Methods and compositions for processing and amplifying nucleic acids

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 424,666, filed on November 11, 2022, and U.S. Provisional Patent Application No. 63 / 501,226, filed on May 10, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] 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

[0004] It is recognized herein that there is a need for improved methods and compositions for processing target nucleic acid molecules with high efficiency and / or simplified optimization of reaction conditions.The methods and compositions described herein can be used to generate initial products for downstream applications such as isothermal amplification.

[0005] In one aspect, the present disclosure provides a method for treating 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 is non-extendable by a polymerase; and (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 cut within the target sequence.

[0006] In some embodiments, in (b), cleavage exposes an extendable 3' end of the target sequence.

[0007] In some embodiments, the method further comprises extending the extendable 3' end using a polymerase.

[0008] In another aspect, 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, 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 to produce an extendable 3′ end under conditions sufficient for the Type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the target sequence; and (c) extending the extendable 3′ end of the target sequence using a polymerase.

[0009] In some embodiments, the guide polynucleotide is a first guide polynucleotide and the guide complex comprises a second guide polynucleotide, wherein the second guide polynucleotide comprises (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 that is configured to hybridize to a target sequence.

[0010] 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 regions of the first guide polynucleotide and the second guide polynucleotide to form a dimer having a double-stranded binding region.

[0011] In some embodiments, the double-stranded binding region comprises a restriction endonuclease recognition sequence.

[0012] In some embodiments, the Type lis restriction enzyme binds to the double-stranded binding region of the dimer.

[0013] In another aspect, the present 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, wherein the guide complex comprises: (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, wherein the double-stranded binding region is bound to an enzyme; (b) cleaving the target sequence using an enzyme to expose the extendable 3' end of the target sequence; (c) extending the extendable 3' end of the target sequence using a polymerase to produce an extension product, wherein the extension product replaces the second guide polynucleotide; (d) cleaving the first guide polynucleotide within the target binding region to expose the extendable 3' end of the first guide polynucleotide; and (e) extending the extendable 3' end of the first guide polynucleotide using a polymerase to produce a complementary molecule of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule.

[0014] In some embodiments, 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 a target sequence.

[0015] In some embodiments, the method further comprises, prior to (b), cleaving the first guide polynucleotide within the target binding region using an enzyme, wherein the guide complex dissociates from the single-stranded nucleic acid molecule.

[0016] In some embodiments, the method further comprises repeating (d) and (e) to generate multiple complementary molecules of the target sequence of the single-stranded nucleic acid molecule.

[0017] In some embodiments, the additional guide complex is associated with a complementary molecule.

[0018] In some embodiments, the method further comprises using the complementary molecule having the additional guide complex bound thereto as a starting template to generate copies of the target molecule.

[0019] 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, a functional fragment thereof, or a combination thereof.

[0020] In some embodiments, the guide polynucleotide comprises a blocked 3' end that is non-extendable by a polymerase. In 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.

[0021] In some embodiments, a single-stranded nucleic acid molecule having a guide polynucleotide for cutting and binding thereto is used as a starting template for amplification. In some embodiments, amplification is isothermal amplification. In some embodiments, the enzyme exhibits high-frequency endonuclease activity. In some embodiments, the high-frequency endonuclease activity is 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 from the small subunit of the enzyme. In some embodiments, the enzyme exhibits at least two different enzyme activity rates. In some embodiments, when cutting two different cleavage sites, at least two different enzyme activity rates include two different endonuclease activity rates.

[0022] In some embodiments, one of the two differential rates of endonuclease activity comprises cleaving a target sequence of a single-stranded nucleic acid molecule at a low frequency. In some embodiments, one of the two differential rates of endonuclease activity comprises cleaving a target binding region of a guide polynucleotide at a high frequency. In some embodiments, the two differential rates of endonuclease activity are asymmetric or unequal.

[0023] In some embodiments, the enzyme comprises BsmAI, Nt.BsmAI, a transcription activator-like effector nuclease, N.Bst9 I, 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.

[0024] In some embodiments, the temperature is varied during the process.

[0025] In some embodiments, a first activity rate of the at least two differential rates of enzyme activity is favored at a first temperature, and a second activity rate of the at least two differential rates of enzyme activity is favored at a second temperature different from the first temperature.

[0026] In some embodiments, the enzyme comprises two distinct active sites or endonuclease domains that confer at least two differential enzymatic activities.

[0027] In some embodiments, the target sequence comprises a recognition site or a first of at least two distinct enzymatic activities of an enzyme that is specifically recognized by the enzyme to induce cleavage.

[0028] 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 distinct enzymatic activities of the enzyme to induce cleavage.

[0029] In some embodiments, the target binding region is at least about 12 to about 25 nucleotides in length.

[0030] 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.

[0031] 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.

[0032] In some embodiments, the single-stranded nucleic acid molecule is single-stranded deoxyribonucleic acid (ssDNA) or single-stranded ribonucleic acid (ssRNA).

[0033] 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 the 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 the first guide polynucleotide.

[0034] 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 the 3' end extension 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 of which comprises a different target sequence. In some embodiments, the two or more single-stranded nucleic acid molecules are contained in a single reaction mixture.

[0035] In some embodiments, a method of amplifying a single-stranded nucleic acid molecule reduces a cycle threshold or result-out time value in nucleic acid amplification compared to an otherwise identical method that amplifies a single-stranded nucleic acid molecule in the absence of a guide complex.

[0036] In some embodiments, the method for amplifying single-stranded nucleic acid molecules shortens the cycle threshold or result time value in nucleic acid amplification compared to the cycle threshold or result time value in existing nucleic acid amplification methods.

[0037] In some embodiments, the existing nucleic acid amplification method is selected from loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA), recombinase polymerase amplification (RPA) and nucleic acid sequence-based amplification (NASBA). In some embodiments, the cycle threshold is at most 30.

[0038] In another aspect, the present disclosure provides a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule bound to a guide complex, wherein the guide complex comprises: (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 comprises a restriction endonuclease recognition sequence for an enzyme that is a type IIs restriction enzyme.

[0039] In another aspect, the present 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 bound to a guide complex comprising a guide polynucleotide, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence of an enzyme, the enzyme being 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 non-extendable by a polymerase; and an enzyme bound to the restriction endonuclease recognition sequence of the non-target binding region.

[0040] In another aspect, the present disclosure provides a kit comprising a guide complex or a guide polynucleotide described herein.

[0041] In some embodiments, the kit further includes a probe or dye for detecting amplification products produced using the kit.

[0042] In some embodiments, the kit further comprises informational material describing instructions for using the kit.

[0043] In another aspect, the present 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, wherein the first single-stranded nucleic acid molecule is bound to a first guide complex comprising a first guide polynucleotide, wherein the first guide polynucleotide comprises: (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 the first target sequence, and (iii) a first blocked 3′ end that is non-extendable by a polymerase; and a second single-stranded nucleic acid molecule. The invention relates to a nucleic acid molecule, wherein the second single-stranded nucleic acid molecule is bound to a second guide complex comprising a second guide polynucleotide, wherein the second guide polynucleotide comprises: (i) a second non-target binding region comprising a second restriction endonuclease recognition sequence for an enzyme that is 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 non-extendable by a polymerase; wherein 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.

[0044] In some embodiments, the system 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, wherein the third guide polynucleotide comprises: (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 non-extendable by a polymerase; wherein the enzyme that is a Type IIs restriction enzyme binds to the third restriction endonuclease recognition sequence of the third non-target binding region.

[0045] In some embodiments, the system 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, wherein the fourth guide polynucleotide comprises: (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 non-extendable by a polymerase; 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.

[0046] 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, different samples include samples obtained from bacteria, viruses, humans, or any combination thereof. In some embodiments, the bacteria are selected from Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis. In some embodiments, the virus is selected from double-stranded DNA virus, single-stranded DNA virus, double-stranded RNA virus, single-stranded RNA virus, positive single-stranded reverse transcriptase virus, and double-stranded DNA reverse transcriptase virus.

[0047] Other aspects and advantages of the present disclosure will readily become 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 appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification 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 restrictive.

[0048] Incorporation by reference

[47] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications, patents, or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the present invention are utilized, and the accompanying drawings (also referred to herein as "Figures" and "FIG."), in which:

[0050] Figure 1A-Figure 1O Shown are examples of precursor steps leading to isothermal amplification cycles according to various embodiments described herein. Figure 1A Depicted is a double-stranded oligonucleotide complex bound to a target nucleic acid strand. Figure 1B The endonucleolytic activity of the double-stranded oligonucleotide / target complex is depicted. Figure 1C The polymerase is depicted extending from the 3' end of the target strand. Figure 1D Depicted is a polymerase that displaces a double-stranded guide molecule. Figure 1E The endonucleolytic activity of the oligonucleotide / extension product complex is depicted. Figure 1FThe polymerase extending from the 3' end of the cleaved oligonucleotide and the displacement of the leader are depicted. Figure 1G The endonuclease activity of the newly synthesized portion complementary to the target strand is depicted. Figure 1H The polymerase extending from the 3' end of the cleavage site and the displacement of the synthesized complement toward the target strand are depicted. Figure 1I Depicted is the displaced complement serving as a new target for the second complementary strand double-stranded oligonucleotide complex. Figure 1J Depicted is a polymerase that displaces a second complementary strand of a double-stranded guide molecule. Figure 1K The completed extension on the new guide molecule is depicted. Figure 1L The endonucleolytic activity of the second complementary strand oligonucleotide / extension product complex is depicted. Figure 1M The polymerase is depicted extending from the 3' end of the cleavage site of the second complementary strand of the oligonucleotide / extension product complex. Figure 1N The endonucleolytic activity of the newly synthesized complementary strand of the second complementary strand guide is depicted. Figure 1O Depicted are displaced single-stranded synthetic fragments that serve as starting material for a strand displacement amplification reaction.

[0051] Figure 2A-2C Shown is a system for generating products suitable for amplification by an isothermal amplification reaction using guide molecules that have point mutations relative to the target sequence. Figure 2A Depicted are a guide molecule with a point mutation binding to target DNA and an endonuclease cleaving the target. Figure 2B Depicted is an endonuclease that extends from the target and cleaves the guide molecule at the 3' end. Figure 2C Depicted is the displacement of a guide complementary to the target following endonucleolytic cleavage, followed by synthesis of a new strand. Reverse strand guided extension will only see bases derived from the target, not bases present in the guide oligonucleotide sequence, as they do not extend from the end of the guide oligonucleotide.

[0052] Figure 3A-3B A control experiment in which there was no mismatch between the guide and the primer is shown. Figure 3A Depicted are the guide oligonucleotide, probe, and target sequences used in a control experiment with no mismatches between the guide oligonucleotide and target. Figure 3B Amplification results for a control reaction without the point mutation are depicted.

[0053] Figure 4A-4B An experiment with an A vs. C mismatch between the guide and primer is shown. Figure 4A Depicted are the guide oligonucleotides, probes, and target sequences used in the mismatch experiments. Figure 4B Amplification is depicted to produce probe signals with target origin; rather than guide oligonucleotide origin indicating asymmetric endonuclease activity.

