Nick initiated cycling emission and replacement reaction

Abstract

This disclosure describes details of polymerase-free, isothermal methods for detecting nucleotide sequences. Methods use target-specific hairpin oligonucleotides, which also specifically interact with probes labelled with fluorescent dye and quencher moieties, to amplify the detection of target polynucleotides by repeating cycles of endonuclease-mediated nicking of the probes to drive the denaturation of fluorophore-labeled probe fragments away from hairpin-probe complexes to disassociate the fluorescent labels from quenchers.

Description

PCT Patent Application Attorney Docket No: 181.0004-WO00 NICK INITIATED CYCLING EMISSION AND REPLACEMENT REACTION FIELD OF THE INVENTION

[0001] The field of this invention relates to isothermal, polymerase-free methods for detecting nucleic acid sequences. BACKGROUND

[0002] Described below are details of a novel approach to dramatically increase the speed and sensitivity of current target nucleic acid detection methods. This is achieved at room temperature. Unlike RT-PCR there is no need for expensive enzymes or nucleotides at depressed temperatures, or for extensive lab equipment temperature cycling / imaging which significantly reduces costs. Limited cold storage and equipment makes this process extremely portable and accessible. In addition, the reagents for this process can be lyophilized to further facilitate accessibility of this technology.

[0003] The increases in speed and sensitivity are leveraged by unique use of a hairpin loop as a site of hydrolysis to indicate the presence of a target and coordinate events that drive signal amplification following specific detection of target sequences. The mechanism lies in linking the restriction site to a target to generate signal through a target toehold or tail hairpin. The result is multiplexing capability defined in part by using single target-specific hairpins, loop sequence-specific nicking endonucleases, and nicking endonuclease functional reaction temperatures. Remarkably the limits of detection are at the single molecule level, which allows for single nucleotide allelic differentiation. Significant fluorescence signal increase is mediated by rapid fluorescence release from unlinked linear or exponential repeated strand displacement from a hairpin loop, nicking endonuclease target sequence.

[0004] The approaches described below can be used with or without target extraction, purification or washing, for single molecule detection of target nucleic acid (with tested sensitivity as low as five copies per reaction). This is achieved using a nicking endonuclease initiated fluorescent dye emission and nucleic acid strand displacement. This technology eliminates the need for complex equipment and reaction setup, multi-enzymatic interactions as well as temperature-controlled cycling. The entire reaction can be conducted at room temperature in less than 20 minutes.

[0005] Another advantage of the methods that follow are that they do not require elevated temperatures for reaction conditions, as is the case for qPCR, LAMP, or EXPAR methods. Moreover, the methods described below also offer improved accuracy over PCR-based detection methods, because, unlike a PCR-based method, there is no risk of introducing amplicon contamination to a reaction or to future reactions.

[0006] Other advantages of the methods include: (i) reduced labor and costs, as sample process and reaction times are significantly reduced; (ii) a greater than a triple increase in sample processing volume;PCT Patent Application Attorney Docket No: 181.0004-WO00 (iii) no requirement for a reverse transcription reaction; and (iv) optional DNA or RNA extractions, as NICER is a method, which can be performed directly from specimen collection to analysis. These foregoing advantages enable NICER methods to be accessible for field applications, such as in developing countries or remote areas. SUMMARY OF INVENTION

[0007] The inventions described herein relate to specific, polymerase-free, isothermal methods for amplifying the detection of target nucleic acid sequences.

[0008] In one aspect, the invention is a method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide contains a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method containing the following steps: (A) contacting the one or more target polynucleotides with a plurality of hairpin oligonucleotides, wherein each hairpin oligonucleotide contains: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the hairpin C sequence), wherein the double-stranded stem region contains a nucleotide sequence (the hairpin B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the hairpin B sequence); and (ii) a single-stranded nucleotide sequence (the hairpin A’ sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the hairpin A’ sequence and the hairpin B’ sequence of one or more of the hairpin oligonucleotides to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of the hairpin oligonucleotide with the target polynucleotide, wherein hybridization of the hairpin B’ sequence to the B sequence of the target polynucleotide opens the stem-loop structure of the hairpin to expose a single-stranded sequence comprising the hairpin C sequence and the hairpin B sequence; (C) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the hairpin C sequence (the C' sequence) and a nucleotide sequence of a nicking endonuclease (NE) site, to the exposed single-stranded C sequence of the hairpin structure to form one or more double- stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; andPCT Patent Application Attorney Docket No: 181.0004-WO00 (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE site is located between the fluorescent and quencher moieties; (D) contacting the one or more double stranded hybridized probe complexes with one or more NEs under reaction conditions that allow the one or more NEs to nick the double stranded hybridized probe complex at the one or more respective NE sites, wherein the nicking reactions generate two probe fragments, each of which denature from the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (i) the fluorescent moiety to emit a fluorescent signal; and (ii) the harpin C sequence to hybridize another probe in the plurality of probes; and (E) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

[0009] In one embodiment, the method further comprises repeating steps (C) and (D) until a desired level of signal amplification is reached or a until a reaction component is exhausted.

