Methods and compositions for processing and amplifying nucleic acids

Abstract

Methods and compositions for processing a target nucleic acid sequence are provided herein. The methods and compositions provided herein include a guide complex for recruiting an enzyme that can introduce a cleavage on the target nucleic acid sequence and a cleavage on the guide complex. The processed target nucleic acid sequence can be used in further applications, such as nucleic acid amplification (e.g., isothermal amplification).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 424,666, filed November 11, 2022, and U.S. Provisional Patent Application No. 63 / 501,226, filed May 10, 2023, each of which is incorporated by reference in its entirety into this specification. [Background technology]

[0002] Nucleic acid amplification techniques, such as the polymerase chain reaction (PCR) and various isothermal amplification techniques, have become an integral part of nucleic acid-based diagnostic and research technologies. Summary of the Invention [Problem to be solved by the invention]

[0003]

[0003] There is herein recognized a need for improved methods and compositions for highly efficient processing of target nucleic acid molecules and / or for simplifying the process of optimizing reaction conditions. The methods and compositions described herein can be used to generate initial products for downstream applications such as isothermal amplification.

[0004]

[0004] In one aspect, the disclosure provides a method for processing a single-stranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions in which the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) a blocked 3' end that cannot be extended by a polymerase; and (b) introducing a Type IIs restriction enzyme under conditions sufficient for the Type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cleave within the target sequence.

[0005] In some embodiments, in (b), the cleavage exposes an extendable 3' end of the target sequence.

[0006] In some embodiments, the method further comprises extending the extendable 3' end using a polymerase.

[0006]

[0007] In another aspect, the disclosure provides a method for amplifying a single-stranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions wherein the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a Type IIs restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) a blocked 3' end that is not extendable by a polymerase; (b) introducing a Type IIs restriction enzyme under conditions sufficient for the Type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cleave within the target sequence to generate an extendable 3' end; and (c) extending the extendable 3' end of the target sequence using a polymerase.

[0007]

[0008] In some embodiments, the guide polynucleotide is a first guide polynucleotide, and the guide complex comprises a second guide polynucleotide, the second guide polynucleotide comprising (i) a non-target binding region that is complementary to the non-target binding region of the first guide polynucleotide, and (ii) a target binding region configured to hybridize to a target sequence.

[0008]

[0009] In some embodiments, when the first guide polynucleotide of the guide complex hybridizes to the target polynucleotide sequence, the target binding region of the second guide polynucleotide does not hybridize to the target sequence. In some embodiments, the first guide polynucleotide and the second guide polynucleotide hybridize to form a dimer. In some embodiments, the first guide polynucleotide and the second guide polynucleotide hybridize via the non-target binding region of the first guide polynucleotide and the second guide polynucleotide to form a dimer having a double-stranded binding region.

[0009]

[0010] In some embodiments, the double-stranded binding region comprises a restriction endonuclease recognition sequence.

[0011] In some embodiments, the type IIs restriction enzyme binds to the double-stranded binding region of the dimer.

[0010]

[0012] In another aspect, the disclosure provides a method for amplifying a single-stranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting a guide complex with the single-stranded nucleic acid molecule, the guide complex comprising: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes to a target sequence of the single-stranded nucleic acid molecule, and (ii) a second guide polynucleotide that hybridizes to the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region binds to an enzyme; (b) amplifying the target sequence using an enzyme; (c) cleaving the first guide polynucleotide within the target binding region to expose an extendable 3' end of the target sequence; (d) cleaving the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide; and (e) using a polymerase to extend the extendable 3' end of the first guide polynucleotide to generate a complement of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule.

[0011]

[0013] In some embodiments, the second guide polynucleotide comprises (i) a non-target binding region that hybridizes to a non-target binding region of the first guide polynucleotide, and (ii) a 5' to 3' target binding region configured to hybridize to a target sequence.

[0012]

[0014] In some embodiments, the method further comprises, prior to (b), cleaving the first guide polynucleotide within the target binding region using an enzyme such that the guide complex dissociates from the single-stranded nucleic acid molecule.

[0013]

[0015] In some embodiments, the method further comprises repeating (d) and (e) to generate multiple complements of the target sequence of the single-stranded nucleic acid molecule.

[0016] In some embodiments, an additional guide complex binds to a complementary molecule.

[0014]

[0017] In some embodiments, the method further comprises using the complementary molecule bound by the additional guide complex as an initiating template for generating copies of the target molecule.

[0015]

[0018] In some embodiments, the enzyme is a type IIs restriction enzyme. In some embodiments, the type IIs restriction enzyme comprises N.BstNBI, N.Bst9 I, N.BspD6I, functional fragments thereof, or combinations thereof.

[0016]

[0019] In some embodiments, the guide polynucleotide comprises a blocked 3' end that is not extendable by a polymerase, hi some embodiments, the blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof.

[0017]

[0020] In some embodiments, the single-stranded nucleic acid molecule to which the cleavage and guide polynucleotides are bound is used as a starting template for amplification. In some embodiments, the amplification is isothermal. In some embodiments, the enzyme exhibits high-frequency endonuclease activity. In some embodiments, the high-frequency endonuclease activity is derived from the large subunit of the enzyme. In some embodiments, the enzyme exhibits low-frequency endonuclease activity. In some embodiments, the low-frequency endonuclease activity is derived from the small subunit of the enzyme. In some embodiments, the enzyme exhibits at least two differential enzyme activity rates. In some embodiments, the at least two differential enzyme activity rates include two differential endonuclease activity rates when cleaving two different cleavage sites.

[0018]

[0021] In some embodiments, one of the two differential endonuclease activity rates comprises a low frequency of cleaving a target sequence of a single-stranded nucleic acid molecule. In some embodiments, one of the two differential endonuclease activity rates comprises a high frequency of cleaving a target binding region of a guide polynucleotide. In some embodiments, the two differential endonuclease activity rates are asymmetric or unequal.

[0019]

[0022] In some embodiments, the enzyme comprises BsmAI, Nt.BsmAI, Transcription Activator-Like Effector Nuclease, N.Bst9I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Mva1269I, Nb.Bpu10I, and Nt.Bpu10I, functional fragments thereof, or combinations thereof.

[0020]

[0023] In some embodiments, the temperature is varied over the course of the method.

[0024] In some embodiments, a first activity rate of the at least two differential enzyme activity rates is favored at a first temperature, and a second activity rate of the at least two differential enzyme activity rates is favored at a second temperature that is different from the first temperature.

[0021]

[0025] In some embodiments, the enzyme comprises two different active sites or endonuclease domains that confer at least two differential enzymatic activities.

[0026] In some embodiments, the target sequence comprises a recognition site that is specifically recognized by an enzyme or a first of at least two differential enzymatic activities of an enzyme to introduce cleavage.

[0022]

[0027] In some embodiments, the target binding region of the guide polynucleotide comprises a recognition site that is specifically recognized by an enzyme, or the second of at least two differential enzymatic activities of an enzyme, to introduce cleavage.

[0023]

[0028] In some embodiments, the target binding region is at least about 12 to about 25 nucleotides in length.

[0029] In some embodiments, the concentration of guide polynucleotide is at least about 0.1 μM, at least about 1 μM, or between about 0.1 μM and about 4 μM.

[0024]

[0030] In some embodiments, the non-target binding region comprises a palindromic sequence. In some embodiments, the non-target binding region is self-complementary. In some embodiments, the non-target binding region is at least about 12 nucleotides in length.

[0025]

[0031] In some embodiments, the single-stranded nucleic acid molecule is a single-stranded deoxyribonucleic acid (ssDNA) or a single-stranded ribonucleic acid (ssRNA).

[0032] In some embodiments, the target binding region comprises at least one peptide nucleic acid (PNA) residue. In some embodiments, the polymerase has strand displacement activity. In some embodiments, the guide polynucleotide or first guide polynucleotide further comprises an additional non-target binding region. In some embodiments, the additional non-target binding region is located at the 5' end of the guide polynucleotide or first guide polynucleotide.

[0026]

[0033] In some embodiments, the additional non-target binding region comprises an additional restriction endonuclease recognition sequence for an additional enzyme. In some embodiments, the additional enzyme is the same as or different from the enzyme. In some embodiments, the additional non-target binding region blocks extension of the 3' end of the guide polynucleotide or the first guide polynucleotide. In some embodiments, the single-stranded nucleic acid molecule comprises two or more single-stranded nucleic acid molecules, each single-stranded nucleic acid molecule comprising a different target sequence. In some embodiments, the two or more single-stranded nucleic acid molecules are contained in a single reaction mixture.

[0027]

[0034] In some embodiments, the method of amplifying single-stranded nucleic acid molecules reduces the time to cycle threshold or result value for nucleic acid amplification compared to the time to cycle threshold or result value for nucleic acid amplification of an otherwise identical method of amplifying single-stranded nucleic acid molecules without using a guide complex.

[0028]

[0035] In some embodiments, the method for amplifying single-stranded nucleic acid molecules reduces the time to cycle threshold or result value in nucleic acid amplification compared to the time to cycle threshold or result value in existing nucleic acid amplification methods.

[0029]

[0036] In some embodiments, the existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinant polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA). In some embodiments, the cycle threshold is at most 30.

[0030]

[0037] In another aspect, the disclosure provides a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule having attached thereto a guide complex, the guide complex comprising: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes to a target sequence of the single-stranded nucleic acid molecule; and ii) a second guide polynucleotide that hybridizes to the non-target binding region of the first guide molecule to form a double-stranded binding region, the double-stranded binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme.

[0031]

[0038] In another aspect, the disclosure provides a system for processing a single-stranded nucleic acid molecule comprising a target sequence, the system comprising: a single-stranded nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide, the guide polynucleotide comprising: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme, (ii) a target binding region configured to hybridize to a target sequence, and (iii) a blocked 3' end that is not extendable by a polymerase; and an enzyme bound to the restriction endonuclease recognition sequence of the non-target binding region.

[0032]

[0039] In another aspect, the present disclosure provides kits comprising the guide complexes or guide polynucleotides described herein.

[0040] In some embodiments, the kit further comprises a probe or dye for detecting amplification products produced using the kit.

[0033]

[0041] In some embodiments, the kit further comprises informational material instructing how to use the kit.

[0042] In another aspect, the disclosure provides a system for processing a plurality of single-stranded nucleic acid molecules, each comprising a different target sequence, the system comprising: a first single-stranded nucleic acid molecule bound to a first guide complex comprising a first guide polynucleotide, the first guide polynucleotide comprising: (i) a first non-target binding region comprising a first restriction endonuclease recognition sequence for an enzyme that is a type IIs restriction enzyme; (ii) a first target binding region configured to hybridize to a first target sequence; and (iii) a first blocked 3' end that is not extendable by a polymerase; and a second single-stranded nucleic acid molecule; and (iii) a second single-stranded nucleic acid molecule that is bound to a second guide complex comprising a second guide polynucleotide, the second guide polynucleotide comprising: (i) a second non-target binding region that comprises a second restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme; (ii) a second target binding region that is configured to hybridize to a second target sequence; and (iii) a second blocked 3' end that is not extendable by a polymerase; and the enzyme that is a Type IIs restriction enzyme binds to the first restriction endonuclease recognition sequence of the first non-target binding region or the second restriction endonuclease recognition sequence of the second non-target binding region.

[0034]

[0043] In some embodiments, the system further comprises a third single-stranded nucleic acid molecule bound to a third guide complex comprising a third guide polynucleotide, the third guide polynucleotide comprising: (i) a third non-target binding region comprising a third restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme; (ii) a third target binding region configured to hybridize to a third target sequence; and (iii) a third blocked 3' end that is not extendable by a polymerase; and the enzyme that is a Type IIs restriction enzyme binds to the third restriction endonuclease recognition sequence in the third non-target binding region.

[0035]

[0044] In some embodiments, the system further comprises a fourth single-stranded nucleic acid molecule bound to a fourth guide complex comprising a fourth guide polynucleotide, the fourth guide polynucleotide comprising: (i) a fourth non-target binding region comprising a fourth restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme; (ii) a fourth target binding region configured to hybridize to a fourth target sequence; and (iii) a fourth blocked 3' end that is not extendable by a polymerase; and the enzyme that is a Type IIs restriction enzyme binds to the fourth restriction endonuclease recognition sequence in the fourth non-target binding region.

[0036]

[0045] In some embodiments, the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule are from different samples. In some embodiments, the different samples include samples obtained from bacteria, viruses, humans, or any combination thereof. In some embodiments, the bacteria is selected from the group consisting of Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis. In some embodiments, the virus is selected from the group consisting of double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, single-stranded RNA viruses, positive-sense single-stranded reverse transcriptase viruses, and double-stranded DNA reverse transcriptase viruses.

[0037]

[0046]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0038] Incorporation by Reference

[0047] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification supersedes and / or takes precedence over such conflicting material.