[0054] Figure 5A-5B A control experiment in which there was no mismatch between the guide and the primer is shown. Figure 5A The guide oligonucleotides, probes, and target sequences used in the control experiments are depicted. Figure 5B Amplification results for a control reaction without the point mutation are depicted.

[0055] Figure 6A-6B An experiment with an A vs. C mismatch between the guide and primer is shown. Figure 6A Depicted are the guide oligonucleotides, probes, and target sequences used in the mismatch experiments. Figure 6B Amplification is depicted to produce probe signals with target origin; rather than guide oligonucleotide origin indicating asymmetric endonuclease activity.

[0056] Figures 7A-7D Shown are experiments using intrinsic fluorescence to detect the formation of double-stranded nucleic acids and using different guides. Figure 7A Depicted are single-stranded DNA (ssDNA) molecules with a 5' quencher and internal fluorescein-T. Figure 7B Depicted is the quenched fluorescence upon strand self-complementarity. Figure 7C Binding of the guide molecule to the target ssDNA is depicted. Figure 7D The cleavage site that will initiate the formation of extension products and fluorescent double-stranded nucleic acids is depicted.

[0057] Figures 8A-8D Amplification / primer extension results using different primers using Bst polymerase are shown. 3' extension of the guide molecule is blocked when a 2' O-methyl RNA base or a phosphorylated base is encountered. Figure 8A Depicted are the results of amplification / primer extension reactions using only Bst polymerase. Figure 8B Depicted are the results of an amplification / primer extension reaction using Bst polymerase and the endonuclease Nt.BsmAI. Figure 8C Depicted are the results of amplification / primer extension reactions using Bst polymerase and the endonucleases Nt.BsmAI and N.BstNBI. Figure 8D Depicted are the results of an amplification / primer extension reaction using Bst polymerase and endonuclease N.BstNBI.

[0058] Figures 9A-9D The results of amplification / primer extension reactions using different primers using Bst polymerase are shown. When a 2' O-methyl RNA base or a phosphorylated base is encountered, 3' extension of the guide molecule is blocked. Figure 9A Depicted are the results of amplification / primer extension reactions using only Bst polymerase. Figure 9B Depicted are the results of an amplification / primer extension reaction using Bst polymerase and the endonuclease Nt.BsmAI. Figure 9CDepicted are the results of amplification / primer extension reactions using Bst polymerase and the endonucleases Nt.BsmAI and N.BstNBI. Figure 9D Depicted are the results of an amplification / primer extension reaction using Bst polymerase and endonuclease N.BstNBI.

[0059] Figures 10A-10B Shown are cycle threshold results for loop-mediated isothermal amplification (LAMP). Figure 10A Depicted are cycle threshold results for LAMP when compared to LAMP plus differentially targeted endonuclease cleavage technology (DTECT) priming and DTECT priming alone. Figure 10B yes Figure 10A A zoomed-in version of the image shows more clearly the difference between the cycle threshold results for LAMP plus DTECT priming and DTECT priming.

[0060] Figures 11A-11D Shown are experiments using intrinsic fluorescence to detect the formation of double-stranded nucleic acids and using different guides. Figure 11A Depicted are single-stranded DNA (ssDNA) molecules with a 5' quencher and internal fluorescein-T. Figure 11B Depicted is the quenched fluorescence upon strand self-complementarity. Figure 11C The 2'O methyl base on the guide molecule is depicted. Figure 11D The cleavage site that will initiate the formation of extension products and fluorescent double-stranded nucleic acids is depicted.

[0061] Figures 12A-12F The primers 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 ) amplification results.

[0062] Figures 13A-13B The results using primer F (which is unblocked and has a methoxylation block on the primer) are shown. Figure 13A ) and an unblocked and extendable primer C ( Figure 13B ) between them.

[0063] Figures 14A-14E Shown are the results of isothermal SDA following restriction digestion of monkeypox target product production. Figure 14A Depicted is a 10-fold dilution series of primers using primers with blocking. Figure 14BDepicted is a 10-fold dilution series of 1x10 using a primer with blocking 6 Copy number / reaction. Figure 14C A summary of the log copies / reaction of monkeypox primer-primed amplification reactions with blocking is depicted. Figure 14D Depicted is the amplification of monkeypox virus using a primer-guided vector with blocking in an NP matrix direct amplification procedure. Figure 14E Depicts from Figure 14D A summary of the data.

[0064] Figures 15A-15B Results of triplicate isothermal amplification reactions are shown. Figure 15A Reaction conditions from a triple isothermal reaction are shown, where Isofast BST is a DNA polymerase, N.BstNBI is a site-specific endonuclease that primarily cleaves only one DNA strand on a double-stranded DNA substrate; AMV rt enz refers to Avian Myeloblastosis Virus reverse transcriptase; dNTP refers to deoxynucleoside triphosphates; NaSO4 is sodium sulfate; MgSO4 is magnesium sulfate; Tris is tris(hydroxymethyl)aminomethane, and (NH4)2SO4 is ammonium sulfate; NG refers to Neisseria gonorrhoeae; CT refers to Chlamydia trachomatis, RPP stands for ribosomal protection protein; rt represents reverse transcriptase; Cy5 refers to cyanine-5; HEX refers to hexachlorofluorescein, and Fam refers to fluorescein amidite. Figure 15B Preliminary performance results are shown for an XCEL triple isothermal reaction using N. gonorrhoeae, C. trachomatis, and human RPP30. For C. trachomatis, approximately 75 IFU / reaction were used, and for N. gonorrhoeae, approximately 150 CFU / reaction were used.

[0065] Figure 16A Results of a quadruple isothermal amplification reaction using Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia trachomatis, and RNase P / MRP subunit p30 (RPP30) are described. RNA titration of M1 purified Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis with 250 pg / reaction purified human RNA. Figure 16BReaction conditions from a quadruple isothermal reaction are shown, where Lucigen BST is a DNA polymerase, N.BstNBI is a site-specific endonuclease that primarily cleaves only one DNA strand on a double-stranded DNA substrate; AMV rt enz refers to avian myeloblastosis virus reverse transcriptase; dNTP refers to deoxynucleoside triphosphates; NaSO4 is sodium sulfate; MgSO4 is magnesium sulfate; Tris is tris(hydroxymethyl)aminomethane, and (NH4)2SO4 is ammonium sulfate; NG refers to Neisseria gonorrhoeae; CT refers to Chlamydia trachomatis, and Tv refers to Trichomonas vaginalis; RPP represents ribosomal protection protein; rt represents reverse transcriptase; Cy5 refers to cyanine-5; HEX refers to hexachlorofluorescein, and Fam refers to fluorescein amidite.

[0066] Figure 17 A computer system is shown that is programmed or otherwise configured to implement the methods provided herein. DETAILED DESCRIPTION

[0067] Although 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. Without departing from the present invention, it will be appreciated by those skilled in the art that many variations, changes, and substitutions may be envisioned. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed. It should be understood that although the vial cap is described in the drawings as having a configuration comprising three gap-filling caps that fill three vials in a linear arrangement, this description is merely illustrative, as the inventive concept described herein contemplates various configurations and quantities of the gap-filling cap.

[0068] Whenever the term "at least," "greater than," or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least," "greater than," or "greater than or equal to" applies to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0069] Whenever the term "not more than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not more than," "less than," or "less than or equal to" applies to every value 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, or less than or equal to 1.

[0070] Certain inventive embodiments herein contemplate numerical ranges. When a range exists, the range includes the range endpoints. In addition, each subrange and value within the range exists as if explicitly written out. The term "about" or "approximately" may mean within an acceptable error range for a particular value, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. For example, according to the practice of this art, "about" may mean within 1 or greater than 1 standard deviation. Alternatively, "about" may 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 may mean within an order of magnitude of a value, within 5 times of a value, or within 2 times of a value. Where a specific value is described in the present application and claims, unless otherwise indicated, it may be assumed that the term "about" means within an acceptable error range for a specific value.

[0071] Overview

[0072] The present disclosure provides methods, systems, compositions and kits for processing target nucleic acid molecules. In some aspects, the present disclosure provides methods for nucleic acid amplification (e.g., isothermal amplification). Such methods may involve steps (e.g., Figures 1A to 1O The method that this paper provides can provide higher amplification efficiency and easier optimization procedure compared with existing amplification (such as isothermal amplification).The target nucleic acid molecules through treatment can be used for various amplification reactions, not limited to the amplification or treatment methods described herein.

[0073] This approach can be used to form structures such as Figure 1A The invention begins with a structure (shown in FIG), where a guide nucleic acid complex (or guide complex) is formed to guide the restriction enzyme to a predetermined site in the nucleic acid. Figure 1A A nucleic acid strand (e.g., a single-stranded DNA strand or an ssDNA strand) (100) comprising a target nucleic acid sequence (101) is depicted. 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. Figure 1A In the present invention, a type IIs restriction enzyme (120) is guided to the vicinity of a target site via the formation of a guide complex. The guide nucleic acid complex is formed by the self-annealing of a single copy of a guide polynucleotide comprising: a non-target binding region comprising a restriction endonuclease recognition sequence for a type IIs restriction enzyme (117), a target binding region configured to hybridize with a target sequence (115), and a blocked 3′ end that is non-extendable by a polymerase (116). Note that in Figure 1A In , self-annealing of the two copies of the guide polynucleotide forms a double-stranded palindromic region that allows the binding of a Type II restriction enzyme near the target site.

[0074] This method can be used in Figure 1B and Figure 1C Continuing in the second stage of the process depicted in . After being 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 is able to cleave single-stranded positions (130, 135) distal to its binding site, which is characteristic of its activity ( Figure 1B ). One of these cleavable single-stranded positions (135) is located on the nucleic acid chain (101) comprising the target nucleic acid sequence (101). Another cleavable single-stranded position (130) is located on the guide polynucleotide itself (130). If selective enzymatic conditions, an engineered polymerase, or BspD6I are used, cleavage at one of the sites (e.g., a single-stranded site on the nucleic acid chain (101) comprising the target nucleic acid sequence (101)) may be advantageous. Cleavage at the single-stranded site on the nucleic acid chain (101) comprising the target nucleic acid sequence (101) generates a free 3′ hydroxyl group, which can then be extended by a strand displacement polymerase present in the reaction.

[0075] This method can be used in Figures 1D to 1F The process described in the third stage continues. Figure 1C ) extension of the free 3′ hydroxyl group produces a region (160) of the nucleic acid chain (101) comprising a target nucleic acid sequence (101) complementary to a restriction endonuclease recognition sequence of a type IIs restriction enzyme (117) from the guide polynucleotide ( Figure 1D ). The extension of the nucleic acid (100) displaces the second copy of the guide polynucleotide (116 / 117, lower molecule) that previously formed half of the guide complex. The extension of the nucleic acid (100) having a region complementary to the restriction endonuclease recognition sequence of the type IIs restriction enzyme (160) forms a new double-stranded structure where the type IIs restriction enzyme (120) can bind ( Figure 1E As in the second stage, type IIs restriction enzymes are able to cleave single-stranded positions distal to their binding sites (130, 135) ( Figure 1E While cleavage at the single-stranded site (135) containing the target nucleic acid site (100) allows the chain (100) to be extended again only by the polymerase, cleavage at the single-stranded site (130) allows a new process to begin ( Figure 1E ). Specifically, the annealed guide polynucleotide Figure 1E Cleavage at position 130 removes the sequence containing the blocked 3′ end (116) and allows the guide polynucleotide to extend to include a sequence (170) complementary to the strand (100) containing the target nucleic acid site (101) ( Figure 1F ).