[0010] In another aspect, the invention is a method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide contains a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method containing the following steps: (A) contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1s), wherein each HP1 contains: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP1 C sequence), wherein the double-stranded stem region contains a nucleotide sequence (the HP1 B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the HP1 B sequence); and (ii) a single-stranded nucleotide sequence (the HP1 A’ sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of HP1 with the target polynucleotide, wherein hybridization of the HP1 B’ sequence to the B sequence of the target polynucleotide opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence;PCT Patent Application Attorney Docket No: 181.0004-WO00 (C) contacting the complex of HP1 with the target polynucleotide with a plurality of second hairpin oligonucleotides (HP2s), wherein each HP2 contains: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region, wherein: (a) the double-stranded stem region contains a nucleotide sequence (the HP2 C’ sequence) complementary to the HP1 C sequence in a double strand with a complementary sequence (the HP2 C sequence); and (b) the single-stranded loop region contains a nucleotide sequence complementary to the HP1 B’ sequence (the HP2 B sequence) flanked by a nucleotide sequence that is complementary to at least a contiguous portion of the HP1 A’ sequence (the HP2 AS sequence) (ii) a single-stranded nucleotide sequence (the HP2 B’S sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence that is complementary to at least a contiguous portion of the HP1 B sequence; (D) hybridizing the HP2 B’S, HP2 C’, the HP2 B, and HP2 AS sequences of one or more of the HP2s to the HP1 B, HP1 C, HP1 B’, and HP1 A’ sequences, respectively, of the complex of HP1 with the target polynucleotide, to form a complex of HP2 and HP1 with the target polynucleotide, wherein hybridization of the HP2 to HP1 opens the stem-loop structure of the HP2 to expose a single- stranded sequence comprising the HP2 C sequence; (E) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the HP1 C sequence (the C' sequence) and a nucleotide sequence of a nicking endonuclease (NE) site, to the exposed HP1 C sequence and exposed single-stranded HP2 sequence of the complex of HP2 and HP1 with the target polynucleotide, to form one or more double-stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; and (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE site is located between the fluorescent and quencher moieties; (F) contacting the one or more double stranded hybridized probe complexes with one or more NEs under reaction conditions that allow the one or more NEs to nick the double stranded hybridized probe complex at the one or more respective NE sites, wherein the nicking reactions generatePCT Patent Application Attorney Docket No: 181.0004-WO00 two probe fragments, each of which denature from the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (i) the fluorescent moiety to emit a fluorescent signal; and (ii) the exposed HP1 and exposed single-stranded HP2 C sequences to hybridize another probe in the plurality of probes; and (G) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

[0011] In some embodiments, the HP2 C’ sequence may contain at least one phosphorothioate modification.

[0012] In another aspect, the invention is a method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide contains a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method containing the following steps: (A) contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1s), wherein each HP1 contains: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP1 C sequence), wherein the double-stranded stem region contains a nucleotide sequence (the HP1 B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the HP1 B sequence), wherein the HP1 B sequence contains a nucleotide sequence of a first nicking endonuclease (NE1) site; and (ii) a single-stranded nucleotide sequence (the HP1 A’ sequence), either 5’ to the double stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of HP1 with the target polynucleotide, wherein hybridization of the HP1 B’ sequence to the B sequence of the target polynucleotide opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence; (C) contacting the complex of HP1 with the target polynucleotide with a plurality of second hairpin oligonucleotides (HP2s), wherein each HP2 contains: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP2 A sequence), wherein the double-stranded stem region contains aPCT Patent Application Attorney Docket No: 181.0004-WO00 nucleotide sequence (the HP2 B’ sequence) complementary to the HP1 B sequence in a double strand with a complementary sequence (the HP2 B sequence); and (ii) a single-stranded nucleotide sequence (the HP2 C’ sequence), 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence that is complementary to the HP1 C sequence and a nucleotide sequence of a second NE (NE2) site; (D) hybridizing the HP2 C’ sequence and HP2 B’ sequence of one or more of the HP2s to the exposed, single-stranded HP1 C sequence and HP1 B sequence, respectively, of the complex of HP1 with the target polynucleotide, to form a complex of HP2 and HP1 with the target polynucleotide, wherein hybridization of the HP2 to HP1 opens the stem-loop structure of the HP2 to expose a single-stranded sequence comprising the HP2 A sequence and the HP2 B sequence; (E) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the HP1 C sequence (the C' sequence) and the nucleotide sequence of the NE2 site, to the exposed HP1 C sequence, to form one or more double-stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; and (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE2 site is located between the fluorescent and quencher moieties; (F) contacting the one or more double stranded hybridized probe complexes with one or more NE2s under reaction conditions that allow the one or more NE2s to: (i) nick the double stranded hybridized probe complex at the one or more respective NE2 sites, wherein the nicking reactions generate two probe fragments, each of which denature from the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (a) the fluorescent moiety to emit a fluorescent signal; and (b) the exposed HP1 and exposed single-stranded HP2 C sequences to hybridize another probe in the plurality of probes; and; (ii) nick the NE2 site of the HP2 C’ sequence of the complex of HP2 and HP1 with the target polynucleotide, thereby allowing: (a) a short 3’ HP2 C’ fragment to denature; andPCT Patent Application Attorney Docket No: 181.0004-WO00 (b) a long HP2 fragment comprising a 5’ HP2 C’ sequence and the HP2 B’, A, and B sequences to be displaced by another HP2 (G) contacting the complex of HP2 and HP1 with the target polynucleotide with one or more NE1s under reaction conditions that allow the one or more NE1s to nick the NE1 site of the HP1 B sequence of the complex of HP2 and HP1 with the target polynucleotide, thereby allowing a nicked HP1 B fragment to denature; (H) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