[0039]

[0048] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figures" and "FIG."). [Brief explanation of the drawings]

[0040] [Figure 1-1]

[0049] 1A-1O illustrate exemplary precursor steps leading to an isothermal amplification cycle according to various embodiments described herein. FIG. 1A depicts a duplex oligo complex binding to a target nucleic acid strand. FIG. 1B depicts endonucleolytic activity on the duplex oligo / target complex. FIG. 1C depicts a polymerase extending from the 3' end of the target strand. [Figure 1-2] Figure ID depicts polymerase displacement of the duplexed guide molecule, and Figure IE depicts endonucleolytic activity on the oligo / extension product complex. [Figure 1-3] Figure 1F depicts the polymerase extending from the 3' end of the cleaved oligo and displacement of the guide. Figure 1G depicts endonuclease activity on the newly synthesized portion complementary to the target strand. Figure 1H depicts the polymerase extending from the 3' end of the cleavage site and displacement of the synthesized complement onto the target strand. [Figure 1-4] Figure 1I depicts the displaced complement serving as a new target for a second complementary duplex oligo complex, Figure 1J depicts the polymerase displacing the second complementary duplex guide molecule, and Figure 1K depicts completed extension on the new guide molecule. [Figure 1-5] Figure 1L depicts endonucleolytic activity on the second complementary strand oligo / extension product complex. Figure 1M depicts a polymerase extending from the 3' end of the cleavage site of the second complementary strand of the oligo / extension product complex. Figure 1N depicts endonucleolytic activity on the newly synthesized complementary strand of the second complementary strand guide. [Figure 1-6] FIG. 10 is a diagram depicting a displaced single-stranded synthetic fragment as the starting material for a strand displacement amplification reaction. [Figure 2A]

[0050] Figures 2A-2C show a system for generating products suitable for amplification by an isothermal amplification reaction using a guide molecule with a point mutation in a target sequence. Figure 2A depicts the guide molecule with a point mutation binding to the target DNA and the endonuclease cleaving the target. [Figure 2B] FIG. 2B is a diagram depicting an endonuclease cleaving the extension and guide molecule from the target at the 3′ end. [Figure 2C] Figure 2C depicts the displacement of the guide complementary to the target after endonucleolytic cleavage and the synthesis of a new strand. Because the guide extension on the opposite strand does not extend beyond the end of the guide oligo, only bases originating from the target are identified, not bases present in the guide oligo sequence. [Figure 3A]

[0051] 3A-3B show a control experiment in which there is no mismatch between the guide and primer. Figure 3A depicts the guide oligo, probe, and target sequences used in the control experiment in which there is no mismatch between the guide oligo and the target. [Figure 3B] FIG. 3B depicts the amplification results of a control reaction without a point mutation. [Figure 4A]

[0052] 4A-4B show experiments in which there is an AC mismatch between the guide and primer. Figure 4A depicts the guide oligo, probe, and target sequence used in the mismatch experiment. [Figure 4B] FIG. 4B depicts amplification resulting in a probe signal derived from the target but not from the guide oligo, demonstrating asymmetric endonuclease activity. [Figure 5A]

[0053] 5A-5B show a control experiment in which there was no mismatch between the guide and primer. Figure 5A depicts the guide oligo, probe, and target sequence used in the control experiment. [Figure 5B] FIG. 5B depicts the amplification results of a control reaction without a point mutation. [Figure 6A]

[0054] 6A-6B show experiments with mismatches A to C between the guide and primer. Figure 6A depicts the guide oligo, probe, and target sequences used in the mismatch experiment. [Figure 6B] FIG. 6B depicts amplification resulting in a probe signal derived from the target but not from the guide oligo, demonstrating asymmetric endonuclease activity. [Figure 7A]

[0055] Figures 7A-7D illustrate experiments using intrinsic fluorescence to detect the formation of double-stranded nucleic acids and the use of different guides: Figure 7A depicts a single-stranded DNA (ssDNA) molecule with a 5' quencher and internal fluorescein-T. [Figure 7B] FIG. 7B is a diagram depicting quenched fluorescence when the strands are self-complementary. [Figure 7C] FIG. 7C is a diagram depicting binding of a guide molecule to a target ssDNA. [Figure 7D] FIG. 7D is a diagram depicting the cleavage site that initiates the formation of extension products and fluorescent double-stranded nucleic acids. [Figure 8A]

[0056] Figures 8A-8D show the amplification / primer extension results of different primers using Bst polymerase. 3' extension of the guide molecule is blocked when a 2'O-methyl RNA base or a phosphorylated base is encountered. Figure 8A depicts the results of an amplification / primer extension reaction using only Bst polymerase. [Figure 8B] FIG. 8B depicts the results of an amplification / primer extension reaction using Bst polymerase and endonuclease Nt.BsmAI. [Figure 8C]FIG. 8C depicts the results of an amplification / primer extension reaction using Bst polymerase and endonucleases Nt.BsmAI and N.BstNBI. [Figure 8D] FIG. 8D depicts the results of an amplification / primer extension reaction using Bst polymerase and endonuclease N.BstNBI. [Figure 9A]

[0057] 9A-9D show the results of amplification / primer extension reactions of different primers using Bst polymerase. 3' extension of the guide molecule is blocked when a 2'O-methyl RNA base or a phosphorylated base is encountered. Figure 9A depicts the results of an amplification / primer extension reaction using only Bst polymerase. [Figure 9B] FIG. 9B depicts the results of an amplification / primer extension reaction using Bst polymerase and endonuclease Nt.BsmAI. [Figure 9C] FIG. 9C depicts the results of an amplification / primer extension reaction using Bst polymerase and endonucleases Nt.BsmAI and N.BstNBI. [Figure 9D] FIG. 9D depicts the results of an amplification / primer extension reaction using Bst polymerase and endonuclease N.BstNBI. [Figure 10A]

[0058] 10A-10B show cycle threshold results for loop-mediated isothermal amplification (LAMP). Figure 10A depicts cycle threshold results for LAMP when comparing LAMP with LAMP with DTECT (differential targeted endonuclease cutting technology) priming and DTECT priming alone. [Figure 10B] FIG. 10B is an expanded version of FIG. 10A, showing more clearly the difference in cycle threshold results between LAMP with DTECT priming and DTECT priming. [Figure 11A]

[0059] 11A-11D illustrate experiments using intrinsic fluorescence to detect the formation of double-stranded nucleic acids and the use of different guides: Figure 11A depicts a single-stranded DNA (ssDNA) molecule with a 5' quencher and an internal fluorescein-T. [Figure 11B] FIG. 11B is a diagram depicting quenched fluorescence when the strands are self-complementary. [Figure 11C] FIG. 11C is a diagram depicting a 2′O methyl base on a guide molecule. [Figure 11D] FIG. 11D is a diagram depicting the cleavage site that initiates the formation of extension products and the formation of fluorescent double-stranded nucleic acids. [Figure 12A]

[0060] Figures 12A to 12F show the amplification results of primer guide C (Figure 12A), primer guide D (Figure 12B), primer guide E (Figure 12C), primer guide H (Figure 12D), primer guide F (Figure 12E), and primer guide G (Figure 12F) under different conditions using Bst polymerase, Bst, and Nt.Bsp. [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 12E] Same as above. [Figure 12F] Same as above. [Figure 13A]

[0061] 13A-13B show a comparison of the use of primer guide F, which is unblocked and has a methoxylated block on the guide (FIG. 13A), and the use of primer guide C, which is unblocked and extendable (FIG. 13B). [Figure 13B] Same as above. [Figure 14A]

[0062] Figures 14A-14E show the results of isothermal SDA following production of restriction digested monkeypox target products. Figure 14A depicts a series of 10-fold dilutions using blocked primer guides. [Figure 14B]FIG. 14B depicts a series of 10-fold dilutions at 1×10 6 copies per reaction using blocked primer guides. [Figure 14C] FIG. 14C depicts a summary of the log copy number per reaction of monkeypox primer guides using blocked amplification reactions. [Figure 14D] FIG. 14D depicts monkeypox virus amplification in the NP matrix direct amplification procedure using blocked primer guides. [Figure 14E] FIG. 14E depicts a summary of the data in FIG. 14D. [Figure 15A]

[0063] 15A to 15B show the results of triple isothermal amplification reactions. Figure 15A shows the reaction conditions from a triplex isothermal reaction. Isofast BST is a DNA polymerase, N.BstNBI is a site-specific endonuclease that primarily cleaves only one strand of DNA on double-stranded DNA substrates; AMV rt enz refers to Avian Myeloblastosis Virus reverse transcriptase; dNTP refers to deoxynucleoside triphosphate; NaSO is sodium sulfate; MgSO is magnesium sulfate; Tris is tris(hydroxymethyl)aminomethane, and (NH)SO is ammonium sulfate; NG refers to Neisseria gonorrhoeae; CT refers to Chlamydia trachomatis; RPP stands for ribosomal protection protein; rt stands for reverse transcriptase; Cy5 stands for cyanine-5, HEX stands for hexachlorofluorescein, and Fam stands for fluorescein amidite. [Figure 15B] Figure 15B shows preliminary performance results of the XCEL triple isothermal reaction using N. gonorrhoeae, C. trachomatis, and human RPP30. Approximately 75 IFU per reaction was used for C. trachomatis, and approximately 150 CFU per reaction was used for N. gonorrhoeae. [Figure 16A]

[0064] Figure 16A depicts the results of quadruple isothermal amplification reactions using Trichomonas vaginalis, N. gonorrhoeae, C. trachomatis, and ribonuclease P / MRP subunit p30 (RPP30). M1 purified Trichomonas vaginalis, N. gonorrhoeae, and C. trachomatis had an RNA titer of 250 picograms per reaction of purified human RNA. [Figure 16B] Figure 16B is a diagram showing the reaction conditions from a quadruple isothermal reaction, in which Lucigen BST is a DNA polymerase, and N.BstNBI is a site-specific endonuclease that primarily cleaves only one strand of DNA on double-stranded DNA substrates; dNTP refers to deoxynucleoside triphosphate; NaSO is sodium sulfate; MgSO is magnesium sulfate; Tris is tris(hydroxymethyl)aminomethane, and (NH)SO is ammonium sulfate; NG refers to Neisseria gonorrhoeae; CT refers to Chlamydia trachomatis, and Tv refers to Trichomonas vaginalis; RPP stands for ribosomal protection protein; rt stands for reverse transcriptase; Cy5 stands for cyanine-5, HEX stands for hexachlorofluorescein, and Fam stands for fluorescein amidite. [Figure 17]

[0065] FIG. 1 illustrates a computer system that is programmed or otherwise configured to implement the methods provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0041]

[0066] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed. While the vial caps are depicted in the figures as having a configuration including three void-fill caps filling three vials in a linear arrangement, it will be understood that such description is merely exemplary, as the inventive concepts described herein contemplate various configurations and numbers of void-fill caps.

[0042]

[0067] Whenever the terms "at least," "greater than," or "equivalent to" appear before the first number (or after the last number) of a series of two or more numbers, the terms "at least," "greater than," or "equivalent to" apply to each number in the series. For example, "1, 2, or 3 or more" is equivalent to "1 or more," "2 or more," or "3 or more."

[0043]

[0068] Whenever the terms "at most," "less," or "less than" appear before the first number (or after the last number) of a series of two or more numbers, the terms "at most," "less than," or "less than" apply to each number in the series. For example, "less than or equal to 3, 2, or 1" is equivalent to "less than or equal to 3," "less than or equal to 2," and "less than or equal to 1."

[0044]

[0069] Certain inventive embodiments herein contemplate numerical ranges. When a range is present, it includes the range's endpoints. Furthermore, all subranges and values ​​within a range are as if expressly written out. The term "about" or "approximately" can mean within an acceptable error range for a particular value, which error range depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, in accordance with the practice of those skilled in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within five-fold, or within two-fold of a value. When a specific value is described in the present specification and claims, unless otherwise specified, the term "about" is intended to mean within an acceptable error range for that particular value.

[0045] overview

[0070] The present disclosure provides methods, systems, compositions, and kits for processing target nucleic acid molecules. In some embodiments, the present disclosure provides nucleic acid amplification methods (e.g., isothermal amplification). Such methods may include cycles of steps as depicted in Figures 1A to 1O. The methods provided herein can provide higher amplification efficiency and easier optimization procedures compared to existing amplification methods (e.g., isothermal amplification). The processed target nucleic acid molecules can be used in various amplification reactions, including but not limited to the amplification or processing methods described herein.

[0046]

[0071] Such methods can begin with the formation of a structure such as that depicted in FIG. 1A, in which a guide nucleic acid complex (or guide complex) is formed to guide a restriction enzyme to a predetermined site in a nucleic acid. FIG. 1A depicts a nucleic acid strand (e.g., a single-stranded DNA strand or ssDNA strand) (100) comprising a target nucleic acid sequence (101). In some cases, the ssDNA strand can be generated by reverse transcribing a target RNA sequence. In some cases, the ssDNA strand can be generated by denaturing a double-stranded DNA (dsDNA) sequence. In FIG. 1A, a type IIs restriction enzyme (120) is guided to the vicinity of the target site via the formation of a guide complex. This guide nucleic acid complex is formed via the self-annealing of a single copy of a guide polynucleotide comprising a non-target binding region (117) comprising a restriction endonuclease recognition sequence for the type IIs restriction enzyme, a target binding region (115) configured to hybridize to the target sequence, and a blocked 3' end (116) that cannot be extended by a polymerase. Note in FIG. 1A that self-annealing of two copies of the guide polynucleotide forms a double-stranded palindromic region that allows binding of a type II restriction enzyme near the target site.

[0047]

[0072] Such methods can continue the process depicted in Figures 1B and 1C in a second step. After the type IIs restriction enzyme (120) is guided to the vicinity of the target site (101) by the double-stranded palindromic region (two copies of 117) formed by self-annealing of the guide polynucleotide, the type IIs restriction enzyme can cleave single-stranded positions (130, 135) distal to its binding site, characteristic of its activity (Figure 1B). One of these cleavable single-stranded positions (135) is located on the nucleic acid strand (101) containing the target nucleic acid sequence (101). The other cleavable single-stranded position (130) is located on the guide polynucleotide itself (130). When selective enzyme conditions, an engineered polymerase, or BspD6I are used, cleavage at one of the sites (e.g., a single-stranded site on the nucleic acid strand (101) containing the target nucleic acid sequence (101)) can be advantageous. Cleavage at a single-stranded site on the nucleic acid strand (101) containing the target nucleic acid sequence (101) generates a free 3' hydroxyl that can then be extended by a strand-displacing polymerase present in the reaction.

[0048]

[0073] Such methods can continue in a third step with the process depicted in Figures 1D-1F. Extension of the free 3' hydroxyl by a strand-displacing polymerase (140, Figure 1C) generates a region (160) of nucleic acid strand (101) from the guide polynucleotide that contains the target nucleic acid sequence (101) complementary to the restriction endonuclease recognition sequence (117) for the type IIs restriction enzyme (Figure 1D). The extension of nucleic acid (100) displaces the second copy of the guide polynucleotide (116 / 117, the lower molecule) that previously formed half of the guide complex. The extension of nucleic acid (100) with a region complementary to the restriction endonuclease recognition sequence for the type IIs restriction enzyme (120) forms a new double-stranded structure to which the type IIs restriction enzyme (120) can bind (Figure 1E). Similar to the second step, a type IIs restriction enzyme can cleave single-stranded positions (130, 135) distal to its binding site (Figure 1E). While cleavage at the single-stranded site (135) containing the target nucleic acid site (100) simply allows the strand (100) to be extended again by the polymerase, cleavage at the single-stranded site (130) allows a new procedure to begin (Figure 1E). In particular, cleavage at site 130 in Figure 1E on the annealed guide polynucleotide removes the sequence containing the blocked 3' end (116) and allows the guide polynucleotide to be extended to include a sequence (170) complementary to the strand (100) containing the target nucleic acid site (101) (Figure 1F).