[0076] This method can be used in Figure 1G and Figure 1H The process described in the fourth stage continues. Figure 1G The double-stranded structure no longer contains a closed 3′ end, so Figure 1G Repeated cleavage at site 130 releases a single strand containing a sequence (170) complementary to the strand (100) containing the target nucleic acid site (101), and then allows extension of a new strand (171) to replace it. Figure 1A Similar to the chain 100, the released chain (170) can further serve as a new template ( Figure 1I ), which allows chain 170 to be Figure 1H shown to be further cut and repeatedly extended ( Figure 1J ). Figure 1K An exemplary completed extension on a new guide molecule is depicted.

[0077] In some cases, the method can continue, e.g. Figure 1L As shown, endonucleolytic activity can occur on the second complementary strand oligonucleotide / extension product complex (170). Figure 1M The polymerase (140) is depicted extending the 3' end of the cleavage site of the second complementary strand of the oligonucleotide / extension product complex. Endocleavage activity occurs on the newly synthesized strand (130). Figure 1N ),and Figure 1O The displaced single-stranded synthetic fragment (42) can serve as the starting material for additional strand displacement amplification reactions.

[0078] In some cases, the methods according to the present disclosure do not involve amplification and utilize Figure 1A The structure shown is used to guide the Figure 1B ) at a single-stranded nucleic acid molecule (100) containing a target site (101).

[0079] definition

[0080] Although 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. Many variations, changes, and substitutions may occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0081] Unless otherwise indicated, the practice of some of the methods disclosed herein employs techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th edition (2012); the series Current Protocols in Molecular Biology (FM Ausubel et al., eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, eds. (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 (RI Freshney, ed. (2010)) (incorporated herein by reference in their entirety).

[0082] 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. In addition, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0083] 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 will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" can mean within one or more standard deviations. 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.

[0084] As used herein, the term "nucleotide" generally refers to a base-sugar-phosphate combination. Nucleotide can include synthetic nucleotides. Nucleotide can include nucleotide analogs. Nucleotide can include synthetic nucleotide analogs. Nucleotide can be a monomeric unit of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). The term nucleotide can 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 can include locked nucleic acids (LNA), bridged nucleic acids (BNA), fluorinated nucleic acids (also known as fluorine-modified nucleic acids) and peptide nucleic acids (PNA). As used herein, the term "locked nucleic acid" ("LNA") generally refers to a nucleic acid analog in which the ribose ring is "locked" using an additional bridge (such as a methylene bridge) connecting the 2'-oxygen atom of the nucleotide to the 4'-carbon atom (see, for example, WO 99 / 14226, which is incorporated herein by reference in its entirety). As used herein, the term "bridged nucleic acid (BNA)" generally refers to a restricted or inaccessible nucleic acid molecule with a fixed bridge structure at the 2' or 4' position. As used herein, "fluorinated nucleic acid" generally refers to a nucleic acid in which a fluorine atom is incorporated, typically at the 2' or 4' position. As used herein, the term "peptide nucleic acid (PNA)" generally refers to a nucleotide analog in which the backbone of the analog (e.g., the sugar backbone in DNA) is a pseudopeptide. The PNA backbone can comprise, for example, a sequence of repeated N-(2-amino-ethyl)-glycine units. Peptide nucleic acid analogs can react like DNA in a given environment and can also bind to complementary nucleic acid sequences and various proteins. In some embodiments, the present invention relates to a nucleic acid molecule comprising a nucleic acid molecule of the present invention and a nucleic acid molecule of the present invention. The nucleic acid molecule of the present invention can be a nucleic acid molecule comprising a nucleic acid molecule of the present invention and a nucleic acid molecule of the present invention. The nucleic acid molecule of the present invention can be a nucleic acid molecule comprising a nucleic acid molecule of the present invention and a nucleic acid molecule of the present invention. The nucleic acid molecule of the present invention can be a nucleic acid molecule comprising a nucleic acid molecule of the present invention and a nucleic acid molecule of the present invention. The nucleic acid molecule of the present invention can be a nucleic acid molecule comprising a nucleic acid molecule of the present invention and a nucleic acid molecule of the present invention. The nucleic acid molecule of the present invention can be a nucleic acid molecule comprising a nucleic acid molecule of the present invention and a nucleic acid molecule of the present invention.

[0085] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably to refer generally to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof, in single-stranded, double-stranded, or multi-stranded form. The polynucleotide may be DNA. The polynucleotide may be RNA. The polynucleotide may comprise one or more nucleotide analogs (e.g., including those with altered backbones, sugars, or nucleobases). If present, the nucleotide structure may be modified before or after polymer assembly. Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acids, heterologous nucleic acids, morpholinos, ethylene glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein attached to a sugar), thiol nucleotides, biotin-attached nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, braided glycosides, wyoside, PNA, and LNA.

[0086] As used herein, term " restriction endonuclease ", " restriction enzyme " or its grammatical equivalent refer generally to the enzyme that originates from bacterial host defense, and is understood to identify the specific sequence on incoming viral DNA, and cuts DNA at recognition sequence or different sequence sites.One group of restriction endonuclease is identified as IIS type.This group can identify asymmetric DNA sequence, and cuts DNA at the site outside the cleavage site at a certain distance from the recognition site.In some cases, IIS type restriction endonuclease cuts DNA between 1 to 20 nucleotide apart from relevant recognition site.

[0087] As used herein, the term "restriction endonuclease recognition sequence" generally refers to a position on a nucleic acid molecule (e.g., a DNA molecule) that contains a specific nucleotide sequence recognized by various restriction enzymes. These sequences can range from 4-8 base pairs to 12-40 base pairs in length. These sites can be palindromic sequences.

[0088] 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. A DNA polymerase binds to the template strand and then moves down the strand, adding nucleotides to the free hydroxyl group at the 3' end of the growing nucleic acid strand. A DNA polymerase synthesizes complementary DNA molecules from a DNA (e.g., a DNA-dependent DNA polymerase) or an RNA template (e.g., an RNA-dependent DNA polymerase or reverse transcriptase), and an RNA polymerase synthesizes RNA molecules from a DNA template (e.g., a DNA-dependent RNA polymerase involved in transcription). DNA polymerases typically use a short, pre-existing RNA or DNA strand (called a primer) to start chain growth; and some DNA polymerases can utilize any free 3' hydroxyl group in the DNA duplex for extension. Some DNA polymerases replicate single-stranded templates, while other DNA polymerases displace the strand upstream of the site where they add bases to the strand.

[0089] As used herein, the term "strand displacement" when used in reference to a polymerase generally refers to the activity of removing a complementary strand from base pairing with a template strand being read by the polymerase. Example polymerases with strand displacement activity include the large fragment of Bacillus stearothermophilus polymerase (Bst polymerase), exo-Klenow polymerase, Bst 2.0 polymerase, Bsp 3.0 polymerase, SD DNA polymerase, phi29 DNA polymerase, sequencing-grade T7 exo-polymerase, and OmniTaq 2LA DNA polymerase.

[0090] As used herein, the terms "amplify," "amplifies," "amplified," and "amplicon" generally refer to any method of replicating nucleic acid. Replication can be performed using a primer-dependent polymerase. Replication can be enzyme-free amplification. In some cases, amplifying or replicating a target nucleic acid strand also includes replicating or amplifying a complementary strand of the target nucleic acid strand. The amplified product can be subjected to subsequence analysis, including but not limited to melting curve analysis, nucleotide sequencing, single-strand conformational polymorphism determination, allele-specific oligonucleotide hybridization, Southern blot analysis, and restriction endonuclease digestion.

[0091] As used herein, the terms "hybridization" and "annealing" generally refer to a reaction in which one or more polynucleotides interact to form a complex stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-sensitive or specific manner. The complex can include two chains forming a double-stranded structure, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination of these. A hybridization reaction can constitute a step in a broader process (such as the initiation of PCR, or the enzyme digestion of polynucleotides by ribozymes). A first sequence that can be stabilized by hydrogen bonding with the bases of the nucleotide residues of the second sequence can generally be "hybridized" with a second sequence. In this case, the second sequence can also be hybridizable with the first sequence.

[0092] As used herein, the terms "complement," "complements," "complementary," and "complementarity" generally refer to a sequence that is fully complementary to and hybridizable to a given sequence. In some cases, a first sequence that is hybridizable to a second sequence or set of sequences can specifically or selectively hybridize to the second sequence or set of sequences, thereby utilizing hybridization to the second sequence or set of sequences. Hybridizable sequences can share a degree of sequence complementarity over all or a portion of their respective lengths, such as between 25%-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.

[0093] The isothermal amplification methods described herein can provide advantages over existing nucleic acid amplification methods. Non-limiting examples of isothermal nucleic acid amplification methods can include helicase-dependent amplification, nickase amplification, recombinase polymerase amplification, loop-mediated isothermal amplification, and nucleic acid sequence-based amplification.

[0094] The methods described herein can utilize DNA polymerases with high chain displacement activity and specially designed primer sets to exponentially amplify target sequences. Compared to the time of existing nucleic acid amplification methods, the methods provided herein can provide faster time for amplifying target nucleic acid molecules. The nucleic acid target processed by the methods described herein (e.g., by a guide complex or enzyme-mediated nick or cut) can be used as the initial template for use with any existing isothermal amplification. Different existing isothermal amplification methods can utilize different DNA polymerases. Loop-mediated isothermal amplification (LAMP) utilizes two sets of specially designed primers (called inner primers and outer primers) and can be carried out at a constant temperature of 50-65°C (122-149°F). The limitation of LAMP can be the use of non-specific detection methods, which may result in the detection of false positives. Helicase-dependent amplification (HDA) utilizes DNA helicase activity to separate the complementary chains of double-stranded DNA molecules, and therefore temperature cycling can be avoided to produce single-stranded templates for primer hybridization and subsequent primer extension by DNA polymerase. The rolling circle amplification (RCA) method utilizes the sequential amplification of a circular DNA template by a strand-displacing DNA polymerase. RCA operates at a constant temperature (e.g., 37°C-42°C, [98.6-107.6°F]) to generate long single-stranded DNA molecules with tandem repeats of the circular template. Limitations of RCA can include challenges in large-scale production, purification, and storage of target molecules. Multiple displacement amplification (MDA) can utilize random exonuclease-resistant primers and a strand-displacing DNA polymerase. DNA polymerases generate target DNA strands at a constant temperature, such as 30°C (86°F). MDA can also be used for whole genome amplification. The recombinase polymerase amplification (RPA) method is a low-temperature (e.g., 37°C [98.6°F]) isothermal amplification that combines isothermal recombinase-driven primer targeting of the target molecule with strand-displacing DNA activity. RPA utilizes a nucleoprotein complex formed by oligonucleotide primers and recombinase proteins to guide and promote binding to the target DNA strand. Nucleic acid sequence-based amplification (NASBA) is an isothermal, transcription-based amplification method designed for amplifying single-stranded RNA or DNA sequences and performed at a constant temperature of 41°C (105.8°F).