[0013] In one embodiment, the method further contains the steps of: (I) contacting the long HP2 fragment comprising a 5’ HP2 C’ sequence and the HP2 B’, A, and B sequences with another HP1 from the plurality of HP1s, thereby hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the HP2 A sequence and the HP2 B sequence, respectively, to form a complex of HP1 with HP2, wherein hybridization of the HP1 B’ sequence to the HP2 B sequence opens the stem-loop structure of the HP1 to expose a single- stranded sequence comprising the HP1 C sequence and the HP1 B sequence; and (J) Repeating steps (D)-(H) until a desired level of signal amplification is reached or a until a reaction component is exhausted.

[0014] In another aspect, the invention is a method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide contains a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method containing the following steps: (A) contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1s), wherein each HP1 contains: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP1 C sequence), wherein the double-stranded stem region contains a nucleotide sequence (the HP1 B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the HP1 B sequence); and (ii) a single-stranded nucleotide sequence (the HP1 A’ sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of HP1 with the target polynucleotide, wherein hybridization of the HP1 B’ sequence toPCT Patent Application Attorney Docket No: 181.0004-WO00 the B sequence of the target polynucleotide opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence; (C) contacting the complex of HP1 with the target polynucleotide with a plurality of second hairpin oligonucleotides (HP2s), wherein each HP2 contains: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP2 A sequence), wherein the double-stranded stem region contains a nucleotide sequence (the HP2 B sequence) complementary to the HP1 B sequence in a double strand with a complementary sequence (the HP2 B’ sequence); (ii) a single-stranded nucleotide sequence (the HP2 C’ sequence), 5’ to the double stranded stem region, wherein the single-stranded nucleotide sequence contains a nucleotide sequence that is complementary to the HP1 C sequence and a nucleotide sequence of a first nicking endonuclease (NE1) site; and (iii) a nucleotide sequence of second NE (NE2) site between the HP2 B sequence and the HP2 C’ sequence; (D) hybridizing the HP2 C’ sequence,HP2 B’ sequence, and HP2 A sequence of one or more of the HP2s to the exposed, single-stranded HP1 C sequence, HP1 B’ sequence, and HP1 A’ sequence respectively, of the complex of HP1 with the target polynucleotide, to denature HP1 from the target polynucleotide and form a complex of HP2 and HP1, wherein hybridization of the HP2 to HP1 opens the stem-loop structure of the HP2 to expose a single-stranded sequence comprising the HP2 B’ sequence; (E) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the HP1 C sequence (the C' sequence) and the nucleotide sequence of the NE1 site, to the exposed HP1 C sequence, to form one or more double-stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; and (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE1 site is located between the fluorescent and quencher moieties; (F) contacting the one or more double stranded hybridized probe complexes with one or more NE1s under reaction conditions that allow the one or more NE1s to: (i) nick the double stranded hybridized probe complex at the one or more respective NE1 sites, wherein the nicking reactions generate two probe fragments, each of which denature fromPCT Patent Application Attorney Docket No: 181.0004-WO00 the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (a) the fluorescent moiety to emit a fluorescent signal; and (b) the exposed HP1 and exposed single-stranded HP2 C sequences to hybridize another probe in the plurality of probes; and; (ii) nick the NE1 site of the HP2 C’ sequence of the complex of HP2 and HP1, thereby allowing a short 5’ HP2 C’ fragment to denature; and (G) contacting the complex of HP2 and HP1 with one or more NE2s under reaction conditions that allow the one or more NE2s to nick the NE2 site between the HP2 B sequence and the HP2 C’ sequence, thereby allowing a nicked 3’ HP2 C’ fragment to denature and forming a complex nicked HP2 and HP1; (H) contacting another HP2 from the plurality of HP2s with the HP1 of the complex of nicked HP2 and HP1, thereby hybridizing the HP2 C’ sequence, HP2 B’ sequence, and HP2 A sequence of the one or more HP2s to the exposed, single-stranded HP1 C sequence, HP1 B’ sequence, and HP1 A’ sequence respectively the displacing the nicked HP2 comprising the HP2 B, HP2 A, and HPB’ sequence; (I) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

