are not limited to, T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the nucleic acid sequenced is from a T cell. T cells of interest include naive T cells (T N ), cytotoxic T cells (T CTL ), memory T cells (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ), effector memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (T EFF ), regulatory T cells (T REG ), helper T cells (T H , T H 1. T H 2. T H 17) CD4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), and gamma delta T cells (T γδ ) are included.

[0062] In certain embodiments, the nucleic acid sequenced by the disclosed methods is a nucleic acid encoding an immune cell receptor (e.g., T cell receptor (TCR), B cell receptor (BCR)) or a portion thereof. For example, in certain embodiments, a method is provided that includes sequencing a nucleic acid encoding one or more CDRs of an alpha or beta chain of a TCR. According to some embodiments, the method includes sequencing a CDR3-encoding portion of a nucleic acid encoding all or a portion of an alpha or beta chain of a TCR. In certain embodiments, such a method uses a synthetic chain comprising a series of molecular discs, each comprising a portion for binding to A, C, G, or T / U, each molecular disc of the series exclusively binds to A, C, G, or T / U, the series designed to hybridize to a known nucleotide sequence (e.g., a constant region sequence) adjacent to the CDR3-encoding portion of a nucleic acid encoding all or a portion of an alpha or beta chain of a TCR, such that the nucleotide sequence of the CDR3-encoding portion may be determined based on the rotational position of the molecular disc adjacent to the series of molecular discs.

[0063] The nucleic acid sequenced by the disclosed method may be present in any nucleic acid sample of interest. In certain embodiments, the nucleic acid is present in a nucleic acid sample isolated from a single cell, a plurality of cells (e.g., cultured cells), a tissue, an organ, or an organism (e.g., bacteria, yeast, etc.). According to some embodiments, the nucleic acid sample is isolated from an animal cell, tissue, organ, etc. In some embodiments, the animal is a mammal, such as a mammal from the genus Homo (e.g., human), rodent (e.g., mouse or rat), dog, cat, horse, cow, or any other mammal of interest. In certain embodiments, the nucleic acid sample is isolated / obtained from a source other than a mammal, such as a bacteria, yeast, insect (e.g., Drosophila), amphibian (e.g., frog (e.g., Xenopus laevis)), virus, plant, or any other non-mammalian nucleic acid sample source.

[0064] The nucleic acids that may be sequenced by the methods of the present disclosure include cell-free nucleic acids, such as cell-free DNA, cell-free RNA, or both. Such cell-free nucleic acids may be obtained from any suitable source. In certain embodiments, the cell-free nucleic acids are from a body fluid sample selected from the group consisting of whole blood, plasma, serum, amniotic fluid, saliva, urine, pleural fluid, bronchial lavage, bronchial aspirate, breast milk, colostrum, tears, semen, peritoneal fluid, pleural fluid, and feces. In certain embodiments, the cell-free nucleic acid is cell-free fetal DNA. According to some embodiments, the cell-free nucleic acid is circulating tumor DNA. In certain embodiments, the cell-free nucleic acid includes infectious agent DNA. According to some embodiments, the cell-free nucleic acid includes DNA from a transplant.

[0065] The term "cell-free nucleic acid" as used herein can refer to nucleic acid isolated from a source that is substantially free of cells. Cell-free nucleic acid may be referred to as "extracellular" nucleic acid, "circulating cell-free" nucleic acid (e.g., CCF fragments, ccf DNA) and / or "cell-free circulating" nucleic acid. Cell-free nucleic acid is present in blood and can be obtained from blood (e.g., from the blood of an animal, from the blood of a human subject). Cell-free nucleic acid is often free of detectable cells and may contain cellular elements or cellular remnants. Non-limiting examples of non-cellular sources for cell-free nucleic acid are described above. Obtaining cell-free nucleic acid may include obtaining a sample directly (e.g., collecting a sample, e.g., a test sample) or obtaining a sample from another person who is collecting a sample. According to some embodiments, cell-free nucleic acid may often be a product of cell apoptosis and cell degradation, providing a basis for cell-free nucleic acid having a range of lengths across a spectrum (e.g., a "ladder"). In some embodiments, the sample nucleic acid from the test subject is circulating cell-free nucleic acid. In some embodiments, the circulating cell-free nucleic acid is from plasma or serum from the test subject.