[0095] Example Implementation

[0096] The present disclosure provides methods and compositions for processing nucleic acid molecules comprising a target sequence. In some aspects, the present disclosure provides a method for processing a single-stranded nucleic acid molecule comprising a target sequence. The method may include contacting the single-stranded nucleic acid molecule with a guide complex comprising a guide polynucleotide under conditions where the guide polynucleotide hybridizes with the single-stranded nucleic acid molecule, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence of an enzyme (e.g., a restriction enzyme). The restriction enzyme may be a type IIs restriction enzyme. The guide polynucleotide may further comprise (ii) a target binding region configured to hybridize with the target sequence. The guide polynucleotide may further comprise (iii) a closed 3′ end that is not extendable by a polymerase. In some embodiments, the guide polynucleotide further comprises (i), (ii), and (iii) in a 5′ to 3′ order. The non-target binding region may be located at the 5′ end of the guide polynucleotide. The target binding region may 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 a restriction endonuclease recognition sequence of a type IIs restriction enzyme, the reverse complement being located at the 3' end of the restriction endonuclease recognition sequence of the type IIs restriction enzyme and the 5' end of the target binding region configured to hybridize with the target sequence. In some embodiments, in (b), the excision exposes the extendable 3' end of the target sequence. In some embodiments, the method further comprises reverse transcribing a single-stranded nucleic acid molecule from the RNA.

[0097] The guide polynucleotides provided herein can be forward guide polynucleotides (e.g., forward guide oligonucleotides) configured to treat a target nucleic acid molecule in a reaction. The reaction can further comprise a reverse guide polynucleotide (e.g., reverse guide oligonucleotide) configured to treat a target nucleic acid molecule or the reverse complement of a target nucleic acid molecule in a reaction.

[0098] The conditions under which a guide polynucleotide hybridizes to a single-stranded nucleic acid molecule can be determined empirically or calculated based on the chemical composition of the guide polynucleotide. Various tools (eg, http: / / www.oligoevaluator.com / LoginServlet) are available to calculate annealing / hybridization temperatures and conditions for a given specific sequence of a polynucleotide.

[0099] The target binding region can have a length sufficient to hybridize to the target site under the conditions required 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 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. 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, 9, 8, 7, 6, 5 or fewer nucleotides in length.

[0100] The enzymes described herein can include type IIs restriction enzymes. Type IIs restriction enzymes can include one or more selected from the group consisting of BsmAI, Nt.BsmAI, transcription activator-like effector nuclease, N.Bst9 I, 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 may include type IIs nicking enzymes such as N.BstNBI, N.BspD6I, N.Bst9 I, and Nt.BstNBI, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutI, or any combination thereof. Alternatively, type IIs restriction enzymes may include BfuAI, BsmAI, BsrDI, or BtsIMutI. Other examples of type IIs restriction enzymes may be found at www.neb.com / tools-and-resources / selection-charts / type-iis-restriction-enzymes, which is incorporated herein by reference.

[0101] In some embodiments, IIs type restriction enzyme comprises the IIs type restriction enzyme of through engineering approaches, and it has nuclease inactivation mutation in one of two subunits, so that nickase is produced by the enzyme that is not natural nickase. In some embodiments, IIs type restriction enzyme comprises the II type restriction enzyme of through engineering approaches, and it has mutation in one of two subunits, and this causes the enzyme activity rate of cutting one chain to be different from the enzyme activity rate of cutting opposite chain. In some cases, enzyme comprises two kinds of enzymes with different activities or activity rates. In some cases, enzyme can comprise the subunit of IIs type restriction enzyme. In some cases, enzyme can comprise the subunit of nickase. In some cases, enzyme can comprise the activity for introducing cutting on target nucleic acid sequence. For example, enzyme can be N.BspD6I. In some cases, enzyme can comprise the activity for introducing cutting on the complementary chain of target nucleic acid sequence. In some cases, enzyme can comprise the activity for introducing cutting on guide polynucleotide (for example, the target binding region of guide polynucleotide). For example, enzyme can be Nt.BstNBI.

[0102] The blocked 3' end can comprise essentially any 3' chemical structure that prevents extension of the guide polynucleotide by DNA polymerase. Such structures include, but are not limited to, 3' phosphate, 3' phosphorothioate, 3'-O-methyl, PNA, modified bases, ddNTPs, solid supports, or spacers.

[0103] 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 additional sites for binding to an enzyme. For example, the additional non-target binding region may comprise additional restriction endonuclease recognition sequences for binding to a restriction enzyme. The enzyme recruited by the additional non-target binding region may be the same 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 act as a blocker to block extension of the 3' end of the guide polynucleotide.

[0104] The method for treating a single-stranded nucleic acid molecule can further comprise introducing a Type IIs restriction enzyme under conditions sufficient to allow the Type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the target sequence. The optimal temperature for a specific Type IIs restriction enzyme can be found, for example, in the Rebase database (available at http: / / rebase.neb.com / rebase / rebase.html).

[0105] The method 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 includes a strand-displacing DNA polymerase. In some embodiments, the polymerase includes a large fragment of Bacillus stearothermophilus polymerase, exo-Klenow polymerase, Bst 2.0 polymerase, phi29 DNA polymerase, T7 exo-polymerase, OmniTaq 2LA DNA polymerase, or any combination thereof. Such methods can further include adding other factors together with the polymerase that are sufficient to add nucleotides to the 3' end, including dNTPs, appropriate buffers, and cofactors (e.g., divalent cations). dNTPs can be natural or non-natural dNTPs. Natural dNTPs can include dATP, dCTP, dGTP, dTTP, and / or dUTP. Non-natural dNTPs can be α-thiol dNTPs (e.g., S-dNTPs). S-dNTPS may include dATPαS, dCTPαS, dGTPαS and / or dTTPαS.

[0106] The target sequence treated by the method provided herein can be used for further downstream applications, such as isothermal amplification. In some cases, the reagents used for amplification can be in the same mixture as the reagents used for target treatment. In some aspects, 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 where 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 closed 3' end that is not extendable by a polymerase; (b) introducing a type IIs restriction enzyme to produce an extendable 3' end under conditions sufficient to allow the type IIs restriction enzyme to bind to the restriction endonuclease recognition sequence and cut within the target sequence; and (c) extending the extendable 3' end of the target sequence using a polymerase. In some embodiments, the guide polynucleotide further comprises (i), (ii), and (iii) in a 5' to 3' order. In some embodiments, the non-target binding region further comprises a sequence comprising the reverse complement of a restriction endonuclease recognition sequence of a type IIs restriction enzyme, the reverse complement being located at the 3′ end of the restriction endonuclease recognition sequence of the type IIs restriction enzyme and the 5′ end of the target binding region configured to hybridize with the target sequence. In some embodiments, the guide polynucleotide is a first guide polynucleotide, and the guide complex comprises a second guide polynucleotide, wherein the second guide polynucleotide comprises (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 with the target sequence. In some cases, when the first guide polynucleotide of the guide complex hybridizes with the target polynucleotide sequence, the target binding region of the second guide polynucleotide of the guide complex does not hybridize with 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 regions of the first guide polynucleotide and the second guide polynucleotide to form 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) may comprise one or more guide polynucleotides, including a first guide polynucleotide and a second guide polynucleotide as described herein. The first guide polynucleotide and the second guide polynucleotide may 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 can comprise the same sequence (e.g., a palindromic sequence), and the target binding region at the 3' end of the first or second guide polynucleotide can be different. In some embodiments, the target binding region can be configured to hybridize to a target sequence. Alternatively, the target binding region can be configured to hybridize to different target sequences.

[0107] In some cases, the reverse guide polynucleotide (or complex) can include 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 can include the same sequence (e.g., a palindromic sequence) at the 5' end 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 can include different sequences.

[0108] In some aspects, the present 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 a single-stranded nucleic acid molecule, wherein the guide complex comprises: (i) a first guide polynucleotide comprising a non-target binding region from 5' to 3' 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, wherein the double-stranded binding region is bound to an enzyme; and (b) cleaving the target sequence using an enzyme to expose the extendable 3' end of the target sequence. In some cases, the extendable 3' end is a 3' hydroxyl group. In some embodiments, if the target molecule is RNA, the method may further include reverse transcribing the single-stranded nucleic acid molecule from the RNA before the target molecule is contacted with the guide complex. For example, a reverse transcriptase can be used to reverse transcribe the target RNA molecule to produce a DNA molecule, which can be further processed using the methods described herein. The DNA molecule can be a single-stranded DNA molecule (ssDNA). In some cases, the reverse transcription reaction can be used to prepare an 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).

[0109] In some cases, the method for amplifying a single-stranded nucleic acid molecule comprising a target sequence further includes extending the extendable 3' end of the target sequence with a polymerase to produce an extension product, wherein the extension product displaces the second guide polynucleotide. In some cases, the polymerase extends the double-stranded product that displaces the second guide polynucleotide. In some embodiments, extension is included in the presence of a DNA polymerase (such as a strand displacement DNA polymerase, including any strand displacement polymerase described herein) and is hatched. Extension can also be included in the presence of a factor (including dNTP, appropriate buffer and cofactor (such as divalent cation)) that is sufficient to add nucleotides to the 3' end together with the polymerase and is hatched. dNTP can be natural or non-natural dNTP. Natural dNTP can include dATP, dCTP, dGTP, dTTP and / or dUTP. Non-natural dNTP can be α-thiol dNTP (such as S-dNTP). S-dNTPS can include dATPαS, dCTPαS, dGTPαS and / or dTTPαS.

[0110] In some cases, the method of 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 the extendable 3' end of the first guide polynucleotide. In some embodiments, the cleavage can comprise 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 group.

[0111] In some cases, the method for amplifying a single-stranded nucleic acid molecule comprising a target sequence further includes extending the extendable 3' end of the first guide polynucleotide using a polymerase to produce a complementary molecule of the target sequence of the single-stranded nucleic acid molecule, thereby amplifying the single-stranded nucleic acid molecule. The polymerase can be a chain displacement DNA polymerase, including any chain displacement polymerase described herein. Extension can also be included in the presence of a factor (including dNTP, appropriate buffer and cofactor (such as divalent cation)) sufficient to add nucleotides to the 3' end together with the polymerase. dNTP can be natural or non-natural dNTP. Natural dNTP can include dATP, dCTP, dGTP, dTTP and / or dUTP. Non-natural dNTP can be α-thiol dNTP (such as S-dNTP). S-dNTPS can include dATPαS, dCTPαS, dGTPαS and / or dTTPαS.