[0015] In some embodiments, the HP2 B’ sequence of the displaced, nicked HP2 from step (H) hybridizes to the exposed, single-stranded HP1 sequence formed in step (B), thereby allowing for hybridization of the HP1 A’ sequence and HP1 B’ sequence of another HP1 from the plurality of HP1s, wherein hybridization of the HP1 B’ sequence to the HP2 B sequence of the HP2 fragment B sequence opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence, the method further comprising repeating steps (D)-(H) until a desired level of signal amplification is reached or a until a reaction component is exhausted.

[0016] In some embodiments, the methods disclosed herein are conducted isothermally.

[0017] In some embodiments, the methods disclosed herein are conducted at room temperature.

[0018] In another aspect, the invention is a polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ or 3’ to 5’, an A’ sequence, a B’ sequence, a C sequence, and a B sequence, wherein: the A’ sequence contains a primary target-specific nucleotide sequence; the B’ sequence contains a secondary target-specific nucleotide sequence; the C sequence contains a nicking endonuclease recognition site nucleotide sequence; the B sequence contains a nucleotide sequence complementary to the B’ sequence, and optionally, contains a nicking endonuclease recognition site nucleotide sequence; andPCT Patent Application Attorney Docket No: 181.0004-WO00 the B’ and B sequences are hybridized to each other to form a stem-loop structure, wherein the loop contains the C sequence.

[0019] In some embodiments of the polynucleotide hairpin, the A’ sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0020] In some embodiments of the polynucleotide hairpin, the B’ and B sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0021] In some embodiments of the polynucleotide hairpin, the C sequence contains 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0022] In some embodiments of the polynucleotide hairpin, the nicking endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

[0023] In another aspect, the invention is a polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ or 3’ to 5’, an B’S sequence, a C’ sequence, a B sequence, an AS sequence, and a C sequence, wherein: (a) the B sequence contains a nucleotide sequence that is identical to a secondary target-specific nucleotide sequence; (b) the B’S sequence contains a sequence complementary to, but contains fewer nucleotides than, the B sequence; (c) the C’ sequence contains a nicking endonuclease recognition site nucleotide sequence and at least one modified nucleotide; (d) the AS sequence contains a nucleotide sequence that is identical to at least a contiguous portion of a primary target-specific sequence; (e) the C’ and C sequences are hybridized to each other to form a stem-loop structure, wherein the loop contains the B and AS sequences; and (f) the C’ sequence contains at least one modified nucleotide that prevents nicking endonuclease activity at the nicking endonuclease recognition site nucleotide.

[0024] In some embodiments of the polynucleotide hairpin, the A sequence contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0025] In some embodiments of the polynucleotide hairpin, the B sequences contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0026] In some embodiments of the polynucleotide hairpin, the C and the C’ sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0027] In some embodiments of the polynucleotide hairpin, modified nucleic acid of (e) is a phosphorothioate-modified nucleic acid, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), aPCT Patent Application Attorney Docket No: 181.0004-WO00 dNTP / ribonucleotide triphosphates (rNTP) hybrid, isoguanosine (isoG); isocytosine (isoC), dUTP, rATP, rCTP, rGTP, or rUTP.

[0028] In some embodiments of the polynucleotide hairpin, the at least one modified nucleic acid is positioned: 1-4 nucleotides from the 5’ end; at an internal nucleotide position; or at or near the 3’ end.

[0029] In another aspect, the invention is a polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ a C’ sequence, a B sequence, an A sequence, and a B’ sequence, wherein: (a) the A sequence contains a primary target-specific nucleotide sequence; (b) the B sequence contains a secondary target-specific nucleotide sequence and a first nicking endonuclease recognition site nucleotide sequence; (c) the B’ sequence contains a nucleotide sequence that is complementary to the B sequence; (d) the B and B’ sequences are hybridized to each other to form a stem-loop structure, wherein the loop contains the A sequence; and (e) the C’ sequence contains a second nicking endonuclease recognition site nucleotide sequence.

[0030] In another aspect, the invention is a polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ a B sequence, an A sequence, a B’ sequence, and a C’ sequence, wherein: (a) the A sequence contains a primary target-specific nucleotide sequence; (b) the B sequence contains a secondary target-specific nucleotide sequence; (c) the B’ sequence contains a nucleotide sequence that is complementary to the B sequence; (d) the B and B’ sequences are hybridized to each other to form a stem-loop structure, wherein the loop contains the A sequence; (e) the interface of the C’ and B sequences contains a second nicking endonuclease recognition site nucleotide sequence; and (f) the C’ sequence contains a second nicking endonuclease recognition site nucleotide sequence.