[0066] Cell-free nucleic acid can contain different nucleic acid species and is therefore referred to herein as "heterogeneous" in certain embodiments. For example, a sample from a subject with cancer can contain nucleic acid from cancer cells (e.g., tumor, neoplasm) and nucleic acid from non-cancerous cells. In another example, a sample from a pregnant woman can contain maternal and fetal nucleic acid. In another example, a sample from a subject with an infection or infectious disease can contain host nucleic acid and nucleic acid from an infectious agent (e.g., bacteria, fungi, protozoa). In another example, a sample from a subject undergoing a transplant can contain host nucleic acid and nucleic acid from a donor organ or tissue. In some examples, the cancer, fetal, infectious agent, or transplant nucleic acid is sometimes about 5% to about 50% of the total nucleic acid (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49% of the total nucleic acid is cancer, fetal, infectious agent, or transplant nucleic acid). In another example, the heterologous cell-free nucleic acid may include nucleic acid from more than one subject.

[0067] Nucleic acids that may be sequenced by the methods of the present disclosure include tumor nucleic acids (e.g., present in a nucleic acid sample isolated from a tumor, e.g., a tumor biopsy sample). As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, various types of head and neck cancer, and the like.

[0068] Approaches, reagents, and kits for isolating, purifying, and / or concentrating DNA and RNA from a source of interest are known in the art and are commercially available. For example, kits for isolating DNA from a source of interest include DNeasy®, RNeasy®, QIAamp®, QIAprep®, and QIAquick® nucleic acid isolation / purification kits from Qiagen, Inc. (Germantown, Md); DNAzol®, ChargeSwitch®, Purelink®, GeneCatcher® nucleic acid isolation / purification kits from Life Technologies, Inc. (Carlsbad, CA); NucleoMag®, NucleoSpin®, and NucleoBond® nucleic acid isolation / purification kits from Clontech Laboratories, Inc. (Mountain View, CA). In certain embodiments, nucleic acids are isolated from fixed biological samples, such as formalin-fixed, paraffin-embedded (FFPE) tissue. Genomic DNA from FFPE tissue may be isolated using commercially available kits, such as the AllPrep® DNA / RNA FFPE kit by Qiagen, Inc. (Germantown, Md), the RecoverAll® Total Nucleic Acid Isolation Kit for FFPE by Life Technologies, Inc. (Carlsbad, Calif.), and the NucleoSpin® FFPE kit by Clontech Laboratories, Inc. (Mountain View, Calif.).

[0069] Nucleic acid sequences determined by the methods of the present disclosure may be analyzed (eg, assembled, etc.) using available sequence analysis software.

[0070] In an alternative embodiment, rather than sequencing a nucleic acid, the method of the present invention comprises sequencing a polypeptide. Amino acids may have conductance fingerprints in a manner similar to nucleotides. Thus, embodiments in which the molecule to be sequenced is a polypeptide are encompassed by the present disclosure. For example, the method of the present invention may comprise causing a relative movement of a first polypeptide through an opening formed at least in part by an electrolyzed second polypeptide, and detecting a change in conductance along the first polypeptide during the relative movement. Alternatively, during the relative movement, the method may comprise detecting a change in conductance between the first polypeptide and an electrode proximate to the first polypeptide.

[0071] The polypeptide to be sequenced may be any polypeptide, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, and the like.

[0072] The term "amino acid" generally refers to any monomeric unit that includes a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, photoactivatable crosslinkers, metal-binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or non-covalently interact with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids, other carbohydrate-modified amino acids, amino acids containing polyethylene glycol or polyethers, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon-linked sugar-containing amino acids, redox-active amino acids, aminothioacid-containing amino acids, and amino acids containing one or more toxic moieties.

[0073] The term "amino acid" includes, but is not limited to, naturally occurring α-amino acids and their stereoisomers. An "stereoisomer" of an amino acid refers to an enantiomer of an amino acid, such as an L-amino acid or a D-amino acid. For example, a stereoisomer of a naturally occurring amino acid refers to an enantiomer of a naturally occurring amino acid (i.e., a D-amino acid).