[0112] In some embodiments, the second guide polynucleotide in the 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 with the non-target binding region of the first guide polynucleotide and (ii) a target binding region that is configured to hybridize with the target sequence. In some embodiments, the method further comprises, before (b), using an enzyme to cut the first guide polynucleotide in the target binding region, wherein the guide complex dissociates from the single-stranded nucleic acid molecule. In some embodiments, the method further comprises cutting the first guide polynucleotide in the target binding region to expose the extendable 3' end of the first guide polynucleotide, and extending the extendable 3' end of the first guide polynucleotide using a polymerase to repeatedly produce complementary molecules of the target sequence of the single-stranded nucleic acid molecule, to produce multiple complementary molecules of the target sequence of the single-stranded nucleic acid molecule. In some embodiments, additional guide complexes are bound to the complementary molecules. In some embodiments, the method further comprises using the complementary molecule having the additional guide complex bound thereto as a starting template to produce a copy of the target molecule. In some embodiments, the enzyme is a type IIs restriction enzyme. In some embodiments, type IIs restriction enzymes include N.BstNBI, N.Bst9 I 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 comprises a 3′ end that is not extendable by a polymerase. The 3′ end of the blockage can include substantially any 3′ chemical structure that prevents extension of the guide polynucleotide by a DNA polymerase, including any structure with such activity as described herein. In some embodiments, the 3′ end of the blockage includes PNA, modified bases, phosphate groups, ddNTPs, solid supports, or spacers. In some embodiments, a single-stranded nucleic acid molecule with a guide polynucleotide bound thereto is used as a starting template for amplification. In some embodiments, amplification is isothermal amplification. In some embodiments, the enzyme has an asymmetric tendency to cut one chain of the DNA duplex. In some embodiments, the enzyme exhibits high-frequency endonuclease activity. In some embodiments, the high frequency endonuclease activity is 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 from the small subunit of the enzyme. In some embodiments, the enzyme exhibits at least two differential enzyme activity rates. In some embodiments, when cutting two different cleavage sites, at least two differential enzyme activity rates include two differential endonuclease activity rates. In some embodiments, one of the two differential endonuclease activity rates includes cutting a target sequence of a single-stranded nucleic acid molecule at a low frequency. In some embodiments, one of the two differential endonuclease activity rates includes cutting a target binding region of a guide polynucleotide at a high frequency.In some embodiments, the two differential endonucleases have an asymmetric or unequal activity rate. In some embodiments, the enzyme comprises N.BstNBI, N.Bst9 I and N.BspD6I, Nt.BsmAI, BfuAI, BsmAI, BsrDI, BtsIMutI, BfuAI, BsmAI, BsrDI, BtsIMutI or a combination thereof.

[0113] In some embodiments, temperature changes during the method. In some embodiments, the first activity rate of at least two different enzyme activity rates is favorable at a first temperature, and the second activity rate of at least two different enzyme activity rates is favorable at a second temperature that is different from the first temperature. In some embodiments, the first temperature wherein the first enzyme activity rate is favorable can be about 15 ℃, about 16 ℃, about 17 ℃, about 18 ℃, about 19 ℃, about 20 ℃, about 21 ℃, about 22 ℃, about 23 ℃, about 24 ℃, about 25 ℃, about 26 ℃, about 27 ℃, about 28 ℃, about 29 ℃, about 30 ℃, about 31 ℃, about 32 ℃, about 33 ℃, about 34 ℃, about 35 ℃, about 36 ℃, about 37 ℃, about 38 ℃, about 39 ℃, about 40 ℃, about 41 ℃, about 42 ℃, about 43 ℃, about 44 ℃, about 45 ℃, about 46 ℃, about 47 ℃, about 48 ℃, about 49 ℃ or about 50 ℃. In some embodiments, the first temperature where the rate of first enzyme activity is favorable is between about 15-50°C, between about 20-45°C, between about 30-45°C, between about 30-40°C, or between about 32-39°C. In some embodiments, the second temperature in which the rate of activity of the second enzyme is favorable 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 where the rate of second enzyme activity is favorable is between about 45-80°C, between about 50-80°C, between about 50-70°C, between about 50-60°C, or between about 52-58°C.

[0114] In some embodiments, temperature can change for a period of time in the process of this method.The time period of temperature change can help the enzyme activity rate between the reaction periods.The temperature change can comprise the first temperature or the second temperature.In some embodiments, the first temperature change or the second temperature change can occur in 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 of persistent period. In some embodiments, the first temperature change or the second temperature change can occur for a duration of at most about 15 minutes, at most about 12 minutes, at most about 10 minutes, at most about 9 minutes, at most about 8 minutes, at most about 7 minutes, at most about 6.5 minutes, at most about 6 minutes, at most about 5.5 minutes, at most about 5 minutes, at most about 4.5 minutes, at most about 4 minutes, at most about 3.5 minutes, at most about 3 minutes, at most about 2.5 minutes, at most about 2 minutes, at most about 1.5 minutes, at most about 1 minute, at most about 30 seconds, or at most about 15 seconds.

[0115] In some embodiments, the first temperature change or the second temperature change can occur over a duration 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 ...2 minutes to about to about 6 minutes, about 2 minutes to about 7 minutes, about 2 minutes to about 7.5 minutes, about 2 minutes to about 10 minutes, about 2 minutes to about 15 minutes, about 2.5 minutes to about 3 minutes, about 2.5 minutes to about 3.5 minutes, about 2.5 minutes to about 4 minutes, about 2.5 minutes to about 5 minutes, about 2.5 minutes to about 6 minutes, about 2.5 minutes to about 7 minutes, about 2.5 minutes to about 7.5 minutes, about 2.5 minutes to about 10 minutes, about 2.5 minutes to about 15 minutes, about 3 minutes to about 3.5 minutes, about 3 minutes to about 4 minutes, about 3 minutes to about 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 The present invention can be applied to a range of about 15 minutes to about 10 minutes, 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.

[0116] In some embodiments, the enzyme comprises two different active sites or endonuclease domains, which give at least two different enzymatic activities. In some embodiments, the target sequence comprises a recognition site specifically identified by an enzyme or a first activity in at least two different enzymatic activities of an enzyme to introduce cutting. In some embodiments, the target binding region of the guide polynucleotide comprises a recognition site specifically identified by an enzyme or a second activity in at least two different enzymatic activities of an enzyme to introduce cutting. The target binding region can have a length sufficient to hybridize with the target site under the conditions (e.g., temperature, pH, ionic strength) required for determining the target sequence. In some embodiments, the length of 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 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.

[0117] 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 reaction conditions. In some embodiments, the length of 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 some embodiments, the length of 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 some embodiments, the single-stranded nucleic acid molecule is a single-stranded deoxyribonucleic acid (ssDNA) or a 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.

[0118] In some embodiments, compared with nucleic acid amplification schemes without programmed restriction enzymes, the methods described herein can result in faster amplification results. The measure of amplification speed can be a cycle threshold. "Cycle threshold" can include the number of cycles required for a signal (e.g., a fluorescent signal) to exceed a background threshold level. A lower cycle threshold can indicate a larger amount of target nucleic acid in a sample. In some embodiments, compared with loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), or other amplification methods known in the art, nucleic acid amplification using the methods described herein can result in a lower cycle threshold. The cycle threshold of the sample processing method described herein can 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 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 value of the sample processing methods described herein can be at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 25%, at most about 20%, at most about 18%, at most about 15%, at most about 12%, at most about 10%, at most about 8%, at most about 5% or at most about 2% lower than the cycle threshold value of LAMP. The cycle threshold value of the sample processing methods described herein can be about 1% to about 50% lower than the cycle threshold value of LAMP.The cycle threshold values ​​of the sample processing methods described herein can be 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 ... 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%.

[0119] In some embodiments, the cycle threshold value 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 value of the sample processing methods described herein can be at most about 40, at most about 35, at most about 30, at most about 25, at most about 20, at most about 18, at most about 15, at most about 12, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, at most about 5, at most about 4, at most about 3, at most about 2, or at most about 1.

[0120] In some aspects, the present disclosure provides a polynucleotide-polypeptide complex comprising: a single-stranded nucleic acid molecule bound to a guide complex, wherein the guide complex comprises: 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 of an enzyme, which is a type IIs restriction enzyme.

[0121] In some aspects, the present 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 bound to a guide complex comprising a guide polynucleotide, wherein the guide polynucleotide comprises: (i) a non-target binding region comprising a restriction endonuclease recognition sequence of an enzyme, wherein the enzyme 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 non-extendable by a polymerase; and an enzyme that binds to the restriction endonuclease recognition sequence of the non-target binding region.

[0122] The method, system or test kit that this paper provided can be used for processing or analyzing a sample or a target nucleic acid molecule or target sequence.Perhaps, the method, system or test kit that this paper provided can be used for processing or analyzing two or more different samples in the same reaction mixture (for example, single reaction), or two or more different target nucleic acid molecules or target sequences.For example, the method, system or test kit that this paper provided can be used for processing or analyzing 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.

[0123] In various embodiments, the guide polynucleotide comprises a target binding region. The sequence of the target binding region can be designed based on the target sequence by following similar primer design rules. 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, dumbbell, and oligonucleotide DNA nearest-neighbor thermal dynamics," Proc. Natl. Acad. Sci. USA, Vol. 95, 1460-1465 (1998), the contents of which are incorporated herein by reference in their entirety), primer composition (e.g., nucleotide composition such as GC content can be determined and filtered and penalized using software, as can the GC content composition of hairpins, the GC content composition of the 3' end of the primer, and specific parameters that can be evaluated are the length of homopolymer nucleotides, hairpin formation, GC content, and amplicon size), predicted dimer-dimer formation, average extension length, etc. In the case of multiplex reactions with two or more target sequences, the target binding region (or primer) can be designed to minimize cross-reactivity. The non-target binding region of the guide polynucleotide can be designed to be non-hybridizable to the target sequence and contain a sequence that can be recognized by an enzyme (e.g., a restriction enzyme) as described herein.

[0124] In some aspects, the present disclosure provides a method or system for multiplexing more than one nucleic acid molecule, each nucleic acid molecule comprising a different target sequence. For each nucleic acid molecule comprising a different target sequence, the method or system may include a nucleic acid molecule that is combined with a guide complex comprising a guide polynucleotide. The guide polynucleotide may include: (i) a non-target binding region comprising a restriction endonuclease recognition sequence of an enzyme, wherein the enzyme is a type IIS restriction enzyme, (ii) a target binding region configured to hybridize with the target sequence, and (iii) a closed 3′ end that is not extendable by a polymerase. The enzyme can bind to the restriction endonuclease recognition sequence of the non-target binding region. In some aspects, the multiplexing of one or more nucleic acid molecules includes using two or more different sets of primers or guide complexes, each set targeting a different target. In some aspects, the multiplexing of one or more nucleic acid molecules includes a reaction mixture comprising two additional different detection probes or fluorophores, each probe or fluorophore targeting a different target sequence. Each of the two or more different detection probes can be connected to a different fluorophore for multiplex detection.