[0049]

[0074] Such a method can continue the process depicted in Figures 1G and 1H in a fourth step. Because the double-stranded structure of Figure 1G no longer contains a blocked 3' end, repeated cleavage at site 130 in Figure 1G liberates a single strand containing sequence (170) complementary to strand (100) containing target nucleic acid site (101), allowing subsequent extension of a new strand (171) in its place. Furthermore, the released strand (170) can further serve as a new template similar to strand 100 in Figure 1A (Figure 1I), allowing strand 170 to be further cleaved and repeatedly extended as in Figure 1H (Figure 1J). Figure 1K depicts an exemplary extension completed on a new guide molecule.

[0050]

[0075] In some cases, the method can continue with endonucleolytic activity occurring on the second complementary strand oligo / extension product complex (170), as seen in Figure 1L. Figure 1M depicts the polymerase (140) extending the 3' end of the cleavage site on the second complementary strand of the oligo / extension product complex. Degradative activity on the newly synthesized strand (130) occurs (Figure 1N), and the displaced single-stranded synthetic fragment (42) in Figure 1O can serve as starting material for further strand displacement amplification reactions.

[0051]

[0076] In some cases, methods according to the present disclosure do not involve amplification and utilize the structure depicted in FIG. 1A to induce cleavage of a single-stranded nucleic acid molecule (100) containing a target site (101) at a specified position (135, FIG. 1B).

[0052] definition

[0077] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0053]

[0078] The practice of some of the methods disclosed herein utilizes, unless otherwise indicated, techniques in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, e.g., Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (eds. F.M.Ausubel et al.); the series Methods in Enzymology (Academic Press), PCR 2: A Practical Approach (eds. M.J. MacPherson, B.D. Hames, and G.R. Taylor (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (ed. R.I. Freshney (2010)), all of which are incorporated herein by reference.

[0054]

[0079] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description and / or claims, such terms are intended to be inclusive in the same manner as the term "comprising."

[0055]

[0080] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within one standard deviation or more than one standard deviation, according to the practice of one of ordinary skill in the art. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0056]

[0081] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. A nucleotide may include a synthetic nucleotide. A nucleotide may include a nucleotide analog. A nucleotide may include a synthetic nucleotide analog. A nucleotide may be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide may include ribonucleoside triphosphates adenosine triphosphate (ATP), uridine triphosphate (UTP), cytosine triphosphate (CTP), guanosine triphosphate (GTP), and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP, or derivatives thereof. Such derivatives may include, for example, [αS]dATP, 7-deaza-dGTP, and 7-deaza-dATP, as well as nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. Synthetic nucleotide analogs may include locked nucleic acids (LNAs), bridged nucleic acids (BNAs), fluorinated nucleic acids (also known as fluorine-modified nucleic acids), and peptide nucleic acids (PNAs). As used herein, the term "locked nucleic acid" ("LNA") typically refers to a nucleic acid analog in which the ribose ring is "locked" with an extra bridge, such as a methylene bridge, connecting the 2'-oxygen atom and the 4'-carbon atom of the nucleotide (see, e.g., International Publication No. 99 / 14226, incorporated herein by reference in its entirety). As used herein, the term "bridged nucleic acid (BNA)" typically refers to a constrained or inaccessible nucleic acid molecule with a bridge structure fixed at the 2'- or 4'-position. As used herein, "fluorinated nucleic acid" typically refers to a nucleic acid incorporating a fluorine atom, often at the 2'- or 4'-position. As used herein, the term "peptide nucleic acid (PNA)" typically refers to a nucleotide analog in which the backbone of the analog, e.g., the sugar backbone of DNA, is a pseudopeptide. The backbone of a PNA may, for example, contain a repeating sequence of N-(2-amino-ethyl)-glycine units. Peptide nucleic acid analogs can react in a given environment in the same way that DNA does, and can further bind to complementary nucleic acid sequences and various proteins.Due to the non-natural backbone, PNAs are insensitive to endonuclease cleavage in situations where endonucleases cleave comparable DNA / RNA sequences, and furthermore, can confer specificity and binding to complementary DNA under various salt conditions. As used herein, the term "nucleotide" may refer to dideoxyribonucleoside triphosphates (ddNTPs) and their derivatives. Exemplary dideoxyribonucleoside triphosphates include, but are not limited to, ddATP, ddCTP, ddGTP, ddITP, and ddTTP. Nucleotides can be unlabeled or detectably labeled, such as with a moiety containing an optically detectable moiety (e.g., a fluorophore). Detectable labels include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

[0057]

[0082] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are typically used interchangeably to refer to a polymer of nucleotides of any length, either single-, double-, or multi-stranded, deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may be DNA. A polynucleotide may be RNA. A polynucleotide may contain one or more nucleotide analogs (including, for example, those with altered backbones, sugars, or nucleobases). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, xeno nucleic acids, morpholinos, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to a sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, wyosine, PNA, and LNA.

[0058]

[0083] As used herein, the terms "restriction endonuclease," "restriction enzyme," or grammatical equivalents refer to enzymes that are typically derived from bacterial host defenses and are understood to recognize specific sequences on invading viral DNA and cleave the DNA either at the recognition sequence or at a distinct sequence site. One group of restriction endonucleases has been identified as Type IIS. This group can recognize asymmetric DNA sequences and cleave the DNA at a cleavage site that is an outer site that is a defined distance from the recognition site. In some cases, Type IIS restriction endonucleases cleave DNA between 1 and 20 nucleotides from the relevant recognition site.

[0059]

[0084] As used herein, the term "restriction endonuclease recognition sequence" generally refers to a location on a nucleic acid molecule (e.g., a DNA molecule) that contains a specific sequence of nucleotides recognized by various restriction enzymes. These sequences range in length from 4-8 base pairs to 12-40 base pairs. These sites may also be palindromic sequences.

[0060]

[0085] As used herein, the term "polymerase" generally refers to an enzyme that uses a nucleic acid as a template strand to produce a complementary copy of a nucleic acid molecule. DNA polymerases bind to the template strand and then translocate it, adding nucleotides to the free hydroxyl group at the 3' end of the growing nucleic acid strand. DNA polymerases synthesize complementary DNA molecules from DNA (e.g., DNA-dependent DNA polymerases) or RNA templates (e.g., RNA-dependent DNA polymerases or reverse transcriptases), while RNA polymerases synthesize RNA molecules from DNA templates (e.g., DNA-dependent RNA polymerases involved in transcription). DNA polymerases typically use a pre-existing short RNA or DNA strand called a primer to initiate strand growth; some DNA polymerases can also utilize any free 3' hydroxyl group within a DNA duplex for extension. Some DNA polymerases replicate a single-stranded template, while others displace the strand upstream of the site where they add a base to the strand.

[0061]

[0086] As used herein, the term "strand displacement," when used with respect to a polymerase, generally refers to the activity of removing a complementary strand from base pairing with the template strand being read by the polymerase. Examples of polymerases with strand displacement activity include the large fragment of Bacillus stearothermophilus polymerase (Bst polymerase), exo-Klenow polymerase, Bst 2.0 polymerase, Bst 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exopolymerase, and OmniTaq 2 LA DNA polymerase.

[0062]

[0087] As used herein, the terms "amplify," "amplifies," "amplified," "amplification," and "amplicon" generally refer to any method for replicating nucleic acids. Replication can be performed using a primer-dependent polymerase. Replication can also be enzymatic amplification. In some cases, amplifying or replicating a target nucleic acid strand also includes replicating or amplifying the complementary strand of the target nucleic acid strand. The amplification product can be subjected to subsequence analysis, including, but not limited to, melting curve analysis, nucleotide sequencing, single-strand conformation polymorphism assay, allele-specific oligonucleotide hybridization, Southern blot analysis, and restriction endonuclease digestion.

[0063]

[0088] As used herein, the terms "hybridizing" and "annealing" generally refer to a reaction in which one or more polynucleotides interact to form a stabilized complex via hydrogen bonding between the bases of nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein binding, or other sequence-sensitive or specific methods. The complex can include two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction can also constitute a step in a more extensive process, such as the initiation of PCR or the enzymatic cleavage of a polynucleotide by a ribozyme. A first sequence that can be stabilized via hydrogen bonding with the bases of nucleotide residues of a second sequence is typically "hybridizable" to the second sequence. In such cases, the second sequence can also be hybridizable to the first sequence.

[0064]

[0089] As used herein, the terms "complement," "complementary," and "complementarity" generally refer to a sequence that is perfectly complementary to and hybridizable with a given sequence. In some cases, a first sequence that is hybridizable with a second sequence or set of sequences is specifically or selectively hybridizable with the second sequence or set of sequences, such that hybridization to the second sequence or set of sequences is used. Hybridizable sequences may share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25% and 100% complementarity, including at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence complementarity.

[0065]

[0090] The isothermal amplification methods described herein can offer advantages over existing nucleic acid amplification methods. Non-limiting examples of isothermal nucleic acid amplification methods can include helicase-dependent amplification, nicking enzyme amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, and nucleic acid sequence-based amplification.

[0066]

[0091] The methods described herein can utilize DNA polymerases with high strand displacement activity and specially designed primer sets to exponentially amplify target sequences. The methods provided herein can provide faster amplification times for target nucleic acid molecules compared to existing nucleic acid amplification methods. Nucleic acid targets processed by the methods described herein (e.g., nicked or cleaved by a guide complex or enzyme) can be used as initial templates for use in any existing isothermal amplification method. Different existing isothermal amplification methods may utilize different DNA polymerases. Loop-mediated isothermal amplification (LAMP) utilizes two pairs of specially designed primers, called inner and outer primers, and can be performed at a constant temperature of 50-65°C (122-149°F). A limitation of the LAMP method is the potential for false positives due to the use of nonspecific detection methods. Helicase-dependent amplification (HDA) utilizes DNA helicase activity to separate complementary strands of double-stranded DNA molecules, thereby avoiding temperature cycling to generate single-stranded templates for primer hybridization and subsequent primer extension by DNA polymerase. Rolling circle amplification (RCA) utilizes sequential amplification of a circular DNA template by a strand-displacing DNA polymerase. RCA functions at a constant temperature (e.g., between 37°C and 42°C, [98.6°F and 107.6°F]) and generates long single-stranded DNA molecules with tandem repeats of the circular template. Limitations of RCA include challenges in large-scale generation, purification, and storage of target molecules. Multiple displacement amplification (MDA) utilizes random exonuclease-resistant primers and φ29 DNA polymerase with strand-displacing activity to generate target DNA strands at a constant temperature, e.g., 30°C (86°F). MDA can also be used for whole genome amplification. Recombinase polymerase amplification (RPA) is a low-temperature (e.g., 37°C [98.6°F]) isothermal amplification method that combines isothermal recombinase-driven primer targeting of target molecules with strand-displacing DNA activity.RPA utilizes a nucleoprotein complex formed by an oligonucleotide primer and a recombinase protein to guide and facilitate binding to the target DNA strand. Nucleic acid sequence-based amplification (NASBA) is an isothermal, transcription-based amplification method designed for the amplification of single-stranded RNA or DNA sequences and is performed at a constant temperature of 41°C (105.8°F).

[0067] Example embodiment

[0092] The present disclosure provides methods and compositions for processing nucleic acid molecules containing a target sequence. In some aspects, the present disclosure provides methods for processing single-stranded nucleic acid molecules containing a target sequence. The method can include contacting a single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions in which the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, the guide polynucleotide comprising: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme (e.g., a restriction enzyme). The restriction enzyme can be a type IIs restriction enzyme. The guide polynucleotide can further comprise (ii) a target binding region configured to hybridize to the target sequence. The guide polynucleotide can further comprise (iii) a blocked 3' end that is not extendable by a polymerase. In some embodiments, the guide polynucleotide further comprises (i), (ii), and (iii) in 5' to 3' order. The non-target binding region can be located at the 5' end of the guide polynucleotide. The target binding region can be located at the 3' end of the guide polynucleotide. In some embodiments, the non-target binding region further comprises a sequence comprising the reverse complement of the restriction endonuclease recognition sequence of the Type IIs restriction enzyme 3' to the restriction endonuclease recognition sequence of the Type IIs restriction enzyme and 5' to the target binding region configured to hybridize to the target sequence. In some embodiments, in (b), cleavage exposes an extendable 3' end of the target sequence. In some embodiments, the method further comprises reverse transcribing the single-stranded nucleic acid molecule from the RNA.

[0068]

[0093] The guide polynucleotides provided herein can be forward guide polynucleotides (e.g., forward guide oligos) configured to process a target nucleic acid molecule in a reaction. The reaction can further include a reverse guide polynucleotide (e.g., reverse guide oligos) configured to process a target nucleic acid molecule or the reverse complement of a target nucleic acid molecule in the reaction.

[0069]

[0094] Conditions for hybridizing a guide polynucleotide to a single-stranded nucleic acid molecule can be determined empirically or calculated based on the chemical composition of the guide polynucleotide. Various tools are available for calculating annealing / hybridization temperatures and conditions from specific sequences of polynucleotides (e.g., http: / / www.oligoevaluator.com / LoginServlet).

[0070]

[0095] The target binding region can be of sufficient length to hybridize to the target site under the conditions desired for the assay (e.g., temperature, pH, ionic strength). In some embodiments, the target binding region is at least about 12 to about 25 nucleotides in length, including 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides in length. In some embodiments, the target binding region is at least about 12 to about 30 nucleotides in length. In some embodiments, the target binding region is at least about 10 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 15 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 10 to about 30 nucleotides in length. In some embodiments, the target binding region is at least about 15 to about 30 nucleotides in length. In some embodiments, the target binding region is at least about 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, 35, 40, 45, 50 or more nucleotides in length, hi some embodiments, the target binding region is up to about 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer nucleotides in length.

[0071]

[0096] The enzymes described herein can include type IIs restriction enzymes, which can include one or more enzymes selected from the group consisting of BsmAI, Nt.BsmAI, transcription activator-like effector nuclease, N.Bst9I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, N.BstNBI, Nt.CviPII, Nb.Mva1269I, Nb.BpulOI, Nt.BpulOI, and any combination thereof. Type IIs restriction enzymes can include type IIs nickases such as N.BstNBI, N.BspD6I, N.Bst9I and Nt.BstNBI, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutI, or any combination thereof. Alternatively, type IIs restriction enzymes can include BfuAI, BsmAI, BsrDI, or BtsIMutI. Additional examples of type IIS restriction enzymes can be found at www.neb.com / tools-and-resources / selection-charts / type-iis-restriction-enzymes, which is incorporated herein by reference.