[0074] Naturally occurring α-amino acids are those encoded by the genetic code, as well as those amino acids that are later modified (e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine). Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, D-alanine (D-Ala), D-cysteine ​​(D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0075] Computer-readable medium and system Aspects of the present disclosure further include systems, such as nucleic acid sequencing systems. In certain embodiments, such systems include one or more processors and one or more non-transitory computer-readable media having instructions stored thereon that cause the system to monitor changing conductance along a first nucleic acid or between the first nucleic acid and an electrode proximate to the first nucleic acid. The changing conductance monitored by the system indicates a continuous interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid. In some embodiments, the changing conductance comprises a conductance fingerprint for different nucleobases in the first nucleic acid, and the one or more non-transitory computer-readable media have instructions stored thereon that cause the system to determine the identity of one or more nucleotides of the first nucleic acid based on the changing conductance. In additional embodiments, the altered conductance comprises a conductance fingerprint for different nucleobases in the first nucleic acid, and the one or more non-transitory computer-readable media comprises instructions stored thereon that cause the system to determine a nucleotide sequence of the first nucleic acid based on the altered conductance.

[0076] Various processor-based systems may be used to implement embodiments of the present disclosure. Such a system may include a system architecture, in which the components of the system are in electrical communication with each other using a bus. The system architecture may include a processing unit (CPU or processor) and a cache, which are variously coupled to a system bus. The bus couples various system components, including system memory (e.g., read-only memory (ROM) and random access memory (RAM)) to the processor.

[0077] The system architecture may include a cache of high-speed memory directly connected to the processor, in close proximity to the processor, or integrated as part of the processor. The system architecture may copy data from the memory and / or storage device to the cache for quick access by the processor. In this way, the cache may provide performance improvements that avoid processor delays while waiting for data. These and other modules may control or be configured to control the processor to perform various actions. Other system memories may also be used. The memory may include multiple different types of memory with different performance characteristics. The processor may include any general-purpose processor, as well as hardware or software modules, such as first, second, and third modules stored in a storage device, configured to control the processor, as well as special purpose processors where software instructions are incorporated into the actual processor design. The processor may be essentially a completely self-contained computing system, including multiple cores or processors, buses, memory controllers, caches, etc. Multi-core processors may be symmetrical or asymmetrical.

[0078] Aspects of the invention also include non-transitory computer readable media. The subject non-transitory computer readable media includes instructions stored thereon that cause the system to monitor the changing conductance along the first nucleic acid or the changing conductance between the first nucleic acid and an electrode proximate the first nucleic acid. As described above, the changing conductance indicates a continuous interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid. In an alternative case, the changing conductance includes a conductance fingerprint for different nucleobases in the first nucleic acid, and the one or more non-transitory computer readable media includes instructions stored thereon that cause the system to determine the identity of one or more nucleotides of the first nucleic acid based on the changing conductance. In an additional case, the changing conductance includes a conductance fingerprint for different nucleobases in the first nucleic acid, and the one or more non-transitory computer readable media includes instructions stored thereon that cause the system to determine the nucleotide sequence of the first nucleic acid based on the changing conductance.

[0079] To enable user interaction with the computing system architecture, the input device may represent any number of input mechanisms, such as, for example, a microphone for voice, a touch-sensitive screen for gesture or graphical input, a keyboard, a mouse, motion input, voice, etc. The output device may also be one or more of several output mechanisms. In some examples, a multimodal system may enable a user to provide multiple types of input to communicate with the computing system architecture. The communication interface may generally control and manage user input and system output. There is no restriction to operating on any particular hardware configuration, and thus the basic features herein may be easily substituted for improved hardware or firmware configurations as they are developed.

[0080] The storage device is typically a non-volatile memory and may be a hard disk or other type of computer-readable medium capable of storing data that is accessible by a computer, such as a magnetic cassette, a flash memory card, a solid-state memory device, a digital versatile disk, a cartridge, a random access memory (RAM), a read-only memory (ROM), and hybrids thereof.

[0081] The storage device may include a software module for controlling the processor. Other hardware or software modules are contemplated. The storage device may be connected to a system bus. In one aspect, a hardware module performing a specific function may include software components stored in a computer-readable medium in association with necessary hardware components, such as a processor, a bus, an output device, etc., to perform various functions of the disclosed technology.