[0125] Amplification product can be detected by various methods. Amplification product can be detected by gel electrophoresis, so as to detect reaction products with specific lengths. Nucleotide can be, for example, labeled, such as, for example, with biotin labeling. The amplified sequence of biotin labeling can be captured using avidin combined with a signal generating enzyme (such as peroxidase). Nucleic acid detection methods can use dyes for specific dyeing double-stranded DNA. Intercalating dyes that show enhanced fluorescence when combined with DNA or RNA can be used. Dyes can be, for example, DNA or RNA intercalating fluorophores, and can include but are not limited to the following examples: acridine orange, ethidium bromide, Hoechst dye (Hoechst dye), PicoGreen, propidium iodide, SYBRI (a kind of asymmetric cyanine dye), SYBRII, TOTO (a kind of thiazole orange dimer) and YOYO (a kind of oxazole yellow dimer) etc. Dyes can provide the opportunity of improving nucleic acid detection sensitivity when used in combination with various detection methods, and can have different optimal use parameters. Nucleic acid detection methods can also use nucleotides that are directly incorporated into the target sequence or incorporated into a probe containing a sequence that is complementary or substantially complementary to the target of interest. In some embodiments, the present invention relates to a method for detecting a target nucleic acid and a nucleic acid sequence. The method comprises the step of detecting a target nucleic acid and a nucleic acid sequence by using a molecular beacon ...

[0126] In accordance with the present disclosure, a wide range of fluorophores and / or dyes can be used in the methods described herein. Useful 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; also known as spiro(isobenzofuran-1(3H),9′-(9H)xanthene)-5-carboxylic acid, 3′,6′-dihydroxy-3-oxo- 6-carboxyfluorescein); 5-hexachlorofluorescein ([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-6-carboxylic acid]); 6-hexachlorofluorescein ([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-5-carboxylic acid]); 5-tetrachlorofluorescein ([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloyl-fluoresceinyl)-5-carboxylic acid] fluorescein ([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-6-carboxylic acid]); 5-TAMRA (5-carboxytetramethylrhodamine; 9-(2,4-dicarboxyphenyl)-3,6-bis(dimethylamino)xantheneium); 6-TAMRA (6-carboxytetramethylrhodamine; 9-(2,5-dicarboxyphenyl)-3, 6-bis(dimethylamino)xanthene); 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); CalOrange (Bioresearch Technologies); and Rox and suitable derivatives thereof. 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 range). Suitable quenchers can also include DABCYL and variants thereof, such as DABSYL, DABMI, and methyl red. Fluorophores can also be used as quenchers because they tend to quench fluorescence when exposed to certain other fluorophores. In some cases, the quencher can be 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.

[0127] In some embodiments, 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 multiple single-stranded nucleic acid molecules can be processed in the same reaction. In some embodiments, each multiple nucleic acid molecule is derived from a different sample.

[0128] The samples described herein may include biological samples. The samples may include single-stranded nucleic acid molecules. Alternatively, the samples may include double-stranded nucleic acid molecules.

[0129] The sample may comprise a fluid sample. Non-limiting examples of fluid samples may 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.

[0130] In some embodiments, the sample includes 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 fecal sample, a sputum sample, and / or a lymph sample for nucleic acid amplification. In some embodiments, the swab sample includes 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 suffers from a disease, illness, or infection. In some embodiments, the sample includes a purified sample. In some embodiments, the sample is a combination of two, three, four, five, or more types of samples. In some embodiments, the sample comprises one, two, three, four, five, six, seven, eight, nine, ten, or more target nucleic acid molecules.

[0131] The sample can be obtained invasively (e.g., a tissue biopsy) or non-invasively (e.g., a venipuncture). The sample can be an environmental sample. The sample can be a water sample (e.g., a water sample obtained from a lake, stream, river, estuary, bay, or ocean). The sample can be a soil sample. The sample can be a tissue or fluid sample from the subject, such as saliva, semen, blood (e.g., whole blood), serum, synovial fluid, tears, urine, or plasma. The sample can be a tissue sample, such as a skin sample or a tumor sample. The sample can be obtained from a part of an organ of the subject. The sample can be a cell sample. The sample can be a cell-free sample (e.g., a plasma sample containing a cell-free analyte or nucleic acid). The sample can be a solid sample or a liquid sample. The sample can be a biological sample or a non-biological sample. The sample can include an in vitro sample or an in vitro sample. Non-limiting examples of samples include amniotic fluid, bile, bacterial samples, breast milk, buffy coat, cells, cerebrospinal fluid, chromatin DNA, ejaculated semen, nucleic acids, plant-derived material, RNA, saliva, semen, blood, serum, soil, synovial fluid, tears, tissue, urine, water, whole blood or plasma and / or any combination and / or any portion thereof. In one example, the sample can be a plasma sample that can contain DNA. In another example, the sample can include a cell sample that can contain cell-free DNA.

[0132] The sample can be a mammalian sample. For example, the sample can be a human sample. Alternatively, the sample can 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.

[0133] The sample can include nucleic acids (e.g., circulating and / or cell-free DNA fragments). Nucleic acids can be derived from eukaryotic cells, prokaryotic cells, or non-cellular sources (e.g., viral particles). Nucleic acids can refer to substances whose molecules are composed of many nucleotides connected in long chains. Non-limiting examples of nucleic acids include artificial nucleic acid analogs (e.g., peptide nucleic acids, morpholino oligomers, locked nucleic acids, ethylene 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 can be double-stranded or single-stranded. The sample can include nucleic acids that can be intracellular. Alternatively, the sample can include nucleic acids that can be extracellular (e.g., cell-free). The sample can include nucleic acids that can be fragmented (e.g., chromatin).

[0134] The sample can be obtained from a virus, a bacterium, an archaea, or a eukaryote. In some embodiments, the sample is obtained from a bacterium. The bacterium can be a spherical bacterium, a rod-shaped bacterium, a spiral-shaped bacterium, a comma-shaped bacterium, or a corkscrew-shaped bacterium. Non-limiting examples of bacteria are Streptococcus pneumoniae, Streptococcus pyogenes, Legionella pneumonia, Bordetella bronchiseptica, Enterobacter aerogenes, Pasteurella multocida, Proteus mirabilis, Staphylococcus aureus, Haemophilus influenzae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Trichomonas vaginalis, Neisseria gonorrhoeae, Chlamydia pneumoniae, 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 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 acids from the sample. In some embodiments, sample preparation can include extracting nucleic acids from the sample by heating the sample. For example, during the heating phase of nucleic acid amplification, target nucleic acids (e.g., target RNA, target DNA) can be extracted or released from a biological sample. As an alternative or supplement to heating, a box system can be used to extract or release target nucleic acids (e.g., target RNA, target DNA) from a biological sample, wherein the sample can be mixed with a lysis buffer and then extracted through a filter to capture the target nucleic acid in the filter. In some cases, the box system can also include a washing step to remove contaminants. An elution buffer can be added to the box to remove the target nucleic acid from the filter for further processing or analysis. The box system can be an automatic box system. In some cases, the box system can be an M1 Sample Box kit (SKU: 3000536, Biomeme, Inc.). In some cases, the sample preparation methods described herein can use a box system for automated sample processing. Details of the sample preparation box 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 various other methods or any commercially available nucleic acid extraction kit or method.

[0135] In some aspects, the present disclosure provides a test kit comprising any guide complex or any guide polynucleotide described herein. In some embodiments, the test kit further comprises a probe or dye for detecting the amplification product produced using the test kit. In some embodiments, the test kit further comprises informational material describing the instructions for use of the test kit. In some embodiments, the information includes the optimal reaction temperature, or its optimal buffer conditions, for amplification using the guide complex or guide polynucleotide. In some embodiments, the test kit further comprises a type II restriction enzyme compatible with the guide polynucleotide or guide complex described herein. In some embodiments, the test kit further comprises a strand displacement polymerase. The test kit can be separated for ease of use and can include one or more containers containing reagents. In some embodiments, all test kit components are packaged together. Alternatively, one or more individual components of the test kit can be provided in a package separate from other test kit components.

[0136] Computer system

[0137] The present disclosure provides a computer system programmed to implement the method of the present disclosure. Figure 17 A computer system 1701 is shown that can be programmed or otherwise configured to analyze polynucleotide-polypeptide complexes. Alternatively or additionally, computer system 1701 can be programmed or otherwise configured to analyze single-stranded nucleic acid molecule processing data. Computer system 1701 can be a user's electronic device or a computer system remotely located relative to the electronic device. The electronic device can be a mobile electronic device.

[0138] Computer system 1701 includes a central processing unit (CPU, also referred to herein as a "processor" and "computer processor") 1705, which can be a single-core or multi-core processor, or multiple processors for parallel processing. 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 peripherals 1725, such as cache memory, other memory, data storage, and / or electronic display adapters. Memory 1710, storage 1715, interface 1720, and peripherals 1725 communicate with CPU 1705 via a communication bus (solid lines), such as a motherboard. Storage 1715 can be a data storage unit (or data repository) for storing data. Computer system 1701 can be operatively coupled to a computer network ("network") 1730 via communication interface 1720. Network 1730 may be the Internet, an internet network, and / or an extranet, or an intranet and / or extranet in communication with the Internet. In some cases, network 1730 is a telecommunications and / or data network. Network 1730 may include one or more computer servers that may implement distributed computing, such as cloud computing. In some cases, network 1730 may implement a peer-to-peer network with the aid of computer system 1701, which may enable devices coupled to computer system 1701 to behave as clients or servers.

[0139] The CPU 1705 can execute a series of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a memory location such as memory 1710. The instructions can be directed to the CPU 1705, which can then program the CPU 1705 or otherwise configure the CPU 1705 to implement the methods of the present disclosure. Examples of operations performed by the CPU 1705 can include fetching, decoding, executing, and writing back.

[0140] CPU 1705 may be part of a circuit such as an integrated circuit. One or more other components of system 1701 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0141] Storage unit 1715 can store files such as drivers, libraries, and saved programs. Storage unit 1715 can store user data, such as user preferences and user programs. In some cases, computer system 1701 may include one or more additional data storage units that are external to computer system 1701, such as on a remote server that communicates with computer system 1701 via an intranet or the Internet.

[0142] 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 personal computers (e.g., portable PCs), tablet or tablet PCs (e.g., iPad, Galaxy Tab), phones, smartphones (e.g. iPhone, Android-enabled devices, ) or personal digital assistant. Users can access computer system 1701 via network 1730.

[0143] The methods described herein can be implemented in the form of machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system 1701, such as, for example, in the memory 1710 or electronic storage unit 1715. The machine executable code or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1705. In some cases, the code can be retrieved from the storage unit 1715 and stored in the memory 1710 for ready access by the processor 1705. In some cases, the electronic storage unit 1715 can be eliminated and the machine executable instructions can be stored in the memory 1710.

[0144] The code may be precompiled and configured for use with a machine having a processor suitable for executing the code, or may be compiled during runtime. The code may be provided in a programming language that may be selected so that the code can be executed in a precompiled or compiled form.