[0031] In some embodiments of the polynucleotide hairpin, the A sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0032] In some embodiments of the polynucleotide hairpin, the B and B’ sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0033] In some embodiments of the polynucleotide hairpin, the C’ sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0034] In some embodiments of the polynucleotide hairpin, the nicking endonuclease recognition sites, or the first and the second nicking endonuclease recognition sites, are independently a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0035] In another aspect, the invention is a linear single-stranded polynucleotide probe comprising a C’ sequence, a dye molecule, and a quencher molecule, wherein: the C’ sequence contains, in order from 5’ to 3’ or 3’ to 5’: an optional X sequence comprising 1-8 nucleotides; a nicking endonuclease recognition site nucleotide sequence; and an optional Y sequence comprising 1-8 nucleotides.

[0036] In some embodiments of the polynucleotide probe, the nicking endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

[0037] In some embodiments of the polynucleotide probe, the probe contains an X sequence.

[0038] In some embodiments of the polynucleotide probe, the X sequence is labeled with a dye, wherein the dye is: internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

[0039] In some embodiments of the polynucleotide probe, the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

[0040] In some embodiments of the polynucleotide probe, the X sequence is labeled with a quencher moiety, wherein the quencher moiety is: internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

[0041] In some embodiments of the polynucleotide probe, the quencher moiety is Black Hole Quencher (BHQ).

[0042] In some embodiments of the polynucleotide probe, the probe contains an Y sequence.

[0043] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a dye, wherein the dye is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence.

[0044] In some embodiments of the polynucleotide probe, the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.

[0045] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a quencher moiety, wherein the quencher moiety is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0046] In some embodiments of the polynucleotide probe, the quencher moiety is Black Hole Quencher (BHQ).

[0047] In another aspect, the invention is a linear single-stranded polynucleotide probe comprising a C’ sequence, a dye molecule, and a quencher molecule, wherein: the C’ sequence contains, in order from 5’ to 3’ or 3’ to 5’: an optional X sequence comprising 1-4 nucleotides; a first nicking endonuclease recognition site nucleotide sequence; an optional Y sequence comprising 1-4 nucleotides; a second nicking endonuclease recognition site nucleotide sequence; and an optional Z sequence comprising 1-4 nucleotides.

[0048] In some embodiments of the polynucleotide probe, the nicking endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

[0049] In some embodiments of the polynucleotide probe, the probe contains an X sequence.

[0050] In some embodiments of the polynucleotide probe, the X sequence is labeled with a dye, wherein the dye is: internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

[0051] In some embodiments of the polynucleotide probe, the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

[0052] In some embodiments of the polynucleotide probe, the X sequence is labeled with a quencher moiety, wherein the quencher moiety is: internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

[0053] In some embodiments of the polynucleotide probe, the quencher moiety is Black Hole Quencher (BHQ).

[0054] In some embodiments of the polynucleotide probe, the probe contains an Y sequence.

[0055] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a dye, wherein the dye is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence.

[0056] In some embodiments of the polynucleotide probe, the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0057] In some embodiments of the polynucleotide probe, the Y sequence is labeled with a quencher moiety, wherein the quencher moiety is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence. In some embodiments of the polynucleotide probe, the quencher moiety is Black Hole Quencher (BHQ). BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 depicts 5’ and 3’ versions of target-specific hairpins used in a linear NICER method (Method 0.0). The A’ sequence (A’) is a primary target site-specific sequence (the A sequence) that is typically 6-24 nucleotides in length. The B’ sequence is a secondary target site-specific sequence (the B sequence), which is located either 3’ or 5’ of the primary target site, A. In a closed hairpin, B’ hybridizes with its complement, B, to form a stem-loop structure in which the loop includes the C sequence (C). C contains a nicking endonuclease nicking site that is either partially located in the stem or fully located in the loop of the hairpin. X and Y are optional linker sequences located between B’ and C or between B and C, respectively, and are typically 1 to 10 nucleotides in length.

[0058] Fig.2 depicts 5’ and 3’ versions of probe signal substrate nucleotide sequences used in linear NICER methods. B’ is optionally, either fully present, partially present, or absent. When B’ is partially or fully present, it hybridizes with the B sequence of the hairpin when B becomes exposed as the hairpin opens after A’ and B’ of the hairpin hybridize with the A and B target sites. X’ and Y’ are optional sequences that are complementary to the X and Y sequences of the hairpin, which are, typically, 1 to 10 nucleotides in length. C’ contains the nicking endonuclease catalytic site, which is indicated by the circumflex (^). Z is an optional sequence that, if present, either flanks B’ or, if the probe does not include B’ or includes a partial B’ sequence, includes nucleotides that are noncomplementary to a B sequence (negative bases). Any one or more, or all, of B’, C’, X’, Y’, or Z can be labelled with fluorescent dyes and / or quenchers that are positioned at or near 5’ ends 3’ or positioned internally, and can, optionally, include modified nucleotides, like, for example, locked nucleic acids (LNA).