[0072]

[0097] In some embodiments, the type IIs restriction enzyme comprises an engineered type IIs restriction enzyme that has a nuclease-inactivating mutation in one of its two subunits, creating a nickase from an enzyme that is not naturally a nickase. In some embodiments, the type IIs restriction enzyme comprises an engineered type IIs restriction enzyme that has a mutation in one of its two subunits, creating a different rate of enzymatic activity for cleaving one strand over the opposite strand. In some embodiments, the enzyme comprises two enzymes with different activities or activity rates. In some embodiments, the enzyme can comprise a type IIs restriction enzyme subunit. In some embodiments, the enzyme can comprise a nicking enzyme subunit. In some embodiments, the enzyme can comprise an activity for introducing a cleavage into a target nucleic acid sequence. For example, the enzyme can be N.BspD6I. In some embodiments, the enzyme can comprise an activity for introducing a cleavage on the complementary strand of a target nucleic acid sequence. In some embodiments, the enzyme can comprise an activity for introducing a cleavage on a guide polynucleotide (e.g., a target binding region of a guide polynucleotide). For example, the enzyme can be Nt.BstNBI.

[0073]

[0098] The blocked 3' end can essentially contain any 3' chemical structure that prevents extension of the guide polynucleotide by a DNA polymerase, including, but not limited to, a 3' phosphate, a 3' thiophosphate, a 3'-O-methyl, a PNA, a modified base, a ddNTP, a solid support, or a spacer.

[0074]

[0099] In some cases, the guide polynucleotide may further comprise an additional non-target binding region located at the 3' end of the guide polynucleotide. The additional non-target binding region may comprise an additional site for binding to an enzyme. For example, the additional non-target binding region may comprise an additional restriction endonuclease recognition sequence for binding to a restriction enzyme. The enzyme recruited by the additional non-target binding region may be the same as or different from the enzyme recruited by the non-target binding region located at the 5' end of the guide polynucleotide. The additional non-target binding region may function as a blocker to block extension of the 3' end of the guide polynucleotide.

[0075]

[0100] The method for processing a single-stranded nucleic acid molecule can further include introducing a type IIs restriction enzyme under conditions sufficient for the type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cleave within the target sequence. Temperature optima for particular type IIs restriction enzymes can be found, for example, in the Rebase database (accessible at http: / / rebase.neb.com / rebase / rebase.html).

[0076]

[0101] Methods for processing single-stranded nucleic acid molecules can further include extending the extendable 3' end using a polymerase. In some embodiments, the polymerase is a DNA polymerase. In some embodiments, the polymerase is a DNA-dependent DNA polymerase. In some embodiments, the polymerase comprises a strand-displacing DNA polymerase. In some embodiments, the polymerase comprises a large fragment of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, phi29 DNA polymerase, T7 exopolymerase, OmniTaq 2 LA DNA polymerase, or any combination thereof. Such methods can further include adding other factors, including dNTPs, appropriate buffering agents, and cofactors (e.g., divalent cations), along with sufficient polymerase to add nucleotides to the 3' end. The dNTPs can be natural or unnatural dNTPs. Natural dNTPs can include dATP, dCTP, dGTP, dTTP, and / or dUTP. The unnatural dNTP can be an α-thiol dNTP (e.g., S-dNTP). The S-dNTPs can include dATPαS, dCTPαS, dGTPαS, and / or dTTPαS.

[0077]

[0102] The target sequence processed by the method provided herein can be used for further downstream applications, such as isothermal amplification. In some cases, the reagents for performing amplification can be in the same mixture as the reagents for target processing. In some embodiments, the present disclosure provides a method for amplifying a single-stranded nucleic acid molecule comprising a target sequence, the method comprising: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions in which the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, the guide polynucleotide comprising: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type IIs restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) a blocked 3' end that cannot be extended by a polymerase; (b) introducing a type IIs restriction enzyme under conditions sufficient for the type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cleave within the target sequence to generate an extendable 3' end; and (c) using a polymerase to extend the extendable 3' end of the target sequence. In some embodiments, the guide polynucleotide further comprises (i), (ii), and (iii) in 5' to 3' order. In some embodiments, the non-target binding region further comprises a sequence comprising the reverse complement of the restriction endonuclease recognition sequence of the Type IIs restriction enzyme 3' to the restriction endonuclease recognition sequence of the Type IIs restriction enzyme and 5' to the target binding region configured to hybridize to the target sequence. In some embodiments, the guide polynucleotide is a first guide polynucleotide, and the guide complex comprises a second guide polynucleotide, the second guide polynucleotide comprising (i) a non-target binding region complementary to the non-target binding region of the first guide polynucleotide, and (ii) a target binding region configured to hybridize to the target sequence. In some cases, when the first guide polynucleotide of the guide complex hybridizes to the target polynucleotide sequence, the target binding region of the second guide polynucleotide of the guide complex does not hybridize to the target sequence. In some embodiments, the first guide polynucleotide and the second guide polynucleotide of the guide complex hybridize to form a dimer.In some embodiments, the first guide polynucleotide and the second guide polynucleotide of the guide complex hybridize at a common 5' region. In some embodiments, the first guide polynucleotide and the second guide polynucleotide hybridize via the non-target binding region of the first guide polynucleotide and the second guide polynucleotide, forming a dimer having a double-stranded binding region. In some embodiments, the double-stranded binding region comprises a restriction endonuclease recognition sequence. In some embodiments, a type IIs restriction enzyme binds to the double-stranded binding region of the dimer. The forward guide polynucleotide (or complex) can comprise one or more guide polynucleotides, including the first guide polynucleotide and the second guide polynucleotide described herein. The first guide polynucleotide and the second guide polynucleotide can be homodimers or heterodimers. For example, the non-target binding region at the 5' end of the first guide polynucleotide and the non-target binding region at the 5' end of the second guide polynucleotide may comprise the same sequence (e.g., a palindromic sequence), while the target binding region at the 3' end of the first or second guide polynucleotide may be different. In some embodiments, the target binding region may be configured to hybridize to a target sequence. Alternatively, the target binding regions may be configured to hybridize to different target sequences.

[0078]

[0103] In some cases, the reverse guide polynucleotide (or complex) may be composed of multiple guide polynucleotides, including a first guide polynucleotide and a second guide polynucleotide. In some cases, the reverse guide polynucleotide and the forward guide polynucleotide may contain the same sequence (e.g., a palindromic sequence) at their 5' ends so that the reverse guide polynucleotide and the forward guide polynucleotide can hybridize to form a heterodimer. The target binding region of the forward guide polynucleotide and the target binding region of the reverse guide polynucleotide may contain different sequences.

[0079]

[0104] In some embodiments, the present disclosure provides a method for amplifying a single-stranded nucleic acid molecule containing a target sequence, the method comprising: (a) contacting a guide complex with the single-stranded nucleic acid molecule, the guide complex comprising: (i) a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes to the target sequence of the single-stranded nucleic acid molecule, and (i) a second guide polynucleotide that hybridizes to the non-target binding region of the first guide molecule to form a double-stranded binding region, the double-stranded binding region binding to an enzyme; and (b) cleaving the target sequence using the enzyme to expose an extendable 3' end of the target sequence. In some cases, the extendable 3' end is a 3' hydroxyl group. In some embodiments, when the target molecule is RNA, the method can further comprise reverse transcribing the single-stranded nucleic acid molecule from the RNA before contacting the target molecule with the guide complex. For example, a target RNA molecule can be reverse transcribed using a reverse transcriptase to generate a DNA molecule, which can then be further processed using the methods described herein. The DNA molecule can be a single-stranded DNA molecule (ssDNA). In some cases, a reverse transcription reaction can be used to generate a ssDNA target from an initial RNA target. In some cases, the reverse transcription reaction can include a reverse transcriptase and a reverse transcription primer. The reverse transcriptase can include avian myeloblastosis virus (AMV) reverse transcriptase (RT), Moloney murine leukemia virus RT (M-MLV RT), telomerase RT, or human immunodeficiency virus type 1 RT (HIV-1 RT).

[0080]

[0105] In some cases, the method for amplifying a single-stranded nucleic acid molecule containing a target sequence further includes extending the extendable 3' end of the target sequence with a polymerase to generate an extension product, where the extension product displaces the second guide polynucleotide. In some cases, the polymerase extension generates a double-stranded product that displaces the second guide polynucleotide. In some embodiments, the extension step includes incubation in the presence of a DNA polymerase, such as a strand-displacing DNA polymerase, including any of the strand-displacing polymerases described herein. The extension step can also include incubation in the presence of factors together with the polymerase sufficient to add nucleotides to the 3' end, including dNTPs, an appropriate buffer, and cofactors (e.g., divalent cations). The dNTPs can be natural or non-natural dNTPs. Natural dNTPs can include dATP, dCTP, dGTP, dTTP, and / or dUTP. The non-natural dNTPs can be α-thiol dNTPs (e.g., S-dNTPs). The S-dNTPS may include dATPαS, dCTPαS, dGTPαS, and / or dTTPαS.

[0081]

[0106] In some cases, the method for amplifying a single-stranded nucleic acid molecule comprising a target sequence further comprises cleaving the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide. In some embodiments, the cleaving step comprises introducing a Type IIs restriction enzyme under conditions sufficient for the Type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cleave the first guide polynucleotide within the target binding region. In some embodiments, the extendable 3' end comprises a 3' hydroxyl.

[0082]

[0107] In some cases, the method for amplifying a single-stranded nucleic acid molecule containing a target sequence further includes using a polymerase to extend the extendable 3' end of the first guide polynucleotide to generate a complementary molecule of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule. The polymerase may be a strand-displacing DNA polymerase, including any of the strand-displacing polymerases described herein. The extension step may also include incubation with the polymerase in the presence of sufficient factors, including dNTPs, a suitable buffer, and cofactors (e.g., divalent cations), to add nucleotides to the 3' end. The dNTPs may be natural or non-natural dNTPs. Natural dNTPs may include dATP, dCTP, dGTP, dTTP, and / or dUTP. Non-natural dNTPs may be α-thiol dNTPs (e.g., S-dNTPs). The S-dNTPS may include dATPαS, dCTPαS, dGTPαS, and / or dTTPαS.

[0083]

[0108] In some embodiments, the second guide polynucleotide in a method for amplifying a single-stranded nucleic acid molecule comprising a target sequence comprises, from 5' to 3', (i) a non-target binding region that hybridizes to the non-target binding region of the first guide polynucleotide, and (ii) a target binding region configured to hybridize to the target sequence. In some embodiments, the method further comprises, prior to (b), cleaving the first guide polynucleotide within the target binding region using an enzyme, whereby the guide complex dissociates from the single-stranded nucleic acid molecule. In some embodiments, the method further comprises cleaving the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide, and extending the extendable 3' end of the first guide polynucleotide using a polymerase to repeatedly generate complementary molecules of the target sequence of the single-stranded nucleic acid molecule, generating multiple complementary molecules of the target sequence of the single-stranded nucleic acid molecule. In some embodiments, additional guide complexes bind to the complementary molecules. In some embodiments, the method further includes using the complementary molecule bound by an additional guide complex as an initiating template to generate copies of the target molecule. In some embodiments, the enzyme is a type IIs restriction enzyme. In some embodiments, the type IIs restriction enzyme includes N.BstNBI, N.Bst9I, and N.BspD6I, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutI, BfuAI, BsmAI, BsrDI, BtsIMutI, functional fragments thereof, or combinations thereof. In some embodiments, the guide polynucleotide includes a blocked 3' end that is not extendable by a polymerase. The blocked 3' end can include essentially any 3' chemical structure that prevents extension of the guide polynucleotide by a DNA polymerase, including any structure with such activity described herein. In some embodiments, the blocked 3' end includes a PNA, a modified base, a phosphate group, a ddNTP, a solid support, or a spacer. In some embodiments, the single-stranded nucleic acid molecule with the cut and the guide polynucleotide attached thereto is used as a starting template for amplification.In some embodiments, the amplification is isothermal amplification. In some embodiments, the enzyme comprises an asymmetric tendency to cleave one strand of a DNA duplex. In some embodiments, the enzyme exhibits a high frequency of endonuclease activity. In some embodiments, the high frequency of endonuclease activity is derived from the large subunit of the enzyme. In some embodiments, the enzyme exhibits a low frequency of endonuclease activity. In some embodiments, the low frequency of endonuclease activity is derived from the small subunit of the enzyme. In some embodiments, the enzyme exhibits at least two differential enzyme activity rates. In some embodiments, the at least two differential enzyme activity rates comprise two differential endonuclease activity rates when cleaving two different cleavage sites. In some embodiments, one of the two differential endonuclease activity rates comprises a low frequency of cleaving a target sequence of a single-stranded nucleic acid molecule. In some embodiments, one of the two differential endonuclease activity rates comprises a high frequency of cleaving a target binding region of a guide polynucleotide. In some embodiments, the two differential endonuclease activity rates are asymmetric or unequal, hi some embodiments, the enzymes include N.BstNBI, N.Bst9I and N.BspD6I, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutI, BfuAI, BsmAI, BsrDI, BtsIMutI, or combinations thereof.

[0084]

[0109] In some embodiments, the temperature is varied over the course of the method, in which a first activity rate of the at least two differential enzyme activity rates is favored at a first temperature and a second activity rate of the at least two differential enzyme activity rates is favored at a second temperature different from the first temperature. In some embodiments, the first temperature at which a first rate of enzyme activity is favored can be about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, or about 50°C. In some embodiments, the first temperature at which the first enzyme activity rate is favored is between about 15°C and 50°C, between about 20°C and 45°C, between about 30°C and 45°C, between about 30°C and 40°C, or between about 32°C and 39°C. In some embodiments, the second temperature at which a second rate of enzyme activity is favored can be about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C, about 53°C, about 54°C, about 55°C, about 56°C, about 57°C, about 58°C, about 59°C, about 60°C, about 61°C, about 62°C, about 63°C, about 64°C, about 65°C, about 66°C, about 67°C, about 68°C, about 69°C, about 70°C, about 71°C, about 72°C, about 73°C, about 74°C, about 75°C, about 76°C, about 77°C, about 78°C, about 79°C, or about 80°C. In some embodiments, the second temperature at which the second enzyme activity rate is favored is between about 45°C and 80°C, between about 50°C and 80°C, between about 50°C and 70°C, between about 50°C and 60°C, or between about 52°C and 58°C.