[0082] Embodiments within the scope of the present disclosure may also include tangible and / or non-transitory computer-readable storage media or devices for carrying or having computer-executable instructions or data structures stored thereon. Such tangible computer-readable storage devices may be any available device that can be accessed by a general-purpose or special-purpose computer, including the functional design of any special-purpose processor as described above. By way of example and not limitation, such tangible computer-readable devices may include RAM, ROM, EEPROM, CD-ROM, or other optical disk storage, magnetic disk storage, or other magnetic storage devices, or any other device that can be used to carry or store desired program code in the form of computer-executable instructions, data structures, or processor chip designs. When information or instructions are provided to a computer over a network or another communications connection (either hardwired, wireless, or a combination thereof), the computer properly views the connection as a computer-readable medium. Thus, any such connection is properly termed a computer-readable medium. Combinations of the above should also be included within the scope of computer-readable storage devices.

[0083] Computer-executable instructions include, for example, instructions and data that cause a general purpose computer, special purpose computer, or special purpose processing device to perform a particular function or group of functions. Computer-executable instructions also include program modules that are executed by a computer in stand-alone or network environments. Generally, program modules include design-specific functions such as routines, programs, components, data structures, objects, and special purpose processors that perform tasks or implement abstract data types. Computer-executable instructions, associated data structures, and program modules represent examples of the program code means for executing steps of the methods disclosed herein. A particular sequence of such executable instructions or associated data structures represents examples of corresponding acts for implementing the functions described in such steps.

[0084] Other embodiments of the present disclosure may be practiced in networked computing environments having many types of computer system configurations, including personal computers, handheld devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, network PCs, minicomputers, mainframe computers, etc. Embodiments may also be practiced in distributed computing environments where tasks are performed by local and remote processing devices that are linked through a communications network (either by hardwired links, wireless links, or a combination thereof). In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0085] In certain aspects, one or more computer-readable media are provided that store instructions for performing any of the steps of the method of the present disclosure using any of the synthetic strands of the present disclosure. According to some embodiments, one or more computer-readable media of any of the systems of the present disclosure are provided. For example, one or more computer-readable media are provided that include instructions stored thereon, which, when executed by one or more processors, cause the one or more processors to use one or more position indicator readers to determine the sequence of nucleic acid by reading the position indicator of the synthetic strand of the present disclosure during or after hybridization of the synthetic strand to the nucleic acid.

[0086] kit Aspects of the present disclosure further include kits. In certain embodiments, the kits find use, for example, in implementing any of the methods of the present disclosure. According to some embodiments, the kits of the present disclosure include any of a plurality of second nucleic acids of the present disclosure. In some embodiments, the kits include a surface having the second nucleic acid disposed thereon (e.g., in any of the configurations described herein, such as a chip).

[0087] The kits of the present disclosure may include one or more reagents that find use in sequencing nucleic acids using synthetic strands. For example, the kits of the present disclosure may include a solution (e.g., a hybridization buffer) having a pH, salt concentration, one or more components (e.g., chelating agents), etc., that are useful for providing suitable conditions for contacting the nucleic acid to be sequenced with a second nucleic acid.

[0088] The kits of the present disclosure may further include instructions for carrying out any of the methods of the present disclosure, for example, instructions for sequencing a first nucleic acid using electrolyzed nucleic acid. The instructions may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be present in the kit as a package insert, on the labeling of the container of the kit or its components (i.e., associated with the packaging or subpackaging), and the like. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer-readable storage medium, for example, a portable flash drive, DVD, CD-ROM, diskette, and the like. In still other embodiments, the actual instructions are not present in the kit, but a means is provided for obtaining the instructions from a remote source, for example, via the Internet. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions are recorded on a suitable substrate.

[0089] Embodiment Regardless of the scope of the appended claims, the present disclosure is also defined by the following embodiments.

[0090] 1. A method comprising: causing relative movement of the first nucleic acid through an opening formed at least in part by the electrolyzed second nucleic acid; and detecting a changing conductance along the first nucleic acid during the relative movement, the change being indicative of a continuous interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid.

[0091] 2. A method comprising: causing relative movement of the first nucleic acid through an opening formed at least in part by the electrolyzed second nucleic acid; and detecting, during relative movement, a changing conductance between the first nucleic acid and an electrode proximate to the first nucleic acid, the changing conductance indicating a continuing interaction between the nucleobases of the first nucleic acid and one or more nucleobases of the electrolyzed second nucleic acid.

[0092] 3. The method of embodiment 1 or embodiment 2, wherein causing relative movement comprises pulling the first nucleic acid through an opening formed at least in part by the electrolyzed second nucleic acid.