[0059] Fig.3 depicts the hybridization of a 5’ tail target-specific hairpin of a linear NICER probe. A’ of the hairpin hybridizes to A of the RNA target, resulting in the invasion and migration of the RNA B strand to B of the hairpin, thereby resulting in the opening of the hairpin to expose C and B of the hairpin.

[0060] Fig.4 depicts Probe 1 hybridization to the exposed C and B sequences of a hairpin following hybridization of A’ and B’ of the hairpin to A and B of the probe. C’ of the probe contains the recognition site for a specific nicking endonuclease that nicks the probe following its hybridization to the exposed A’ and B’ sequences of the hairpin. The star represents a fluorophore and Q represents a quencher.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0061] Fig.5 depicts the release of the C’ probe fragment in a linear NICER method following nicking of the hybridized probe by to cause the fluorophore-linked nicked portion of the probe to denature from C of the hairpin, thereby separating the fluorophore from the quencher, which, in turn, results in fluorescence.

[0062] Fig.6 depicts the hybridization of an intact fluorophore-labeled probe to the exposed single- stranded portion of hairpin C following a nicking reaction as depicted in Fig.5. Hybridization of the new probe causes strand invasion and displacement of the previously nicked probe from the complex hairpin- target complex.

[0063] Fig.7 depicts the increase in fluorescent signal as the events of nicking and strand displacement are cycled repeatedly until the reaction is terminated.

[0064] Fig.8A is a signal generation plot showing the specific detection of SARS-CoV-2 N1 (nucleocapsid) RNA using a linear probe NICER method. Samples were diluted to concentrations of 40, 20, 18, 16, 14, 12, 10, 5, and 1 virus genomes / genome per µL.

[0065] Fig.8B s is a signal generation plot corresponding to Fig.8A using reactions with no target RNA. three negative control replicate experiments

[0066] Fig.8C shows limit of detection (LoD) triplicate plots for 40 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0067] Fig.8D shows LoD triplicate plots for 20 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0068] Fig.8E shows LoD triplicate plots for 18 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0069] Fig.8F shows LoD triplicate plots for 16 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0070] Fig.8G shows LoD triplicate plots for 14 cp / µl of SARS-CoV-2N1 (nucleocapsid) target DNA / µL.

[0071] Fig.8H shows LoD triplicate plots for 12 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0072] Fig.8I shows LoD triplicate plots for 10 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0073] Fig.8J shows LoD triplicate plots for 5 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0074] Fig.8K shows LoD triplicate plots for 1 cp / µl of SARS-CoV-2 N1 (nucleocapsid) target DNA / µL.

[0075] Fig.9A shows signal generation plots of control experiments without target nucleic acid to determine proper concentration of hairpin to minimize the potential for oligonucleotide impurities contributing to background noise. Reaction conditions were 25 °C at a hairpin reaction concentration of 50 nM.

[0076] Fig.9B shows signal generation plots of control experiments without target nucleic acid to determine proper concentration of hairpin to minimize the potential for oligonucleotide impurities to cause background noise. Reaction conditions were 25 °C at a hairpin reaction concentration of 25 nM.

[0077] Fig.9C shows signal generation plots of control experiments without target nucleic acid to determine proper concentration of hairpin to minimize the potential for oligonucleotide impurities to cause background noise. Reaction conditions were 25 °C at a hairpin reaction concentration of 10 nM.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0078] Fig.9D shows signal generation plots of control experiments without target nucleic acid to determine proper concentration of hairpin to minimize the potential for oligonucleotide impurities to cause background noise. Reaction conditions were 25 °C at a hairpin reaction concentration of 5 nM.

[0079] Fig.10A is a signal generation plot showing failed fluorescent detection of SARS-CoV-2 RNA using a NICER hairpin specific to SARS-CoV-2 B.1.1.7 - N501Y variant. The signal pattern is atypical, and the signal generation collapses over time to generate a dome shaped plot.

[0080] Fig.10B is a signal generation plot showing the negative control results corresponding to Fig.10A using reactions with no target RNA.

[0081] Fig.10C is a consolidated signal generation plot of the data in Figs.8A, 8B, 10A, and 10B.

[0082] Fig.11 depicts a variation of the NICER approach (Method 2) that further includes a linear probe, as described for Figs.1-10. Either the linear probe or hairpin probe can hybridize to the open hairpin cause displacement. This method amplifies signal exponentially.

[0083] Fig.12 depicts the acceleration of signal amplification as consequence of utilizing a probe that contains a target sequence to increase the number of hybridization and nicking events. Fig.15 illustrates a universal probe structure in which the nicking nuclease site (NS) is flanked by X and Y domains which can be used to differentiate probes in a multiplex version of NICER. The Xand Y domains typically contain 0-8 nucleotides each. Universal probes are typically modified by attachments of 5’, internally positioned, 3’ dyes, and / or quenchers.