[0085]

[0110] In some embodiments, the temperature can be varied over a period of time throughout the course of the method. The period over which the temperature is varied can be beneficial to the rate of enzyme activity during the reaction. The temperature change can include a first temperature or a second temperature. In some embodiments, the first temperature change or the second temperature change can occur over a duration of at least about 15 seconds, at least about 30 seconds, at least about 1 minute, at least about 1.5 minutes, at least about 2 minutes, at least about 2.5 minutes, at least about 3 minutes, at least about 3.5 minutes, at least about 4 minutes, at least about 4.5 minutes, at least about 5 minutes, at least about 5.5 minutes, at least about 6 minutes, at least about 6.5 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 12 minutes, or at least about 15 minutes. In some embodiments, the first temperature change or the second temperature change may occur for a duration of up to about 15 minutes, up to about 12 minutes, up to about 10 minutes, up to about 9 minutes, up to about 8 minutes, up to about 7 minutes, up to about 6.5 minutes, up to about 6 minutes, up to about 5.5 minutes, up to about 5 minutes, up to about 4.5 minutes, up to about 4 minutes, up to about 3.5 minutes, up to about 3 minutes, up to about 2.5 minutes, up to about 2 minutes, up to about 1.5 minutes, up to about 1 minute, up to about 30 seconds, or up to about 15 seconds.

[0086]

[0111] In some embodiments, the first temperature change or the second temperature change can occur over a period of about 1 minute to about 15 minutes. In some embodiments, the sample can be heated for about 1 minute to about 2 minutes, about 1 minute to about 2.5 minutes, about 1 minute to about 3 minutes, about 1 minute to about 3.5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 1 minute to about 6 minutes, about 1 minute to about 7 minutes, about 1 minute to about 7.5 minutes, about 1 minute to about 10 minutes, about 1 minute to about 15 minutes, about 2 minutes to about 2.5 minutes, about 2 minutes to about 3 minutes, about 2 minutes to about 3.5 minutes, about 2 minutes to about 4 minutes, about 2 minutes to about 5 minutes, about 2 minutes to about 2 minutes. ~approx. 6 minutes, ~approx. 2 minutes to ~approx. 7 minutes, ~approx. 2 minutes to ~approx. 7.5 minutes, ~approx. 2 minutes to ~approx. 10 minutes, ~approx. 2 minutes to ~approx. 15 minutes, ~approx. 2.5 minutes to ~approx. 3 minutes, ~approx. 2.5 minutes to ~approx. 3.5 minutes, ~approx. 2.5 minutes to ~approx. 4 minutes, ~approx. 2.5 minutes to ~approx. 5 minutes, ~approx. 2.5 minutes to ~approx. 6 minutes, ~approx. 2.5 minutes to ~approx. 7 minutes, ~approx. 2.5 minutes to ~approx. 7.5 minutes, ~approx. 2.5 minutes to ~approx. 10 minutes, ~approx. 2.5 minutes to ~approx. 15 minutes, ~approx. 3 minutes to ~approx. 3.5 minutes, ~approx. 3 minutes to ~approx. 4 minutes, ~approx. 3 minutes to ~approx. 5 minutes , about 3 minutes to about 6 minutes, about 3 minutes to about 7 minutes, about 3 minutes to about 7.5 minutes, about 3 minutes to about 10 minutes, about 3 minutes to about 15 minutes, about 3.5 minutes to about 4 minutes, about 3.5 minutes to about 5 minutes, about 3.5 minutes to about 6 minutes, about 3.5 minutes to about 7 minutes, about 3.5 minutes to about 7.5 minutes, about 3.5 minutes to about 10 minutes, about 3.5 minutes to about 15 minutes, about 4 minutes to about 5 minutes, about 4 minutes to about 6 minutes, about 4 minutes to about 7 minutes, about 4 minutes to about 7.5 minutes, about 4 minutes to about 10 minutes It can be heated for about 4 minutes to about 15 minutes, about 5 minutes to about 6 minutes, about 5 minutes to about 7 minutes, about 5 minutes to about 7.5 minutes, about 5 minutes to about 10 minutes, about 5 minutes to about 15 minutes, about 6 minutes to about 7 minutes, about 6 minutes to about 7.5 minutes, about 6 minutes to about 10 minutes, about 6 minutes to about 15 minutes, about 7 minutes to about 7.5 minutes, about 7 minutes to about 10 minutes, about 7 minutes to about 15 minutes, about 7.5 minutes to about 10 minutes, about 7.5 minutes to about 15 minutes, or about 10 minutes to about 15 minutes.

[0087]

[0112] In some embodiments, the enzyme comprises two distinct active sites or endonuclease domains that confer at least two differential enzymatic activities. In some embodiments, the target sequence comprises a recognition site specifically recognized by the enzyme or a first of the at least two differential enzymatic activities of the enzyme for introducing cleavage. In some embodiments, the target binding region of the guide polynucleotide comprises a recognition site specifically recognized by the enzyme or a second of the at least two differential enzymatic activities of the enzyme for introducing cleavage. The target binding region can be of sufficient length to hybridize to the target site under the conditions desired for the assay (e.g., temperature, pH, ionic strength). In some embodiments, the target binding region is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the target binding region is at most about 50, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or fewer nucleotides in length. In some embodiments, the target binding region is at least about 15 to about 25 nucleotides in length, including 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides in length. In some embodiments, the target binding region is at least about 15 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 10 to about 25 nucleotides in length. In some embodiments, the target binding region is at least about 12 to about 25 nucleotides in length.

[0088]

[0113] In some embodiments, the concentration of the guide polynucleotide is at least about 0.1 μM, at least about 1 μM, or about 0.1 μM to about 4 μM. In some embodiments, the concentration of the guide polynucleotide is at least about 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4 μM, or more. In some embodiments, the non-target binding region comprises a palindromic sequence. In some embodiments, the non-target binding region is self-complementary or forms a self-annealing dimer under the reaction conditions. In some embodiments, the non-target binding region is at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides in length. In some embodiments, the non-target binding region is at most about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or fewer nucleotides in length. In some embodiments, the single-stranded nucleic acid molecule is single-stranded deoxyribonucleic acid (ssDNA) or single-stranded ribonucleic acid (ssRNA). In some embodiments, the method further comprises reverse transcribing the single-stranded nucleic acid molecule from RNA. In some embodiments, the target binding region comprises at least one peptide nucleic acid (PNA) residue. In some embodiments, the polymerase has strand displacement activity.

[0089]

[0114] In some embodiments, the methods described herein may result in faster amplification results compared to nucleic acid amplification protocols that do not use programmed restriction enzymes. An indicator of the rate of amplification may be the cycle threshold. The "cycle threshold" may include the number of cycles required for a signal (e.g., a fluorescent signal) to exceed a background threshold level. A lower cycle threshold may indicate a higher amount of target nucleic acid in a sample. In some embodiments, nucleic acid amplification using the methods described herein may result in a lower cycle threshold compared to loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), or other amplification methods known in the art. The cycle threshold of the sample processing methods described herein may be at least about 2%, at least about 5%, at least about 8%, at least about 10%, at least about 12%, at least about 15%, at least about 18%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% lower than the cycle threshold of LAMP. The cycle threshold of the sample processing methods described herein may be up to about 60%, up to about 50%, up to about 40%, up to about 30%, up to about 25%, up to about 20%, up to about 18%, up to about 15%, up to about 12%, up to about 10%, up to about 8%, up to about 5%, or up to about 2% lower than the cycle threshold of LAMP. The cycle threshold of the sample processing methods described herein may be about 1% to about 50% lower than the cycle threshold of LAMP.The cycle threshold of the sample processing method described herein is about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 8%, about 1% to about 10%, about 1% to about 12%, about 1% to about 15%, about 1% to about 20%, about 1% to about 25%, about 1% to about 50%, about 2% to about 3%, about 2% to about 4%, about 2% to about 5%, about 2% to about 8%, about 2% to about 10%, about 2% to about 12%, about 2% to about 15%, about 2% to about 20%, about 2% to about 25%, about 2% to about 50%, about 3% to about 4%, about 3% to about 5%, about 3% to about 8%, about 3% to about 10%, about 3% to about 12%, about 3% to about 15%, about 3% to about 20%, about 3% to about 25%, about 3% to about 50%, about 4% to about 5%, about 4% to about 8%, about 4% to about 10%, About 4% to about 12%, about 4% to about 15%, about 4% to about 20%, about 4% to about 25%, about 4% to about 50%, about 5% to about 8%, about 5% to about 10%, about 5% to about 12%, about 5% to about 15%, about 5% to about 20%, about 5% to about 25%, about 5% to about 50%, about 8% to about 10%, about 8% to about 12%, about 8% to about 15%, about 8% to about 20%, about 8% to about 25%, about 8% to about 50% , about 10% to about 12%, about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 50%, about 12% to about 15%, about 12% to about 20%, about 12% to about 25%, about 12% to about 50%, about 15% to about 20%, about 15% to about 25%, about 15% to about 50%, about 20% to about 25%, about 20% to about 50%, or about 25% to about 50% lower.

[0090]

[0115] In some embodiments, the cycle threshold of the sample processing methods described herein can be at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 15, at least about 18, at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40. In some embodiments, the cycle threshold of the sample processing methods described herein can be up to about 40, up to about 35, up to about 30, up to about 25, up to about 20, up to about 18, up to about 15, up to about 12, up to about 10, up to about 9, up to about 8, up to about 7, up to about 6, up to about 5, up to about 4, up to about 3, up to about 2, or up to about 1.

[0091]

[0116] In some embodiments, the disclosure provides a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule having attached thereto a guide complex, the guide complex comprising: a first guide polynucleotide comprising, from 5' to 3', a non-target binding region and a target binding region that hybridizes to a target sequence of the single-stranded nucleic acid molecule; and a second guide polynucleotide that hybridizes to the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme.

[0092]

[0117] In some embodiments, the disclosure provides a system for processing a single-stranded nucleic acid molecule comprising a target sequence, the system comprising: a single-stranded nucleic acid molecule having bound thereto a guide complex comprising a guide polynucleotide, the guide polynucleotide comprising: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) a blocked 3' end that is not extendable by a polymerase; and an enzyme bound to the restriction endonuclease recognition sequence of the non-target binding region.

[0093]

[0118] The methods, systems, or kits provided herein can be used to process or analyze one sample or one target nucleic acid molecule or target sequence. Alternatively, the methods, systems, or kits provided herein can be used to process or analyze two or more different samples or two or more different target nucleic acid molecules or target sequences in the same reaction mixture (e.g., a single reaction). For example, the methods, systems, or kits provided herein can be used to process or analyze 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different target nucleic acid sequences in the same reaction mixture. In some embodiments, the reaction mixture is lyophilized. In some embodiments, the reaction mixture is not lyophilized.

[0094]

[0119] In various embodiments, the guide polynucleotide comprises a target binding region. The sequence of the target binding region can be designed according to the target sequence, following similar rules for primer design. For example, primer design can be based on various parameters, including primer melting temperature (which can be calculated using the nearest neighbor algorithm described in John Santa Lucia, Jr., "A unified view of polymers, dumbbells, and oligonucleotide DNA nearest-neighbor thermal dynamics," Proc. Natl. Acad. Sci. USA, vol. 95, pp. 1460-1465 (1998) (the entire contents of which are incorporated herein by reference)), primer composition (e.g., nucleotide composition, such as GC content, can be determined and filtered using software, and penalized, as can the GC content of hairpins and the composition of the 3' end of the primer. Specific parameters that can be evaluated are homopolymer nucleotide length, hairpin formation, GC content, and amplicon size), predicted dimer-dimer formation, average extension length, etc. For multiplex reactions using two or more target sequences, the target binding regions (or primers) can be designed to minimize cross-reactivity. The non-target binding regions of the guide polynucleotide can be designed not to hybridize with the target sequences and contain sequences that can be recognized by enzymes (e.g., restriction enzymes) described herein.

[0095]

[0120] In some embodiments, the present disclosure provides a method or system for multiplexing the processing of multiple nucleic acid molecules, each nucleic acid molecule comprising a different target sequence. The method or system can include, for each nucleic acid molecule comprising a different target sequence, a guide complex comprising a guide polynucleotide bound thereto. The guide polynucleotide can include (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type IIs restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) a blocked 3' end that cannot be extended by a polymerase. The enzyme can bind to the restriction endonuclease recognition sequence in the non-target binding region. In some embodiments, the multiplexing of one or more nucleic acid molecules includes using two or more different sets of primers or guide complexes, each targeting a different target. In some embodiments, the multiplexing of one or more nucleic acid molecules includes a reaction mixture comprising two or more different detection probes or fluorophores, each targeting a different target sequence. Each of the two or more different detection probes can be linked to a different fluorophore for multiplexed detection.

[0096]

[0121] Amplification products can be detected in a variety of ways. Amplification products may be detected by gel electrophoresis, thereby detecting reaction products of specific lengths. Nucleotides may be labeled, for example, with biotin. Biotin-labeled amplified sequences may be captured using avidin conjugated to a signal-generating enzyme, such as peroxidase. Nucleic acid detection methods may employ the use of dyes that specifically stain double-stranded DNA. Intercalating dyes that exhibit enhanced fluorescence upon binding to DNA or RNA may also be used. Dyes may be, for example, DNA or RNA intercalating fluorophores; examples include, but are not limited to, acridine orange, ethidium bromide, Hoechst dye, PicoGreen, propidium iodide, SYBRI (asymmetric cyanine dye), SYBR II, TOTO (thiazole orange dimer), and YOYO (oxazole yellow dimer). Dyes can provide opportunities for improving the sensitivity of nucleic acid detection by using them in combination with various detection methods, and optimal usage parameters may vary. Nucleic acid detection methods can also employ the use of labeled nucleotides incorporated directly into target sequences or into probes containing sequences complementary or substantially complementary to the target of interest. Such labels may be radioactive and / or fluorescent in nature. Labeled nucleotides that can be detected but otherwise function as natural nucleotides can be distinguished from modified nucleotides that do not function as natural nucleotides. The production or presence of target nucleic acids and nucleic acid sequences can be detected and monitored by molecular beacons.

[0097] The production or presence of target nucleic acids and nucleic acid sequences can also be detected and monitored by fluorescence resonance energy transfer (FRET).