[0093] 4. The method of embodiment 3, wherein the first nucleic acid is attached to an elongated structure, and pulling the first nucleic acid through the opening comprises pulling the elongated structure in a direction in which the first nucleic acid is to be pulled.

[0094] 5. The method of embodiment 4, wherein the elongated structure comprises a nanotube, a nanowire, or a biopolymer.

[0095] 6. The method of embodiment 5, wherein the biopolymer is a nucleic acid.

[0096] 7. The method of embodiment 6, wherein the first nucleic acid and the nucleic acid comprise complementary ends and are hybridized to each other during pulling.

[0097] 8. The method of embodiment 1 or embodiment 2, wherein the electrolyzed second nucleic acid is disposed in the channel and causing relative movement comprises translocating the first nucleic acid into the channel through an opening formed at least in part by the electrolyzed second nucleic acid.

[0098] 9. The method of any one of embodiments 1 to 8, wherein the electrolyzed second nucleic acid comprises first and second discontinuous regions attached to the surface such that the electrolyzed second nucleic acid forms a bridge structure, and the relative movement of the first nucleic acid is through an opening in the bridge structure.

[0099] 10. The method of embodiment 9, wherein the surface is functionalized with oligonucleotides comprising sequences complementary to the first and second discontinuous regions, and the first and second discontinuous regions are attached to the surface via hybridization to the oligonucleotides.

[0100] 11. The method of any one of embodiments 1 to 8, wherein the electrolyzed second nucleic acid comprises a first end attached to a surface and forms a stem-loop structure, and the relative movement of the first nucleic acid is through the opening of the loop portion of the stem-loop structure.

[0101] 12. The method of any one of embodiments 1 to 8, wherein the electrolyzed second nucleic acid comprises a first end attached to a surface and first and second discontinuous regions hybridized to a third nucleic acid molecule such that the electrolyzed second nucleic acid and the third nucleic acid molecule form a loop structure, and the relative movement of the first nucleic acid is through the opening of the loop structure.

[0102] 13. The method of embodiment 11 or embodiment 12, wherein the first end of the electrolyzed second nucleic acid is attached to the surface via a biotin-streptavidin interaction or via magnetic attraction.

[0103] 14. The method of any one of embodiments 9 to 13, wherein causing relative movement comprises moving the surface relative to the first nucleic acid.

[0104] 15. The method of embodiment 14, wherein the first nucleic acid is immobilized during relative movement.

[0105] 16. The method of any one of embodiments 1 to 15, wherein the electrolyzed second nucleic acid is one of a plurality of electrolyzed nucleic acids, and the method includes causing relative movement of the first nucleic acid through a plurality of openings formed at least in part by the plurality of electrolyzed nucleic acids.

[0106] 17. The method of embodiment 16, wherein one or more of the plurality of openings independently comprise a single type of nucleobase selected from nucleobases that base pair with adenine, nucleobases that base pair with thymine or uracil, nucleobases that base pair with guanine, and nucleobases that base pair with cytosine.

[0107] 18. The method of embodiment 16 or embodiment 17, wherein one or more of the plurality of openings comprises an abasic nucleotide.

[0108] 19. The method of any one of embodiments 1 to 18, wherein the varying conductance comprises a conductance fingerprint for different nucleic acid bases in the first nucleic acid.

[0109] 20. The method of embodiment 19, further comprising determining the identity of one or more nucleotides of the first nucleic acid based on the changed conductance.

[0110] 21. The method of embodiment 19 or embodiment 20, further comprising determining the nucleotide sequence of the first nucleic acid based on the changed conductance.

[0111] 22. The method of any one of embodiments 1 to 21, wherein the first nucleic acid is selected from genomic DNA, complementary DNA (cDNA), or RNA.

[0112] 23. A system comprising: one or more processors; One or more non-transitory computer-readable media, and one or more non-transitory computer readable media comprising instructions stored thereon that cause the system to monitor changing conductance along a first nucleic acid or between the first nucleic acid and an electrode proximate to the first nucleic acid, the changing conductance being indicative of a continuous interaction between a nucleobase of the first nucleic acid and one or more nucleobases of an electrolyzed second nucleic acid.

[0113] 24. The system of embodiment 23, wherein the changing conductance comprises a conductance fingerprint for different nucleobases in the first nucleic acid, and the one or more non-transitory computer-readable media comprises instructions stored thereon that cause the system to determine the identity of one or more nucleotides of the first nucleic acid based on the changing conductance.