[0145] Various aspects of the systems and methods (such as computer system 1701) provided herein can be embodied in programming. Various aspects of the present technology can be considered to be "products" or "articles" in the form of machine (or processor) executable code and / or associated data, typically carried or embodied on a type of machine-readable medium. Machine executable code can be stored in an electronic storage unit such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. "Storage" type media can include any or all tangible memories 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 part of the software can sometimes be communicated over the Internet or various other telecommunications networks. For example, such communication can enable software to be loaded from one computer or processor to another, such as from a management server or host to a computer platform of an application server. Therefore, another type of medium that can carry software elements includes light waves, radio waves, and electromagnetic waves, such as across physical interfaces between local devices, through wired and optical landline networks, and through various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered to be the medium that carries the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0146] Thus, machine-readable media such as computer executable code may take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media include, for example, optical or magnetic disks (such as any storage device in any computer, etc.), such as those that may be used to implement the databases shown in the accompanying drawings, etc. 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 optical fiber, including the wires that make up a bus within a computer system. Carrier transmission media may take the form of electrical 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, a floppy disk, a flexible disk, a hard disk, magnetic tape, any other magnetic medium, a CD-ROM, a DVD or DVD-ROM, any other optical medium, punched card stock tape, any other physical storage medium having a pattern of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave that transmits data or instructions, a cable or link that transmits such a carrier wave, 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 carrying one or more sequences of one or more instructions to a processor for execution.

[0147] The computer system 1701 may include or communicate with an electronic display 1735 that includes 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.

[0148] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit 1705. For example, the algorithms may analyze single-stranded nucleic acid molecule processing data.

[0149] Example

[0150] Example 1: Programmed Restriction Enzyme (PRE) Composition

[0151] In this experiment, a guide molecule directs strand cleavage at a specific programmed site on a single-stranded nucleic acid sequence, e.g. Figures 1A-1J As shown. The PRE compositions or methods provided herein may be referred to as DTECT, and the two terms may be used interchangeably herein. Figure 1AIn the present invention, a double-stranded oligonucleotide (110) is formed by two separate oligonucleotides (115), each of which contains 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 oligonucleotide (115). The guide molecule PNA sequence (116) has a blocking portion at its 3' end. The guide molecule nucleic acid sequence (117) is located at the 5' end of the oligonucleotide (115). The guide molecule nucleic acid sequence (117) is self-complementary at a non-target complement region (e.g., a non-target binding region). The double-stranded oligonucleotide (110) forms a complex with a restriction endonuclease (120) at a selected site on the guide molecule nucleic acid sequence (117) of each oligonucleotide (115). The double-stranded oligonucleotide-restriction endonuclease complex binds to the target single-stranded nucleic acid sequence (100) at the target region (101).

[0152] Figure 1B The cleavage sites for the high frequency endonuclease (130) and the low frequency endonuclease (135) are shown. If the high frequency endonuclease cleaves, the double-stranded oligonucleotide-restriction endonuclease complex will dissociate from the target. If the low frequency endonuclease cleaves, it will create an open and extendable 3' end on the target strand. Figure 1C The polymerase (140) is shown extending from the 3' end provided by the low frequency endonuclease. Figure 1D The polymerase (140) is shown dissociating after completion of synthesis of a strand (160), which has displaced one of the oligonucleotides (115) from the double-stranded oligonucleotide (110).

[0153] Figure 1E The cleavage site for the high frequency endonuclease (130) and the cleavage site for the low frequency endonuclease (135) are shown again. At this stage, if the low frequency endonuclease cuts, the structure will be regenerated. However, if the high frequency endonuclease cuts, it will produce an open and extendable 3' end on the oligonucleotide chain. Figure 1F The polymerase (140) is shown extending from the 3' end provided by the high frequency endonuclease. The polymerase (140) displaces the guide molecule PNA sequence (116) and produces the target synthetic strand (170).

[0154] Figure 1G The cleavage site of the high frequency endonuclease (130) is shown. Figure 1H The high frequency endonuclease is shown to cleave and create an open and extendable 3' end on the target strand, where the polymerase (140) binds and extends to produce another target synthetic strand (171), displacing the previous target synthetic strand (170).

[0155] Figure 1I and1J It is shown that the target synthetic strand (170), which is the complement of the target region (101) of the target single-stranded nucleic acid sequence (100), serves as a new target for forming an additional synthetic strand (172), which represents a copy of the target single-stranded nucleic acid sequence (100). The synthetic strand, which is a copy of the target single-stranded nucleic acid sequence (100), is the starting material for strand displacement amplification.

[0156] Figure 1K An exemplary completed extension on a new guide molecule is depicted.

[0157] In some experiments, the method continued, e.g. Figure 1L As shown, endonucleolytic activity occurs on the second complementary strand oligonucleotide / extension product complex (170). Figure 1M The polymerase (140) is depicted extending the 3' end of the cleavage site of the second complementary strand of the oligonucleotide / extension product complex. Endocleavage activity occurs on the newly synthesized strand (42). Figure 1N ),and Figure 1O The displaced single-stranded synthetic fragment (42) serves as the starting material for additional strand displacement amplification reactions.

[0158] Example 2: Asymmetric programmed restriction enzyme 1

[0159] In this experiment, the guide molecules were designed to have a single point mutation so that they could still bind to the target DNA. Using the method of Example 1, an additional chain was generated that did not contain the point mutation of the guide, but instead retained a product with the correct complementary sequence of the target (different from the first synthesized chain).

[0160] exist Figure 2A In the process, a primer / adapter double-stranded oligonucleotide with a single mismatch (C to T mismatch) is complexed with the target (template) single-stranded DNA. The low-frequency endonuclease cuts the target DNA and digests it within the target region of the template to generate an extension of the new 3′ end of the template ( Figure 2B High frequency endonuclease site activity leads to cleavage, displacement of the guide molecule, and synthesis of new chains ( Figure 2C ). This first strand has thymidines, but all subsequently synthesized sequences have cytosines that match the complement of the original template region of the target strand.

[0161] In this experiment, low-frequency endonuclease activity is the key step, allowing the generation of a product that feeds into the strand displacement reaction.

[0162] Molecular beacon single nucleotide polymorphism (SNP) analysis was performed to distinguish between primer extension (using Cy5 fluorescent dye; black triangles) and guide oligonucleotide exonuclease activity (using FAM fluorescent dye; grey circles).

[0163] In the first control experiment, the guide and primers contained no mismatches. The results showed that amplification resulted in an increase in the fluorescence of the probe containing the same sequence as the DNA target (which bound to the amplified complementary target). Figure 3B However, when a mismatch is introduced into the guide sequence, the fluorescence is enhanced for the probe containing the same sequence as the DNA target, rather than for the probe containing the complement of the guide sequence ( Figure 4B ). The sequences used in control experiment 1 and mismatch experiment 1 can be found in Table 1, Figure 3A and Figure 4A Found in.

[0164] In a second control experiment, the guide and primers contained no mismatches. The results showed that amplification resulted in an increase in the fluorescence of the probe containing the same sequence as the DNA target (which bound to the amplified complementary target). Figure 5B However, when a mismatch is introduced into the guide sequence, the fluorescence is enhanced for the probe containing the same sequence as the DNA target, rather than for the probe containing the complement of the guide sequence ( Figure 6B ). The sequences used in control experiment 2 and mismatch experiment 2 can be found in Table 1, Figure 5A and Figure 6A This example demonstrates that the oligonucleotide acts as a guide for endonuclease activity, rather than a primer. In addition, this example demonstrates that endonuclease activity occurs on both strands of the hybridized oligonucleotide in the complex.

[0165] Table 1: Sequences used in Example 2

[0166]

[0167]

[0168]

[0169] Example 3: Asymmetric programmed restriction enzyme 2

[0170] In this experiment, a detection molecule with an internal fluorophore-quencher pair is used as the target. Figures 7A-7B As shown in Table 2, when unpaired, the target molecule is self-complementary and self-quenching. However, the molecule is fluorescent when double-stranded. Using the method of Example 1, different end-guides were tested for extension by Bst polymerase. The different end-guides shown in Table 2 each contain a target non-complementary endonuclease recognition site.

[0171] Figures 7C-7D The extension of the guide molecule and the endonuclease recognition site are shown. N.BstNBI endonuclease has an optimal temperature of approximately 55°C, while Nt.BsmAI endonuclease has an optimal temperature of approximately 37°C. The test conditions included Bst polymerase favoring ( Figure 8A ), Bst polymerase and Nt.BsmAI temperature is favorable ( Figure 8B ), Bst polymerase and N.BstNBI temperature is favorable ( Figure 8D ), and Bst polymerase is equally favorable with N.BstNBI and Nt.BsmAI ( Figure 8C The thermocycler protocol was 15 cycles (3.5 minutes) at 40°C (Nt.BsmAI preference) followed by 160 cycles at 58°C (N.BstNBI preference) under reaction conditions that favor Nt.BsmAI activity over N.BstNBI activity, resulting in two stages of asymmetric enzyme activity.

[0172] In reactions where N.BstNBI performs the primary cut, the oligonucleotide falls off at the reaction temperature due to the overlap of only four bases. In reactions where Nt.BsmAI performs the primary cut, Bst polymerase uses the guide molecule as a target, activating the N.BstNBI cutting activity. The resulting extension of the cut guide and the opening of the probe enhance Fam fluorescence. In reactions where both N.BstNBI and Nt.BsmAI cut, the oligonucleotide will decompose due to the overlap of only one base.

[0173] Table 3 summarizes Figures 8A-8D results. Bst polymerase can extend from the 3′ end of the DNA base, but blocks extension via 2′-O-methyl RNA bases or phosphorylated bases. Nt.BsmAI has no effect on Bst extension. Systems with both Nt.BsmAI and N.BstNBI show that the two enzymes work synergistically to speed up the reaction rate. This 2-enzyme system maximizes enzyme activity using temperature regulation over time to asymmetrically cut the target, producing defined / designed oligonucleotides that can be used for subsequent amplification reactions (such as SDA). In this dual enzyme system, target cutting by Nt.BsmAI is the rate-limiting step. N.BstNBI seems to behave like a 2-enzyme asymmetric restriction enzyme system; this can be understood as the small subunit of N.BstNBI acting as a lower activity restriction endonuclease, and the large subunit acting as a higher activity restriction endonuclease.

[0174] Table 2: Guide molecules

[0175]

[0176] Table 3: Amplification Summary

[0177]

[0178]

[0179] Example 4: Asymmetric programmed restriction enzyme 3

[0180] This experiment used the method of Example 3, using different guides. The different end guides are shown in Table 4. The test conditions included Bst polymerase being favorable ( Figure 9A ), Bst polymerase and Nt.BsmAI temperature is favorable ( Figure 9B ), Bst polymerase and N.BstNBI temperature is favorable ( Figure 9D ), and Bst polymerase is equally favorable with N.BstNBI and Nt.BsmAI ( Figure 9C ).