[0098]

[0122] A wide range of fluorophores and / or dyes can be used in the methods described herein in accordance with the present disclosure. Available fluorophores include coumarin, fluorescein, tetrachlorofluorescein, hexachlorofluorescein, Lucifer Yellow, rhodamine, BODIPY, tetramethylrhodamine, Cy3, Cy5, Cy7, eosin, Texas Red, SYBR Green I, SYBR Gold, 5-FAM (also known as 5-carboxyfluorescein; spiro(isobenzofuran-1(3H),9'-(9H)xanthene)-5-carboxylic acid, 3',6'-dihydroxy-3-oxo-6-carboxyfluorescein); 5-hexachloro-fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloyl-fluoresceinyl )-6-carboxylic acid]); 6-hexachloro-fluorescein ([4,7,2',4',5',7'-hexachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 5-tetrachloro-fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-5-carboxylic acid]); 6-tetrachloro-fluorescein ([4,7,2' ,7'-Tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6-carboxylic acid]; 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium, 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethylamino); 6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid); DABCYL (4-((4-(dimethylamino)phenyl)azo)benzoic acid); Cy5 (indodicarbocyanine-5); Cy3 (indodicarbocyanine-3); BODIPY FL (2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid); Quasar-670 (Bioresearch Technologies);Examples of suitable quenchers include CalOrange (Bioresearch Technologies); and Rox and their suitable derivatives. Combination fluorophores, such as fluorescein-rhodamine dimers, may also be suitable. Fluorophores can be selected to absorb and emit in the visible spectrum or outside the visible spectrum, such as in the ultraviolet or infrared regions. Suitable quenchers include DABCYL and its variants, such as DABSYL, DABMI, and Methyl Red. Fluorophores can also be used as quenchers because they tend to quench the fluorescence of other fluorophores when exposed to them. In some cases, the quencher is a chromophore, such as DABCYL or malachite green, or a fluorophore that does not fluoresce within the detection range when the probe is in the open conformation.

[0099]

[0123] In some embodiments, a plurality of single-stranded nucleic acid molecules, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more, can be processed in the same reaction, in some embodiments, each of the plurality of multiplexed nucleic acid molecules is derived from a different sample.

[0100]

[0124] The sample described herein may include a biological sample. The sample may include single-stranded nucleic acid molecules. Alternatively, the sample may include double-stranded nucleic acid molecules.

[0125] The sample can include a fluid sample, non-limiting examples of which include blood, plasma, urine, feces, saliva, sweat, tears, pericardial fluid, peritoneal fluid, pleural fluid, cerebrospinal fluid, gastric fluid, respiratory secretions, semen, synovial fluid, or amniotic fluid.

[0101]

[0126] In some embodiments, the sample comprises a blood sample, a swab sample, a saliva sample, a urine sample, a cerebrospinal fluid sample, a pleural fluid sample, a rectal sample, a vaginal sample, a stool sample, a sputum sample, and / or a lymphatic sample for nucleic acid amplification. In some embodiments, the swab sample comprises a vaginal swab, an oral swab, and / or a rectal swab. In some embodiments, the sample is a solid sample. In some embodiments, the sample is a liquid sample. In some embodiments, the sample is obtained from a subject. In some embodiments, the subject has a disease, condition, or infection. In some embodiments, the sample comprises a purified sample. In some embodiments, the sample is a combination of two, three, four, five, or more samples. In some embodiments, the sample comprises one, two, three, four, five, six, seven, eight, nine, ten, or more target nucleic acid molecules.

[0102]

[0127] The sample may be obtained invasively (e.g., tissue biopsy) or non-invasively (e.g., venipuncture). The sample may be an environmental sample. The sample may be a water sample (e.g., a water sample obtained from a lake, stream, river, estuary, bay, or ocean). The sample may be a soil sample. The sample may be a tissue or liquid sample from a subject, such as saliva, semen, blood (e.g., whole blood), serum, synovial fluid, tears, urine, or plasma. The sample may be a tissue sample, such as a skin sample or a tumor sample. The sample may be obtained from part of an organ of a subject. The sample may be a cell sample. The sample may be an acellular sample (e.g., a plasma sample containing acellular analytes or nucleic acids). The sample may be a solid sample or a liquid sample. The sample may be a biological sample or a non-biological sample. The sample may include an in vitro sample or an ex vivo sample. Non-limiting examples of samples include amniotic fluid, bile, bacterial samples, breast milk, buffy coat, cells, cerebrospinal fluid, chromatin DNA, ejaculate, nucleic acid, plant material, RNA, saliva, semen, blood, serum, soil, synovial fluid, tears, tissue, urine, water, whole blood, or plasma, and / or any combination and / or fraction thereof. In one example, the sample may be a plasma sample, which may contain DNA. In another example, the sample may include a cell sample, which may contain cell-free DNA.

[0103]

[0128] The sample may be a mammalian sample. For example, the sample may be a human sample. Alternatively, the sample may be a non-human animal sample. Non-limiting examples of non-human samples include cat samples, dog samples, goat samples, guinea pig samples, hamster samples, mouse samples, pig samples, non-human primate samples (e.g., gorilla samples, ape samples, orangutan samples, lemur samples, or baboon samples), rat samples, sheep samples, cow samples, and zebrafish samples.

[0104]

[0129] A sample may contain nucleic acids (e.g., circulating DNA fragments and / or cell-free DNA fragments). Nucleic acids may be derived from eukaryotic cells, prokaryotic cells, or non-cellular sources (e.g., virus particles). Nucleic acids may refer to molecules consisting of many nucleotides linked together in a long chain. Non-limiting examples of nucleic acids include artificial nucleic acid analogs (e.g., peptide nucleic acids, morpholino oligomers, locked nucleic acids, glycol nucleic acids, or threose nucleic acids), chromatin, niRNA, cDNA, DNA, single-stranded DNA, double-stranded DNA, genomic DNA, plasmid DNA, or RNA. Nucleic acids may be double-stranded or single-stranded. A sample may contain nucleic acids that may be intracellular. Alternatively, a sample may contain nucleic acids that may be extracellular (e.g., cell-free). A sample may contain nucleic acids (e.g., chromatin) that may be fragmented.

[0105]

[0130] The sample may be obtained from a virus, a bacterium, an archaea, or a eukaryote. In some embodiments, the sample is obtained from a bacterium. The bacterium may be spherical, rod-shaped, spiral, comma-shaped, or cork-shaped. Non-limiting examples of bacteria include Streptococcus pneumoniae, Streptococcus pyogenes, Legionella pneumoniae, Bordetella bronchiseptica, Enterobacter aerogenes, Pasteurella multocida, Proteus mirabilis, Staphylococcus aureus, Haemophilus influenzae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Trichomonas vaginalis, Examples of suitable viruses include Neisseria gonorrhoeae, Chlamydia pneumoniae, and Chlamydia trachomatis. In some embodiments, the sample is obtained from a virus. The virus can be a double-stranded DNA virus, a single-stranded DNA virus, a double-stranded RNA virus, a single-stranded RNA virus, a positive-sense single-stranded reverse transcriptase virus, or a double-stranded DNA reverse transcriptase virus. In some cases, the sample contains a human gene such as RPP30. In some embodiments, sample preparation can include extracting nucleic acid from the sample. In some embodiments, sample preparation can include extracting nucleic acid from the sample by heating the sample. For example, target nucleic acid (e.g., target RNA, target DNA) can be extracted or released from the biological sample during the heating stage of nucleic acid amplification.Alternatively, or in addition to heating, target nucleic acids (e.g., target RNA, target DNA) can be extracted or released from biological samples using a cartridge system that can mix the sample with a lysis buffer and then aspirate through a filter, thereby capturing the target nucleic acid in the filter. In some cases, the cartridge system can also include a wash step to remove contaminants. An elution buffer can be added to the cartridge to remove the target nucleic acid from the filter for further processing or analysis. The cartridge system can be an automated cartridge system. In some cases, the cartridge system can be the M1 Sample Prep® Cartridge Kit (SKU: 3000536, Biomeme, Inc.). In some cases, the sample preparation methods described herein can use the cartridge system for automated sample processing. Details of sample preparation cartridges and related methods are described in U.S. Application No. 16 / 817,733, the entire contents of which are incorporated herein by reference. It should be understood that the samples described herein can be processed by a variety of other methods or commercially available nucleic acid extraction kits or methods.

[0106]

[0131] In some aspects, the present disclosure provides kits comprising any of the guide complexes or guide polynucleotides described herein. In some embodiments, the kits further comprise a probe or dye for detecting amplification products generated using the kit. In some embodiments, the kits further comprise informational material describing how to use the kit. In some embodiments, the information includes optimal reaction temperatures or optimal buffer conditions for amplification using the guide complexes or guide polynucleotides. In some embodiments, the kits further comprise a type II restriction enzyme compatible with the guide polynucleotides or guide complexes described herein. In some embodiments, the kits further comprise a strand-displacing polymerase. The kits may be compartmentalized for ease of use and may include one or more containers with reagents. In some embodiments, all components of the kit are packaged together. Alternatively, one or more individual components of the kit may be provided in a separate package from the other kit components.

[0107] Computer Systems

[0132] The present disclosure provides a computer system programmed to perform the methods of the present disclosure. Figure 17 shows a computer system 1701 that can be programmed or otherwise configured to analyze polynucleotide-polypeptide complexes. Alternatively or additionally, the computer system 1701 can be programmed or otherwise configured to analyze single-stranded nucleic acid molecule processing data. The computer system 1701 can be a user's electronic device or a computer system located remotely relative to the electronic device. The electronic device can be a mobile electronic device.

[0108]

[0133] The computer system 1701 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1705, which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1701 also includes memory or memory locations 1710 (e.g., random access memory, read-only memory, flash memory), electronic storage 1715 (e.g., a hard disk), a communication interface 1720 (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices 1725, such as cache, other memory, data storage, and / or electronic display adapters. The memory 1710, storage 1715, interface 1720, and peripheral devices 1725 communicate with the CPU 1705 via a communication bus (solid lines), such as a motherboard. The storage 1715 may be a data storage device (or data repository) for storing data. The computer system 1701 may be operably coupled to a computer network ("network") 1730 with the aid of the communication interface 1720. Network 1730 may be the Internet, an Internet and / or extranet, or an intranet and / or extranet in communication with the Internet. Network 1730 may, in some cases, be a telecommunications network and / or a data network. Network 1730 may include one or more computer servers that enable distributed computing, such as cloud computing. Network 1730 may, in some cases, implement a peer-to-peer network with the help of computer system 1701, thereby enabling devices coupled to computer system 1701 to operate as clients or servers.

[0109]

[0134] CPU 1705 can execute sequences of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as memory 1710. The instructions may instruct CPU 1705, which may then program or otherwise configure CPU 1705 to implement the methods of the present disclosure. Examples of operations performed by CPU 1705 may include fetch, decode, execute, and writeback.

[0110]

[0135] The CPU 1705 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1701 may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0111]

[0136] Storage device 1715 may store files such as drivers, libraries, and saved programs. Storage device 1715 may store user data, such as user preferences and user programs. In some cases, computer system 1701 may include one or more additional data storage devices external to computer system 1701, such as located on remote servers in communication with computer system 1701 via an intranet or the Internet.

[0112]

[0137] Computer system 1701 can communicate with one or more remote computer systems via network 1730. For example, computer system 1701 can communicate with a user's remote computer system. Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad®, a Samsung® Galaxy Tab), a telephone, a smartphone (e.g., an Apple® iPhone®, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access computer system 1701 via network 1730.

[0113]

[0138] The methods described herein may be implemented by machine (e.g., a computer processor) executable code stored in an electronic storage location of computer system 1701, such as memory 1710 or electronic storage 1715. The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by processor 1705. In some cases, the code may be retrieved from storage 1715 and stored in memory 1710 for ready access by processor 1705. In some cases, electronic storage 1715 may not be used and machine-executable instructions may be stored in memory 1710.

[0114]

[0139] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during run-time. The code may be supplied in a programming language that can be selected so that the code can be executed pre-compiled or as-compiled.

[0115]

[0140] Aspects of the systems and methods provided herein, such as computer system 1701, can be embodied in programming. Various aspects of the present technology can be thought of as a "product" or "article of manufacture," typically in the form of machine (or processor) executable code and / or associated data carried or embodied on a type of machine-readable medium. The machine-executable code can be stored in electronic storage, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. A "storage" type medium can include any or all of the tangible memory of a computer, processor, etc., or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or portions of the software may be communicated over the Internet or various other telecommunications networks. Such communication enables loading of the software from one computer or processor to another, such as, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may propagate software elements include optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, via wired and optical fixed line networks, various air links, etc. Physical elements that transmit such waves, such as wired or wireless links, optical links, etc., may also be considered media that propagate software. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0116]

[0141] Thus, a machine-readable medium such as a computer-executable code may take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices of any computer(s), such as may be used to implement the databases, etc., shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched card paper tape, any other physical storage media with a pattern of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves transmitting data or instructions, cables or links transmitting such carrier waves, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer readable media may be involved in transmitting one or more sequences of one or more instructions to a processor for execution.

[0117]

[0142] The computer system 1701 can include or be in communication with an electronic display 1735 that constitutes a user interface (UI) 1740 for providing, for example, analysis of single-stranded nucleic acid molecule processing data. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0118]

[0143] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented by software when executed by the central processing unit 1705. The algorithms can, for example, analyze single-stranded nucleic acid molecule processing data. [Example]

[0119] Example 1: Programmed Restriction Enzyme (PRE) Composition

[0144] In this experiment, as shown in Figures 1A-1J, a guide molecule induces strand cleavage at specific, programmed sites on a single-stranded nucleic acid sequence. The PRE compositions or methods provided herein may be referred to as DTECT, and the two terms may be used interchangeably herein. In Figure 1A, a double-stranded oligo (110) is formed from two individual oligos (115) comprising a guide molecule PNA sequence (116) and a guide molecule nucleic acid sequence (117). The guide molecule PNA sequence (116) is located at the 3' end of the oligo (115). The guide molecule PNA sequence (116) has a blocking moiety at its 3' end. The guide molecule nucleic acid sequence (117) is located at the 5' end of the oligo (115). The guide molecule nucleic acid sequence (117) is self-complementary in a non-target complementary region (e.g., a non-target binding region). The double-stranded oligos (110) form complexes with a restriction endonuclease (120) at selected sites on the guide molecule nucleic acid sequence (117) of each oligo (115). The double-stranded oligo-restriction endonuclease complex binds to the target single-stranded nucleic acid sequence (100) at the target region (101).