[0114] 25. The system of embodiment 23 or embodiment 24, wherein the changing conductance comprises a conductance fingerprint for different nucleobases in the first nucleic acid, and the one or more non-transitory computer-readable media comprises instructions stored thereon that cause the system to determine a nucleotide sequence of the first nucleic acid based on the changing conductance.

[0115] 26. One or more non-transitory computer-readable media, One or more non-transitory computer readable media comprising instructions stored thereon that cause the system to monitor changing conductance along a first nucleic acid or between the first nucleic acid and an electrode proximate to the first nucleic acid, the changing conductance being indicative of a continuous interaction between the nucleobases of the first nucleic acid and one or more nucleobases of an electrolyzed second nucleic acid.

[0116] 27. One or more non-transitory computer-readable media according to embodiment 26, wherein the changing conductance comprises a conductance fingerprint for different nucleobases in the first nucleic acid, and the one or more non-transitory computer-readable media comprises instructions stored thereon that cause the system to determine the identity of one or more nucleotides of the first nucleic acid based on the changing conductance.

[0117] 28. One or more non-transitory computer-readable media of embodiment 26 or embodiment 27, wherein the changing conductance comprises a conductance fingerprint for different nucleic acid bases in the first nucleic acid, and the one or more non-transitory computer-readable media comprises instructions stored thereon that cause the system to determine a nucleotide sequence of the first nucleic acid based on the changing conductance.

[0118] The following are offered by way of example and not by way of limitation.

[0119] experiment A chip for monitoring the changing conductance associated with the conductance fingerprint was constructed as shown in Figures 9A-9B. The right and left electrodes and the corresponding gate electrode (i.e., according to the structure shown in Figure 9B) were imaged using a scanning helium ion microscope. The resulting micrograph is presented in Figure 10. As shown in Figure 10, the left electrode 1001l and the right electrode 1001r are insulated from the gate electrode 1002g via an insulating layer 1003.

[0120] 11A-11C show additional helium ion micrographs taken from a chip constructed as shown in FIG. 9A-9B. FIG. 11A shows multiple left and right electrodes arranged as discussed in FIG. 9B and their respective gate electrodes. FIG. 11B and FIG. 11C show exemplary distances between the left and right electrodes. In FIG. 11B, the distance is 34 nm. In FIG. 11C, the distance is 48 nm. FIG. 12 presents a prototype of a chip constructed as shown in FIG. 9A-9B.

[0121] Figures 13A-13D present images of the tip-electrode obtained via atomic force microscopy (AFM). The images in Figures 13A and 13C were generated via AFM tapping mode, while the images in Figures 13B and 13D were generated via conductive AFM and show that the deposited electrode is capable of conducting current.

[0122] Figures 14A-14E show the topographical and conductive aspects of a chip constructed as shown in Figures 9A-9B. Figure 14A shows a 3D topographical AFM image of the electrodes, while Figure 14B presents a 2D image of it. Figures 14C-E show testing of the conductivity of the gold electrodes using conductive AFM. The topographical tapping mode image easily shows the electrodes, but misses the gates, as these structures are below the surface. Nevertheless, the conductive AFM is able to resolve the gates as conductive (white = current = conductive).

[0123] 15A-15B present AFM images showing single stranded DNA immobilized on a gold substrate via a thio bond. The "worm-like" structures are DNA. These images show that DNA may be immobilized on a gold electrode in a manner that allows for the formation of electrolytic bridge structures that find use in the practice of methods according to embodiments of the present disclosure.

[0124] Thus, the above description merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art can devise various configurations that embody the principles of the present invention and are within its spirit and scope, even though not explicitly described or illustrated herein. Furthermore, all examples and conditional language recited herein are intended primarily to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to promote the art, and should not be interpreted as limitations to such specifically recited examples and conditions. Furthermore, all descriptions herein reciting the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Thus, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

1. 1. A method comprising: causing relative movement of the first nucleic acid through an opening formed at least in part by the electrolyzed second nucleic acid; and detecting, during said relative movement, a changing conductance between said first nucleic acid and an electrode proximate to said first nucleic acid, said changing conductance indicating a continuous interaction between a nucleobase of said first nucleic acid and one or more nucleobases of said electrolyzed second nucleic acid.