[0084] Fig.13 depicts a dual enzyme probe structure wherein C’ is composed of X’ wherein 0-4 nucleotides; NS1 comprises the nicking site for first Nicking Endonuclease; Y’ comprises 0-4 nucleotides; NS2 comprises the nicking site for the second endonuclease; and Z’ comprises 0-4 nucleotides. There is potential for greater than 5 nucleotides at X’, Y’, or Z’. The probe herein could contain 5’; internally positioned, or 3’ dyes, quenchers, or the combination of performance.

[0085] Fig.14 depicts a dual enzyme probe structure wherein C’ is composed of X’ wherein 0-4 nucleotides; NS1 comprises the nicking site for first Nicking Endonuclease; Y’ comprises 0-4 nucleotides; NS2 comprises the nicking site for the second endonuclease; and Z’ comprises 0-4 nucleotides. There is potential for greater than 5 nucleotides at X’, Y’, or Z’. The probe herein could contain 5’; internally positioned, or 3’ dyes, quenchers, or the combination of performance.

[0086] Fig.15 depicts a version of NICER that uses a universal probe (NICER method 0.5). The target- specific A’ and B’ hairpin sequences hybridize to the A and B target sequences, which, in turn, causes the stem to open and leave C and B exposed so that C’ of the probe hybridizes to C of the hairpin. Following a nicking reaction and disassociation of the labeled C’ probe fragment, a new universal probe hybridizes to the exposed portion of hairpin C and the cycle repeats.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0087] Fig.16 depicts a two hairpin, accelerated version of NICER (NICER Method 1). Hairpin 1 (HP1) contains a A, B, B’, and C sequences, wherein B and B’ flank C and hybridize to each other to form a stem structure. Hairpin 2 (HP2) contains a shortened B’ sequence (B’S), C, C’, and B sequences, and a shortened A sequence (AS). C and C’ flank B and As, and hybridize to each other to form a stem structure. A’ of HP1 hybridizes to A of the target template sequence and the hairpin performs strand displacement while B’ of HP 1 hybridizes to B of the target template sequence. C and B of HP1 become single stranded, which allows for C’ of a universal probe to hybridize with the exposed C sequence of HP1. Following the denaturing off of the probe fragment generated by the nicking reaction, a new, un-nicked probe to hybridize to the exposed HP1 C sequence. The HP2 B’S sequence hybridizes to HP1 B, which results in HP1 and HP2 experiencing strand displacement. HP1 denatures from the template sequence and HP2 hybridizes to HP1. In turn, HP2 hybridizes to C, B’, and A’ of HP1. The original template becomes available for hybridization to a new HP1 and the cycle of steps repeats.

[0088] Fig.17 depicts an exponential version of NICER (“Method 4.2”) that includes dual hairpins, a universal probe, and two endonuclease sites / enzymes. Method 4.2 NICER includes a first hairpin with a 5’ overhang (HP1) a second hairpin with a 3’ overhang (HP2). In the method, (i) A’ of hairpin 1 (HP1) hybridizes to target template sequence A; (ii) HP1 performs strand displacement; (iii) HP1 B’ hybridizes to target sequence B; (iv) C and B of HP1 become single stranded; (v) probe C’ hybridizes to the exposed C sequence of HP1; (vi) a first nicking endonuclease nicks at a first recognition site on the probe to generate two fragments that denature off of HP1; and (vii) new un-nicked probe hybridizes to the exposed C sequence of HP1 and (v)-(vii) repeat and cycle; (viii) C’ of hairpin 2 (HP2) hybridizes to the exposed C sequence of HP1; (ix) HP2 performs strand displacement, B’ of HP2 hybridizes to B of HP1; (x) The exposed A and B sequences of HP2 act as target sequences for a non-hybridized HP1; (xi) the first endonuclease nicks at the recognition in site C’ of HP2 and a new un-nicked HP2 displaces the nicked HP2; and (xii) a second endonuclease nicks HP1 at the second nicking endonuclease recognition site in B’, allowing a short, double-stranded sequence resulting from nicking in C’ of HP1 and B’ of HP2 to denature, resulting in a free target template sequence on the resulting large HP2 fragment.