[0181] Table 5 summarizes Figures 9A-9D Results. Bst polymerase can extend from the 3' end of DNA bases but blocks extension from 2'-O-methyl RNA bases or phosphorylated bases. Nt.BsmAI has no effect on Bst extension. In a system containing both Nt.BsmAI and N.BstNBI, the two enzymes work synergistically to overcome the 3' block. The N.BstNBI system shows a slow release of the extension block.

[0182] Table 4: Guide molecules

[0183]

[0184] Table 5: Amplification Summary

[0185]

[0186]

[0187] Example 5: LAMP enhancement using PRE

[0188] This experiment compared loop-mediated isothermal amplification (LAMP), PRE (also known as DTECT), and a combination of LAMP and PRE priming. PRE was performed as described in Example 1. The results showed that PRE-enhanced LAMP had a lower cycle threshold ( ) than PRE alone or LAMP alone. Figures 10A-10B ). Amplification enhancement using PRE used approximately 100 copies of hRNA / reaction. Table 6 shows the primers used in this experiment.

[0189] This experiment demonstrates that symmetric endonuclease activity can generate starting products for isothermal amplification systems such as LAMP and shorten time to results. The increased reaction rate is not limited to SDA.

[0190] Table 6: LAMP guide molecules

[0191]

[0192] Example 6: Modification of enzyme activity by guide design

[0193] This experiment used the same detection molecule with an internal fluorophore-quencher pair as in Example 3 ( Figures 11A-11B However, the assay also includes further modified leads that modify enzyme activity ( Figure 11C ).

[0194] This experiment uses the enzyme BspQI, which has a primary cleavage site next to its recognition site (boxed) and a forced cut site on the guide. Nt.BspQI (also used as a control in this experiment) is a triple-mutant form of BspQI that has top-strand DNA nicking activity.

[0195] In the reaction where BspQI cuts at its primary cleavage site, the oligonucleotide falls off at the reaction temperature due to only one base overlap. In the reaction where BspQI is forced to perform asymmetric secondary cleavage, Bst polymerase targets the guide molecule, activating BspQI's primary cleavage activity. The resulting extension of the cleavage guide and the opening of the probe enhance Fam fluorescence. In the reaction where BspQI cuts at two cleavage sites, the oligonucleotide will decompose due to only three base overlaps. The guides used in this experiment are shown in Table 7.

[0196] Primer C shows an increase in fluorescence from the extra copy of the endonuclease, which means that either the Bst polymerase activity is faster than the endonuclease activity or the endonuclease holds the target together after cleavage to allow Bst extension ( Figure 12A ). Guide D showed fluorescence enhancement with endonuclease and Bst extension was blocked in the absence of endonuclease, which means that asymmetric endonuclease activity allows bypassing the guide blockade and Nt.BspQI has bottom-strand nuclease activity ( Figure 12B ). Guide E shows fluorescence enhancement with endonuclease; Bst extension is blocked in the absence of endonuclease; and fluorescence enhancement with Nt.BspQI ( Figure 12C ). This suggests that asymmetric endonuclease activity allows for bypassing guide blockade; Nt.BspQI possesses bottom-strand nuclease activity; and that enzyme activity can be modulated using different guide chemistries. Guide H exhibits fluorescence enhancement with nickase; Bst extension is blocked in the absence of endonuclease; and 2'O-MeO on the opposing bottom endonuclease cleavage prevents the fluorescent reporter ( Figure 12DThis means that inhibition of BspQI bottom cleavage allows the guide to cleave only at the top, thus "shortening" the system. Additionally, top-strand cleavage by Nt.BspQI may not be as efficient under these conditions. Guide F showed fluorescence enhancement from the extra copy of the endonuclease, which means that either Bst polymerase activity is faster than endonuclease activity or the endonuclease holds the target together after cleavage to allow Bst extension ( Figure 12E ). Guide G showed minimal fluorescence enhancement, which means that either both endonucleases were inhibited or the nuclease activity was enhanced relative to the polymerase activity ( Figure 12F ).

[0197] Figures 13A-13B Comparison of leader F with leader C shows a slight increase in signal when the methoxylation on the leader is close to the cleavage site.

[0198] This experiment demonstrates that guide molecule extension can be blocked by various moieties and that restriction enzyme activity can be modified to exhibit asymmetry, accelerating cleavage activity on one side over the other. Modification of the guide molecule can direct endonuclease activity to a specific, desired location on the target, while also enabling the removal of guide blockade by modifying either the endonuclease activity or the strand-displacing polymerase activity in the system.

[0199] Table 7: Guide molecules

[0200]

[0201] Example 7: Isothermal SDA after restriction digestion target product production

[0202] This experiment used the method of Example 1 and then performed isothermal SDA to observe the effect on the resulting amplification of the target product. A monkeypox titration 10-fold dilution series ( Figures 14A-14B ). The amplification results are as follows Figure 14C As shown. Monkeypox virus amplification was also performed in a NP matrix direct amplification reaction ( Figures 14D-14E ).

[0203] Example 8: Multiplexed Isothermal Amplification

[0204] This experiment used the method of Example 1 and performed isothermal SDA on Neisseria gonorrhoeae, Chlamydia trachomatis and RPP30 samples. The reaction conditions of the triple experiment were as follows: Figure 15A M1Sample Target quantification is based on M1 Sample 100% recovery from the sample culture was assumed. The performance of the triple isothermal reaction showed amplification of all three reactions ( Figure 15B ).

[0205] This experiment also used the method of Example 1 and performed isothermal SDA on Neisseria gonorrhoeae, Chlamydia trachomatis, RPP30, and Trichomonas vaginalis samples. As in the triple experiment, the quadruple isothermal reaction showed competitive amplification under the four reaction conditions ( Figure 16A The reaction conditions for the quadruple experiment are as follows. Figure 16B shown.

[0206] Although 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. Without departing from the present invention, those skilled in the art will now contemplate various modifications, changes, and substitutions. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to practice the present invention. The following claims are intended to define the scope of the present invention and thus encompass methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for treating 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 the 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) blocked 3′ end that is non-extendable by polymerase; as well as (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 cut within the target sequence. 2 . The method of claim 1 , wherein in (b), the cleavage exposes the extendable 3′ end of the target sequence. The method of claim 2 , further comprising extending the extendable 3′ end using a polymerase.

4. 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 the 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 a Type IIs restriction enzyme, (ii) a target binding region configured to hybridize to the target sequence, and (iii) blocked 3′ end that is non-extendable by polymerase; (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 cut within the target sequence to produce an extendable 3' end; and (c) extending the extendable 3' end of the target sequence using a polymerase.

5. The method of any one of claims 1 to 4, wherein the guide polynucleotide is a first guide polynucleotide and the guide complex comprises a second guide polynucleotide, wherein the second guide polynucleotide comprises (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 that is configured to hybridize to the target sequence.

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

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

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. The method of claim 11 or 12, further comprising, before (b), cleaving the first guide polynucleotide within the target binding region using the enzyme, wherein the guide complex dissociates from the single-stranded nucleic acid molecule.

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

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

16. The method of claim 15, further comprising using the complementary molecule with the additional guide complex bound thereto as a starting template to produce 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. The method according to any one of claims 1-10 and 17, wherein the type IIs restriction enzyme comprises N.BstNBI, N.Bst9 I, N.BspD6I, a functional fragment thereof, or a combination thereof.

19. The method of any one of claims 11-18, wherein the guide polynucleotide comprises a blocked 3' end that is non-extendable by a polymerase.

20. The method of any one of claims 1-10 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. The method of any one of claims 1-19, wherein the single-stranded nucleic acid molecule having the cleavage and the guide polynucleotide bound thereto is used as a starting template for amplification.

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

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

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

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

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

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

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

29. 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 at a low frequency.

30. The method of claim 27, wherein one of the two differential rates of endonuclease activity comprises cleaving the target binding region of the guide polynucleotide at a high frequency.

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

32. The method of claim 27, wherein the enzyme comprises BsmAI, Nt.BsmAI, a transcription activator-like effector nuclease, N.Bst9 I, 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. The method of any one of claims 27-32, wherein the temperature is varied during the process.

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

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

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

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

39. The method of any one of claims 1-38, wherein 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.

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

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

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

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

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

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

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

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

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

51. The method of any one of claims 1-50, 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. The method of claim 51, wherein the two or more single-stranded nucleic acid molecules are contained in a single reaction mixture.

53. The method of any one of claims 4-52, wherein the method of amplifying a single-stranded nucleic acid molecule shortens a cycle threshold value or a result-out time value in nucleic acid amplification as compared to an otherwise identical method that amplifies the single-stranded nucleic acid molecule in the absence of the guide complex.

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

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

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

57. A polynucleotide-polypeptide complex comprising: A single-stranded nucleic acid molecule associated with a guide complex, wherein the guide complex comprises: (i) a first guide polynucleotide comprising a non-target binding region from 5′ to 3′ and a region adjacent to the target site; a target binding region that hybridizes to a target sequence of a 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 comprises an enzyme restriction endonuclease. The enzyme is a type IIs restriction enzyme.

58. A system for processing a single-stranded nucleic acid molecule comprising a target sequence, the system comprising: The single-stranded nucleic acid molecule to which is bound a guide complex comprising a guide polynucleotide, 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 is non-extendable by a polymerase; and The enzyme binds to the restriction endonuclease recognition sequence of the non-target binding region.

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

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

61. The kit of claim 59 or 60, wherein the kit further comprises informational material describing instructions for using the kit.

62. A system for processing a plurality of single-stranded nucleic acid molecules, each single-stranded nucleic acid molecule comprising a different target sequence, the system comprising: a first single-stranded nucleic acid molecule, wherein the first single-stranded nucleic acid molecule is associated with a first guide complex comprising a first guide polynucleotide, wherein the first guide polynucleotide comprises: (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 non-extendable by a polymerase; and a second single-stranded nucleic acid molecule, wherein the second single-stranded nucleic acid molecule is associated with a second guide complex comprising a second guide polynucleotide, wherein the second guide polynucleotide comprises: (i) a second non-target binding region comprising a second restriction endonuclease recognition sequence for the enzyme, which is 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 non-extendable by a polymerase; 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 system of claim 62, further comprising a third single-stranded nucleic acid molecule, wherein the third single-stranded nucleic acid molecule is associated with a third guide complex comprising a third guide polynucleotide, wherein the third guide polynucleotide comprises: (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 non-extendable by a polymerase; wherein the enzyme being a type IIs restriction enzyme binds to the third restriction endonuclease recognition sequence of the third non-target binding region.

64. The system of claim 62 or 63, further comprising a fourth single-stranded nucleic acid molecule, wherein the fourth single-stranded nucleic acid molecule is associated with a fourth guide complex comprising a fourth guide polynucleotide, wherein the fourth guide polynucleotide comprises: (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 non-extendable by a polymerase; wherein the enzyme being 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 62-64, wherein the first single-stranded nucleic acid molecule and the second single-stranded nucleic acid molecule are from different samples.

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

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

68. The system of claim 66, wherein the virus is selected from the group consisting of 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 retrotranscriptase virus, and a double-stranded DNA retrotranscriptase virus.

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