[0120]

[0145] Figure 1B shows the cleavage sites of the high-frequency endonuclease (130) and the low-frequency endonuclease (135). Upon cleavage by the high-frequency endonuclease, the double-stranded oligo-restriction endonuclease complex dissociates from the target. Upon cleavage by the low-frequency endonuclease, the 3' end of the target strand becomes open and available for extension. Figure 1C shows the polymerase (140) extending from the 3' end made available by the low-frequency endonuclease. Figure 1D shows the polymerase (140) dissociating after completion of the synthetic strand (160), which displaces one of the oligos (115) from the double-stranded oligo (110).

[0121]

[0146] Figure 1E again shows the cleavage sites of the high-frequency endonuclease (130) and the low-frequency endonuclease (135). At this stage, cleavage by the low-frequency endonuclease regenerates the structure. However, cleavage by the high-frequency endonuclease creates an open, extendable 3' end on the oligo strand. Figure 1F shows the polymerase (140) extending from the 3' end made available by the high-frequency endonuclease. The polymerase (140) displaces the guide molecule PNA sequence (116) to create the target synthetic strand (170).

[0122]

[0147] Figure 1G shows the cleavage site of the frequent endonuclease (130), and Figure 1H shows that the frequent endonuclease cleaves on the target strand, leaving an open, extendible 3' end to which a polymerase (140) binds and extends to create another target synthetic strand (171), displacing the previous target synthetic strand (170).

[0123]

[0148] Figures 1I and 1J show that the target synthesized strand (170) that was the complement of the target region (101) of the target single-stranded nucleic acid sequence (100) served as a new target for forming an additional synthesized strand (172) representing a copy of the target single-stranded nucleic acid sequence (100). The synthesized strand that was a copy of the target single-stranded nucleic acid sequence (100) was the starting material for strand displacement amplification.

[0124]

[0149] FIG. 1K depicts an example of a completed extension on a new guide molecule.

[0150] In some experiments, the process continued as shown in Figure 1L, with endonucleolytic activity occurring on the second complementary strand oligo / extension product complex (170). Figure 1M depicts the polymerase (140) extending the 3' end of the cleavage site on the second complementary strand of the oligo / extension product complex. Degradative activity on the newly synthesized strand (42) occurred (Figure 1N), and the displaced single-stranded synthetic fragment (42) in Figure 1O served as starting material for an additional strand displacement amplification reaction.

[0125] Example 2: Asymmetrically programmed restriction enzyme 1

[0151] In this experiment, the guide molecule was designed with a single point mutation to enable it to bind to the target DNA. Using the method of Example 1, an additional strand was generated that did not contain the guide point mutation, but instead maintained the product (other than the originally synthesized strand) with the correct complementary sequence of the target.

[0126]

[0152] In Figure 2A, a guide / adapter duplex oligo containing a single mismatch (C to T) complexes with a target (template) single-stranded DNA. The low-frequency endonuclease cleaves the target DNA, digesting within the target region of the template region and creating a new 3'-end extension of the template (Figure 2B). Site activity of the high-frequency endonuclease results in cleavage, displacement, and synthesis of a new strand of the guide molecule (Figure 2C). This first strand contained thymine, but all subsequently synthesized sequences contained cytosines, matching the complement of the original template region of the target strand.

[0127]

[0153] In this experiment, low frequency endonuclease activity was the key step that allowed the generation of product to feed into the strand displacement reaction.

[0154] Molecular beacon single nucleotide polymorphism (SNP) analysis was performed to distinguish between primer extension (using Cy5 fluorescent dye; black triangles) and guide oligo exonuclease activity (using FAM fluorescent dye; gray circles).

[0128]

[0155] In the first control experiment, the guide and primers contained no mismatches. As a result, amplification increased the fluorescence of probes containing the same sequence as the DNA target that bound to the amplified complementary target (Figure 3B). However, when mismatches were introduced into the guide sequence, the increase in fluorescence was due to the probe containing the same sequence as the DNA target, as opposed to the probe containing the complement of the guide sequence (Figure 4B). The sequences used in control experiment 1 and mismatch experiment 1 are listed in Table 1, Figures 3A and 4A.

[0129]

[0156] In a second control experiment, the guide and primer contained no mismatches. As a result, amplification increased the fluorescence of probes containing the same sequence as the DNA target that bound to the amplified complementary target (Figure 5B). However, when mismatches were introduced into the guide sequence, the increase in fluorescence was due to the probe containing the same sequence as the DNA target, as opposed to the probe containing the complement of the guide sequence (Figure 6B). The sequences used in control experiment 2 and mismatch experiment 2 are listed in Table 1, Figures 5A and 6A. This example demonstrated that the oligonucleotide functions as a guide for endonucleolytic activity, as opposed to a primer. Furthermore, this example demonstrated that endonucleolytic activity occurs on both strands of the hybridized oligonucleotide in the complex.

[0130] [Table 1-1]

[0131] [Table 1-2]

[0132] Example 3: Asymmetrically programmed restriction enzyme 2

[0157] In this experiment, a detection molecule containing an internal fluorophore-quencher pair is used as the target. As seen in Figures 7A-7B, the target molecule is self-complementary and self-quenching when unpaired. However, the molecule becomes fluorescent when double-stranded. Using the method described in Example 1, different end guides are tested for extension with Bst polymerase. The different end guides shown in Table 2 each contain a target-noncomplementary endonuclease recognition site.

[0133]

[0158] Figures 7C-7D show the extension of the guide molecule and endonuclease recognition site. The N.BstNBI endonuclease exhibited a temperature optimum at approximately 55°C, while the Nt.BsmAI endonuclease exhibited a temperature optimum at approximately 37°C. Test conditions included favoring Bst polymerase (Figure 8A), favoring Bst polymerase with temperature for Nt.BsmAI (Figure 8B), favoring Bst polymerase with temperature for N.BstNBI (Figure 8D), and equally favoring Bst polymerase with N.BstNBI and Nt.BsmAI (Figure 8C). In reaction conditions favoring Nt.BsmAI activity before N.BstNBI activity, resulting in two stages of asymmetric enzyme activity, the thermocycler protocol was 15 cycles (3.5 min) at 40°C (favoring Nt.BsmAI), followed by 160 cycles at a temperature of 58°C (favoring N.BstNBI).

[0134]

[0159] In reactions where N.BstNBI performed the primary cleavage, the oligos were dropped off at the reaction temperature because there was only a four-base overlap. In reactions where Nt.BsmAI performed the primary cleavage, Bst polymerase used the guide molecule as a target to activate the cleavage activity of N.BstNBI. As a result, Fam fluorescence increased due to extension from the cleavage guide and opening of the probe. In reactions where both N.BstNBI and Nt.BsmAI cleaved, the oligos were dispersed because there was only a one-base overlap.

[0135]

[0160] Table 3 summarizes the results shown in Figures 8A–8D. Bst polymerase can extend from the 3' end of DNA bases but is blocked from extending through 2'-O-methyl RNA bases or phosphorylated bases. Nt.BsmAI did not affect Bst extension. In a system using both Nt.BsmAI and N.BstNBI, the two enzymes were shown to work together to increase the reaction rate. In this two-enzyme system, temperature control was performed over time to maximize enzyme activity, resulting in asymmetric cleavage of the target, generating defined / designed oligonucleotides that can be used in subsequent amplification reactions (e.g., SDA). In this two-enzyme system, target cleavage by Nt.BsmAI is the rate-limiting step. N.BstNBI appears to behave like a two-enzyme asymmetric restriction enzyme system; this can be understood as the small subunit of N.BstNBI acting as a less active restriction endonuclease and the large subunit acting as a more active restriction endonuclease.

[0136] [Table 2]

[0137] [Table 3]

[0138] Example 4: Asymmetrically programmed restriction enzyme 3

[0161] In this experiment, the method of Example 3 is used, using different guides. The different terminal guides are shown in Table 4. Test conditions included favoring Bst polymerase (Figure 9A), favoring Bst polymerase with temperature of Nt.BsmAI (Figure 9B), favoring Bst polymerase with temperature of N.BstNBI (Figure 9D), and favoring Bst polymerase equally with N.BstNBI and Nt.BsmAI (Figure 9C).

[0139]

[0162] Table 5 summarizes the results shown in Figures 9A-9D. Bst polymerase can extend from the 3' end of DNA bases, but extension from 2'O-methyl RNA bases or phosphorylated bases is inhibited. Nt.BsmAI did not affect Bst extension. In systems using both Nt.BsmAI and N.BstNBI, the two enzymes worked in tandem to overcome the 3' block. The N.BstNBI system showed delayed release of the extension block.

[0140] [Table 4]

[0141] [Table 5]

[0142] Example 5: LAMP enhancement by PRE priming

[0163] In this experiment, we compared LAMP (Loop-Mediated Isothermal Amplification), PRE (also known as DTECT), and LAMP combined with PRE priming. PRE was performed as described in Example 1. Results show that PRE-enhanced LAMP has a lower cycle threshold than PRE alone or LAMP alone (Figures 10A-10B). For PRE-enhanced amplification, approximately 100 copies of hRNA were used per reaction. Table 6 shows the guidelines used in this experiment.

[0143]

[0164] This experiment demonstrates that symmetric endonuclease activity can generate starting products for isothermal amplification systems such as LAMP, shortening the time to results. The increased reaction rate is not limited to SDA.

[0144] [Table 6]

[0145] Example 6: Modification of enzyme activity by guide design

[0165] This experiment uses the same detector molecule as in Example 3 (Figures 11A-11B), which has an internal fluorophore-quencher pair, but also includes an additional modification guide that modifies the enzyme activity (Figure 11C).

[0146]

[0166] In this experiment, we used the enzyme BspQI, which has a primary cleavage site (boxed) next to its recognition site and a forced cleavage site on the guide. We also used Nt.BspQI as a control, which is a triple mutant of BspQI with top-strand DNA nicking activity.

[0147]

[0167] In reactions in which BspQI cleaved at its primary cleavage site, the oligos overlapped by only one base and were dropped off at the reaction temperature. In reactions in which BspQI was forced to perform an asymmetric secondary cleavage, Bst polymerase used the guide molecule as a target to activate the primary cleavage activity of BspQI. This resulted in an increase in Fam fluorescence due to extension from the cleaved guide and opening of the probe. In reactions in which BspQI cleaved at both cleavage sites, the oligos dispersed due to only a three-base overlap. The guides used in this experiment are found in Table 7.

[0148]

[0168] Guide C showed increased fluorescence from the additional copies upon endonuclease, suggesting either that Bst polymerase activity was faster than endonuclease activity or that the endonuclease pulled the target together after cleavage, allowing Bst extension (Figure 12A). Guide D showed increased fluorescence upon endonuclease, and Bst extension was inhibited without endonuclease, suggesting that asymmetric endonuclease activity allowed bypass of the guide blockage and that Nt.BspQI possesses bottom-strand nuclease activity (Figure 12B). Guide E showed increased fluorescence upon endonuclease, and Bst extension was blocked without endonuclease, suggesting that Nt.BspQI increased fluorescence (Figure 12C). This suggests that asymmetric endonuclease activity allowed bypass of the guide blockage; that Nt.BspQI possesses bottom-strand nuclease activity; and that enzyme activity can be tuned using different guide chemistries. Guide H showed an increase in fluorescence with nickase; Bst extension was blocked without endonuclease; and 2'O-MeO on the opposite bottom endonuclease cleavage prevented fluorescence from reporting (Figure 12D). This suggests that inhibition of BspQI bottom cleavage allowed the guide to be cleaved only at the top, "shorting" the system. Furthermore, top-strand cleavage by Nt.BspQI may be less efficient under these conditions. Guide F showed an increase in fluorescence with additional copies by endonuclease, suggesting either that Bst polymerase activity was faster than that of the endonuclease, or that the endonuclease brought the target together after cleavage, allowing Bst extension (Figure 12E). Guide G showed a minimal increase in fluorescence, suggesting that either both endonucleases were inhibited or that nuclease activity was enhanced over polymerase activity (Figure 12F).

[0149]

[0169] Figures 13A-13B compare guide F with guide C and show that when methoxylation on the guide is close to the cleavage site, the signal is slightly enhanced.

[0170] This experiment demonstrated that extension of the guide molecule can be inhibited by various moieties, and that restriction enzyme activity can be engineered to behave asymmetrically, promoting one cleavage activity over the other. Modification of the guide molecule allows endonuclease activity to occur on the target at a desired specific location, while unblocking the guide molecule can be achieved by modifying the activity of the endonuclease in the system or by modifying the activity of the strand-displacing polymerase in the system.

[0150] [Table 7]

[0151] Example 7: Isothermal SDA after production of restriction digest target product

[0171] In this experiment, the method of Example 1 was used, followed by isothermal SDA to examine the effect on amplification of the resulting target product. A series of monkeypox titration 10-fold dilutions was performed (Figures 14A-14B). The amplification results are shown in Figure 14C. Monkeypox virus amplification was also performed in NP matrix direct amplification reactions (Figures 14D-14E).

[0152] Example 8: Multiplexed isothermal amplification

[0172] In this experiment, isothermal SDA was performed on samples of Neisseria gonorrhoeae, Chlamydia trachomatis, and RPP30 using the method described in Example 1. The reaction conditions for the triplicate experiment are shown in Figure 15A. M1 Sample Prep® was performed using 400 μL of frozen sample culture medium resuspended in approximately 800 μL of total culture medium. Target quantification was based on M1 Sample Prep® assuming 100% recovery from the sample culture medium. Performance of the triplicate isothermal reactions showed amplification in all three reactions (Figure 15B).

[0153]

[0173] In this experiment, isothermal SDA was performed on samples of Neisseria gonorrhoeae, Chlamydia trachomatis, RPP30, and Trichomonas vaginalis using the method described in Example 1. Similar to the triplicate experiment, the quadruplicate isothermal reaction demonstrated competitive amplification under four reaction conditions (Figure 16A). The reaction conditions for the quadruplicate experiment are shown in Figure 16B.