2. 2. The method of claim 1, wherein causing the relative movement comprises translocating the first nucleic acid through the opening formed at least in part by the electrolyzed second nucleic acid.

3. 3. The method of claim 2, wherein the first nucleic acid is attached to an elongated structure, and translocating the first nucleic acid through the opening comprises pulling the elongated structure in a direction in which the first nucleic acid is to translocate.

4. The method of claim 3 , wherein the elongated structure comprises a nanotube, a nanowire, or a biopolymer.

5. The method of claim 4 , wherein the biopolymer is a nucleic acid.

6. 6. The method of claim 5, wherein the first nucleic acid and the nucleic acid comprise complementary ends and are hybridized to each other during the pulling.

7. 2. The method of claim 1, wherein the electrolyzed second nucleic acid is disposed in a channel, and causing the relative movement comprises translocating the first nucleic acid into the channel through the opening formed at least in part by the electrolyzed second nucleic acid.

8. 8. The method of claim 1, wherein the electrolyzed second nucleic acid comprises first and second discrete regions attached to a surface such that the electrolyzed second nucleic acid forms a bridge structure, and the relative movement of the first nucleic acid is through an opening in the bridge structure.

9. 9. The method of claim 8, wherein the surface is functionalized with oligonucleotides comprising sequences complementary to the first and second discontinuous regions, and the first and second discontinuous regions are attached to the surface via hybridization to the oligonucleotides.

10. 8. The method of claim 1, wherein the electrolyzed second nucleic acid comprises a first end attached to a surface and forms a stem-loop structure, and the relative movement of the first nucleic acid is through an opening in a loop portion of the stem-loop structure.

11. 8. The method of claim 1, wherein the electrolyzed second nucleic acid comprises a first end attached to a surface and first and second discontinuous regions hybridized to a third nucleic acid molecule such that the electrolyzed second nucleic acid and the third nucleic acid molecule form a loop structure, and the relative movement of the first nucleic acid is through an opening in the loop structure.

12. 8. The method of any one of claims 1 to 7, wherein the electrolyzed second nucleic acid is one of a plurality of electrolyzed nucleic acids, and the method comprises causing relative movement of the first nucleic acid through a plurality of openings formed at least in part by the plurality of electrolyzed nucleic acids.

13. 13. The method of claim 12, wherein one or more of the plurality of openings independently comprise a single type of nucleobase selected from nucleobases that base pair with adenine, nucleobases that base pair with thymine or uracil, nucleobases that base pair with guanine, and nucleobases that base pair with cytosine.

14. 8. The method of any one of claims 1 to 7, wherein the varying conductance comprises a conductance fingerprint for different nucleobases in the first nucleic acid.

15. 15. The method of claim 14, further comprising determining the identity of one or more nucleotides of the first nucleic acid based on the altered conductance.

16. The method of any one of claims 1 to 7, wherein the first nucleic acid is selected from genomic DNA, complementary DNA (cDNA), or RNA.

17. 1. A system comprising: one or more processors; One or more non-transitory computer-readable media, one or more non-transitory computer-readable media having stored thereon instructions that cause the system to monitor a changing conductance along a first nucleic acid or a changing conductance between the first nucleic acid and an electrode proximate to the first nucleic acid, the changing conductance being indicative of a continuous interaction between a nucleobase of the first nucleic acid and one or more nucleobases of an electrolyzed second nucleic acid.

18. 18. The system of Claim 17, wherein the varying conductance comprises conductance fingerprints for different nucleobases in the first nucleic acid, and wherein the one or more non-transitory computer-readable media comprise stored instructions that cause the system to determine the identity of one or more nucleotides of the first nucleic acid based on the varying conductance.

19. 19. The system of Claim 17 or 18, wherein the varying conductance comprises conductance fingerprints for different nucleobases in the first nucleic acid, and the one or more non-transitory computer-readable media comprise stored instructions that cause the system to determine a nucleotide sequence of the first nucleic acid based on the varying conductance.

20. A method comprising: causing relative movement of the first nucleic acid through an opening formed at least in part by the electrolyzed second nucleic acid; and detecting, during said relative movement, a changing conductance along said first nucleic acid indicative of a continuous interaction between the nucleobases of said first nucleic acid and one or more nucleobases of said electrolyzed second nucleic acid.

Images