[0154]

[0174] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method for processing a single-stranded nucleic acid molecule containing a target sequence, comprising: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising the guide polynucleotide under conditions such that the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type IIs restriction enzyme; (ii) a target binding region configured to hybridize to the target sequence; and (iii) a blocked 3' end that cannot be extended by a polymerase a process comprising: (b) introducing the Type IIs restriction enzyme under conditions sufficient for the Type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cleave within the target sequence. A method comprising:

2. The method of claim 1, wherein in b), the cleavage exposes an extendable 3' end of the target sequence.

3. The method of claim 2, further comprising extending the extendable 3' end using a polymerase.

4. 1. A method for amplifying a single-stranded nucleic acid molecule comprising a target sequence, comprising: (a) contacting the single-stranded nucleic acid molecule with a guide complex comprising the guide polynucleotide under conditions such that the guide polynucleotide hybridizes to the single-stranded nucleic acid molecule, wherein the guide polynucleotide: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for a type IIs restriction enzyme; (ii) a target binding region configured to hybridize to the target sequence; and (iii) a blocked 3' end that cannot be extended by a polymerase a process comprising: (b) introducing the Type IIs restriction enzyme under conditions sufficient for the Type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cleave within the target sequence to generate an extendable 3' end; and (c) extending the extendable 3' end of the target sequence using a polymerase. A method comprising:

5. 5. The method of any one of claims 1-4, wherein the guide polynucleotide is a first guide polynucleotide, and the guide complex comprises a second guide polynucleotide, the second guide polynucleotide comprising (i) a non-target binding region that is complementary to the non-target binding region of the first guide polynucleotide, and (ii) a target binding region configured to hybridize to the target sequence.

6. 6. The method of Claim 5, wherein when the first guide polynucleotide of the guide complex hybridizes to the target polynucleotide sequence, the target binding region of the second guide polynucleotide does not hybridize to the target sequence.

7. 7. The method of claim 5 or 6, wherein the first guide polynucleotide and the second guide polynucleotide hybridize to form a dimer.

8. 8. The method of claim 7, wherein the first guide polynucleotide and the second guide polynucleotide hybridize via the non-target binding regions of the first guide polynucleotide and the second guide polynucleotide to form the dimer having a double-stranded binding region.

9. The method of claim 8 , wherein the double-stranded binding region comprises the restriction endonuclease recognition sequence.

10. The method of claim 8 , wherein the type IIs restriction enzyme binds to the double-stranded binding region of the dimer.

11. 1. A method for amplifying a single-stranded nucleic acid molecule comprising a target sequence, comprising: (a) contacting a guide complex with the single-stranded nucleic acid molecule, wherein the guide complex: (i) a first guide polynucleotide comprising, from 5′ to 3′, a non-target binding region and a target binding region that hybridizes to the target sequence of the single-stranded nucleic acid molecule; and (ii) a second guide polynucleotide that hybridizes to the non-target binding region of the first guide molecule to form a double-stranded binding region, the double-stranded binding region comprising a second guide polynucleotide that binds to an enzyme; (b) cleaving the target sequence using the enzyme to expose an extendable 3' end of the target sequence; (c) extending the extendable 3' end of the target sequence with a polymerase to generate an extension product that displaces the second guide polynucleotide; (d) cleaving the first guide polynucleotide within the target binding region to expose an extendable 3' end of the first guide polynucleotide; and (e) using the polymerase to extend the extendable 3' end of the first guide polynucleotide to generate a complement of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule. A method comprising:

12. 12. The method of Claim 11, wherein the second guide polynucleotide comprises, from 5' to 3', (i) a non-target binding region that hybridizes to the non-target binding region of the first guide polynucleotide, and (ii) a target binding region configured to hybridize to the target sequence.

13. 13. The method of Claim 11 or 12, further comprising, prior to (b), using the enzyme to cleave the first guide polynucleotide within the target binding region, such that the guide complex dissociates from the single-stranded nucleic acid molecule.

14. 14. The method of any one of claims 11 to 13, further comprising repeating (d) and (e) to generate multiple complements of the target sequence of the single-stranded nucleic acid molecule.

15. 15. The method of any one of claims 11 to 14, wherein an additional guide complex binds to the complementary molecule.

16. 16. The method of Claim 15, further comprising using the complementary molecule bound by the additional guide complex as an initiating template for generating copies of the target molecule.

17. The method according to any one of claims 11 to 16, wherein the enzyme is a type IIs restriction enzyme.

18. 18. The method of any one of claims 1 to 10 and 17, wherein the type IIs restriction enzyme comprises N.BstNBI, N.Bst9I, N.BspD6I, functional fragments thereof, or combinations thereof.

19. 19. The method of any one of claims 11 to 18, wherein the guide polynucleotide comprises a blocked 3' end that is not extendable by a polymerase.

20. 20. The method of any one of claims 1 to 10 and 19, wherein the blocked 3' end comprises a PNA, a modified base, a phosphate group, a ddNTP, a solid support, a spacer, or any combination thereof.

21. 20. The method of any one of claims 1 to 19, wherein the single-stranded nucleic acid molecule with the cleavage and the guide polynucleotide attached is used as a starting template for amplification.

22. 22. The method of claim 21, wherein the amplification is an isothermal amplification.

23. The method of any one of claims 1 to 22, wherein the enzyme exhibits high frequency endonuclease activity.

24. 24. The method of claim 23, wherein the high frequency endonuclease activity is derived from the large subunit of the enzyme.

25. The method of any one of claims 1 to 24, wherein the enzyme exhibits low frequency endonuclease activity.

26. 26. The method of claim 25, wherein the low frequency endonuclease activity is derived from a small subunit of the enzyme.

27. 27. The method of any one of claims 1 to 26, wherein the enzyme exhibits at least two differential enzyme activity rates.

28. 28. The method of claim 27, wherein the at least two differential enzyme activity rates comprise two differential endonuclease activity rates when cleaving two different cleavage sites.

29. 28. The method of claim 27, wherein one of the two differential rates of endonuclease activity comprises cleaving the target sequence of the single-stranded nucleic acid molecule less frequently.

30. 28. The method of Claim 27, wherein one of the two differential endonuclease activity rates comprises frequently cleaving the target binding region of the guide polynucleotide.

31. 28. The method of claim 27, wherein the two differential endonuclease activity rates are asymmetric or unequal.

32. 28. The method of claim 27, wherein the enzyme comprises BsmAI, Nt.BsmAI, transcription activator-like effector nuclease, N.Bst9I, N.BspD6I, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Mva1269I, Nb.BpulOI, and Nt.BpulOI, functional fragments thereof, or combinations thereof.

33. A method according to any one of claims 27 to 32, wherein the temperature is varied over the course of the method.

34. 34. The method of claim 33, wherein a first activity rate of the at least two differential enzyme activity rates is favored at a first temperature and a second activity rate of the at least two differential enzyme activity rates is favored at a second temperature different from the first temperature.

35. 35. The method of any one of claims 1 to 34, wherein the enzyme comprises two different active sites or endonuclease domains that confer at least two differential enzymatic activities.

36. 36. The method of claim 35, wherein the target sequence comprises a recognition site that is specifically recognized by the enzyme or a first of the at least two differential enzymatic activities of the enzyme to introduce cleavage.

37. 37. The method of Claim 35 or 36, wherein the target binding region of the guide polynucleotide comprises a recognition site that is specifically recognized by the enzyme or a second of the at least two differential enzymatic activities of the enzyme to introduce a cleavage.

38. 38. The method of any one of claims 1 to 37, wherein the target binding region is at least about 12 to about 25 nucleotides in length.

39. 39. The method of any one of claims 1 to 38, wherein the concentration of the guide polynucleotide is at least about 0.1 μM, at least about 1 μM, or from about 0.1 μM to about 4 μM.

40. 40. The method of any one of claims 1 to 39, wherein the non-target binding region comprises a palindromic sequence.

41. 41. The method of any one of claims 1 to 40, wherein the non-target binding region is self-complementary.

42. 42. The method of any one of claims 1 to 41, wherein the non-target binding region is at least about 12 nucleotides in length.

43. 43. The method of any one of claims 1 to 42, wherein the single-stranded nucleic acid molecule is a single-stranded deoxyribonucleic acid (ssDNA) or a single-stranded ribonucleic acid (ssRNA).

44. 44. The method of any one of claims 1 to 43, wherein the target binding region comprises at least one peptide nucleic acid (PNA) residue.

45. The method of any one of claims 3 to 44, wherein the polymerase has strand displacement activity.

46. 46. ​​The method of any one of claims 1 to 45, wherein the guide polynucleotide or the first guide polynucleotide further comprises an additional non-target binding region.

47. 47. The method of Claim 46, wherein the additional non-target binding region is located at the 5' end of the guide polynucleotide or the first guide polynucleotide.

48. 48. The method of claim 46 or 47, wherein the additional non-target binding region comprises an additional restriction endonuclease recognition sequence for an additional enzyme.

49. 49. The method of claim 48, wherein the additional enzyme is the same as or different from the enzyme.

50. 50. The method of any one of claims 46 to 49, wherein the additional non-target binding region inhibits extension of the 3' end of the guide polynucleotide or the first guide polynucleotide.

51. 51. The method of any one of claims 1 to 50, wherein the single-stranded nucleic acid molecule comprises two or more single-stranded nucleic acid molecules, each single-stranded nucleic acid molecule comprising a different target sequence.

52. 52. The method of claim 51 , wherein the two or more single-stranded nucleic acid molecules are contained in a single reaction mixture.

53. 53. The method of any one of claims 4 to 52, wherein the method of amplifying single-stranded nucleic acid molecules reduces the time to cycle threshold or result value for nucleic acid amplification compared to the time to cycle threshold or result value for nucleic acid amplification of an otherwise identical method of amplifying the single-stranded nucleic acid molecule without the guide complex.

54. 53. The method of any one of claims 4 to 52, wherein the method for amplifying single-stranded nucleic acid molecules reduces the time to cycle threshold or result value in nucleic acid amplification compared to the time to cycle threshold or result value in existing nucleic acid amplification methods.

55. 55. The method of claim 54, wherein the existing nucleic acid amplification method is selected from the group consisting of loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinant polymerase amplification (RPA), and nucleic acid sequence-based amplification (NASBA).

56. 56. The method of any one of claims 53 to 55, wherein the cycle threshold is at most 30.

57. 1. A polynucleotide-polypeptide complex comprising a single-stranded nucleic acid molecule having attached thereto a guide complex, said guide complex comprising: (i) a first guide polynucleotide comprising, from 5′ to 3′, a non-target binding region and a target binding region that hybridizes to a target sequence of the single-stranded nucleic acid molecule; and (ii) a polynucleotide-polypeptide complex comprising a second guide polynucleotide that hybridizes to the non-target binding region of the first guide molecule to form a double-stranded binding region, wherein the double-stranded binding region comprises a restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme.

58. 1. A system for processing a single-stranded nucleic acid molecule comprising a target sequence, the system comprising: The single-stranded nucleic acid molecule binding a guide complex comprising a guide polynucleotide, wherein the guide polynucleotide is: (i) a non-target binding region comprising a restriction endonuclease recognition sequence for an enzyme that is a type IIs restriction enzyme; (ii) a target binding region configured to hybridize to the target sequence; and (iii) a blocked 3' end that cannot be extended by a polymerase the single-stranded nucleic acid molecule comprising: the enzyme bound to the restriction endonuclease recognition sequence of the non-target binding region.

59. A kit comprising a guide complex or guide polynucleotide according to any one of claims 1 to 58.

60. 60. The kit of claim 59, further comprising a probe or dye for detecting amplification products produced using the kit.

61. 61. The kit of claim 59 or 60, further comprising informational material instructing how to use the kit.

62. 1. A system for processing a plurality of single-stranded nucleic acid molecules, each containing a different target sequence, said system comprising: A first single-stranded nucleic acid molecule, said first single-stranded nucleic acid molecule bound to a first guide complex comprising a first guide polynucleotide, said first guide polynucleotide comprising: (i) a first non-target binding region comprising a first restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme; (ii) a first target binding region configured to hybridize to a first target sequence; and (iii) a first blocked 3′ end that is not extendable by a polymerase; a first single-stranded nucleic acid molecule comprising: a second single-stranded nucleic acid molecule, said second single-stranded nucleic acid molecule bound to a second guide complex comprising a second guide polynucleotide, said second guide polynucleotide comprising: (i) a second non-target binding region comprising a second restriction endonuclease recognition sequence for said enzyme, said enzyme being a Type IIs restriction enzyme; (ii) a second target binding region configured to hybridize to a second target sequence; and (iii) a second blocked 3' end that is not extendable by a polymerase; a second single-stranded nucleic acid molecule comprising: The system, wherein the enzyme, which is a type IIs restriction enzyme, binds to the first restriction endonuclease recognition sequence of the first non-target binding region or the second restriction endonuclease recognition sequence of the second non-target binding region.

63. The method further comprises a third single-stranded nucleic acid molecule, wherein the third single-stranded nucleic acid molecule is bound to a third guide complex comprising a third guide polynucleotide, the third guide polynucleotide comprising: (i) a third non-target binding region comprising a third restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme; (ii) a third target binding region configured to hybridize to a third target sequence; and (iii) comprises a third blocked 3' end that is not extendable by a polymerase; 63. The system of claim 62, wherein the enzyme is a type IIs restriction enzyme and binds to the third restriction endonuclease recognition sequence of the third non-target binding region.

64. The method further comprises a fourth single-stranded nucleic acid molecule, wherein the fourth single-stranded nucleic acid molecule is bound to a fourth guide complex comprising a fourth guide polynucleotide, the fourth guide polynucleotide comprising: (i) a fourth non-target binding region comprising a fourth restriction endonuclease recognition sequence for an enzyme that is a Type IIs restriction enzyme; (ii) a fourth target binding region configured to hybridize to a fourth target sequence; and (iii) comprises a fourth blocked 3' end that is not extendable by a polymerase; 64. The system of claim 62 or 63, wherein the enzyme that is a Type IIs restriction enzyme binds to the fourth restriction endonuclease recognition sequence of the fourth non-target binding region.

65. The system of any one of claims 62 to 64, wherein the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule are derived from different samples.

66. 66. The system of claim 65, wherein the different samples comprise samples obtained from bacteria, viruses, humans, or any combination thereof.

67. 67. The system of claim 66, wherein the bacteria is selected from the group consisting of Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis.

68. 67. The system of claim 66, wherein the virus is selected from the group consisting of double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, single-stranded RNA viruses, positive-sense single-stranded reverse transcriptase viruses, and double-stranded DNA reverse transcriptase viruses.

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