[0089] Fig.18 depicts an alternative iteration of an exponential NICER method (NICER Method 4.1). The method uses a first and a second hairpin, both with 5’ overhangs. In Method 4.1 reactions, (i) A’ of HP1 hybridizes to A of target template sequence; (ii) HP1 performs strand displacement, B’ of HP1 hybridizes to B of target template sequence; (iii) C and B of HP1 becomes single stranded; (iv) C’ probe can bind to exposed C portion of HP1; (v) a first nicking endonuclease nicks at recognition site on probe to generate two fragments, which (vi) denature off of HP1; and (vii) new un-nicked probe binds to exposed C portion of HP1 and (iv)-(vi) repeat and cycle; (viii) C’ of HP2 hybridizes to C of HP1; (ix) HP1 and HP2 experience strand displacement and HP1 denatures from template sequence and HP2 hybridizes to HP1, and B and A of HP2PCT Patent Application Attorney Docket No: 181.0004-WO00 hybridize to B’ and A’ of HP1; (x) original template sequence presented on HP2 becomes available for a new HP1 to hybridize (xi) C’ of HP2 may be nicked by both the first and second endonuclease, creating small fragments, each with a similar Tm below 25 °C that may denature off of HP1; (xii) a new, un-nicked HP2 may invade and hybridize to HP1, displacing the nicked HP2; and (xiii) the displaced large HP2 fragment may either form a hairpin, or B’ of HP2 may hybridize to an exposed B of HP1, keeping the nicked HP2 open and providing a new target template sequence for unhybridized HP1 to invade.

[0090] Fig.19 is a signal generation plot that shows the signal generation between a probe with an internal quencher and a probe with an internal dye.

[0091] Fig.20 illustrates the signal generation between Method 0 (linear method, hairpin specific probe), and Method 0.5 (linear, universal probe).

[0092] Fig.21 is a signal generation plot showing NICER Method 0.5 results from comparing various template concentrations tested with a lyophilized NICER master mix.

[0093] Fig.22 contains a signal generation plot for Method 0.5 that shows differences in signal generation relative to different universal probe concentrations (top panel). The bottom panel is an signal generation plot for Method 0.5 that shows differences in signal generation relative to different hairpin concentrations.

[0094] Fig.23 shows multiplexing NICER signal amplification data for two different targets, including a mismatched hairpin and probe combination to demonstrate specificity. (1) Template present with matching FAM hairpin & probe; (2) Template present with matching ROX hairpin & probe; (3) Template present with matching FAM hairpin & probe; (4) Template present with matching ROX hairpin & probe;(5) Template present with matching FAM hairpin & probe; (6) Template present with mismatched ROX hairpin & FAM probe. (7) Template present with matching ROX hairpin & probe; (8) Template present with mismatched FAM hairpin & ROX probe.

[0095] Fig.24 shows data obtained from comparing NICER 0.5 methods in which the reaction mix contained different amounts of 6000 MW PEG. Experimental conditions include CFX96, 1s x 250 cycles (not including imaging), and a Master Mix in which all components are combined then added to template. The bar graph shows how template concentration and PEG concentrations affect signal amplification.

[0096] Fig.25 shows data obtained from comparing NICER 0.5 methods in which the reaction mix contained either 6000 or 8000 MW PEG or no PEG. The bar graph shows how template concentration and PEG concentrations affect signal amplification.

[0097] Fig.26 is a signal generation plot showing a comparison in signal generation between method 0.5 and method 1 at CFX96, 25˚C, 1s cycle x 250 cycles not (including imaging) using 50nM template, 50nM hairpin, 50nM probe.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0098] Fig.27 is a signal generation plot showing a comparison of background signals detected when using a probe with a terminal quencher and dye or a probe with internal quencher and terminal dye.

[0099] Fig.28 contains a signal generation plot comparing background signal generated using a modified hairpin 2 in a Method 1 NICER (top panel). The background signal in the sample without template (gray) is indistinguishable from the sample with template (black). The bottom panel is an signal generation plot of a Method 1 NICER performed using a hairpin 2 that had been modified to protect the nicking site in its C’ domain. Noticeable distinction between background signal in sample without template (gray) and sample with template (black). DETAILED DESCRIPTION

[0100] This disclosure provides details of the invention of polymerase-free isothermal methods for amplifying the number of signalling events generated following the specific detection of target nucleic acid sequences and subsequent repeated cycles of endonuclease-mediated nicking events that result in the disassociation of a dye moieties from quencher moieties, thereby amplifying the generation of signal events over multiple cycles.

[0101] Some methods of the invention use pluralities of single target-specific hairpin oligonucleotides, universal probes, and nicking endonucleases to detect a target single-stranded nucleotide sequence in a sample and then linearly amplify the number of detectable fluorescence signalling events over the course of multiple cycles of the reaction. In such methods of the invention, generally referred to herein as a Method 0.5 methods, a method detects target nucleotide sequences in a sample by contacting a target polynucleotide in a sample, wherein the target polynucleotide contains the target nucleotide sequence, with an oligonucleotide comprising a hairpin structure (“the hairpin”) that is characterized by having at least one single-stranded region that is adjacently positioned either 5’ or 3’ to the hairpin. The single- stranded sequence contains an “A’ sequence”, which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is a portion of the target sequence designated as the primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is contiguous with the A sequence and is described herein as secondary (or second) target sequence. The B’ sequence of a hairpin oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing to a complementary B sequence. The loop structure of the hairpin contains a “C sequence”, which is flanked by the B’ and B sequences and contains the sequence of a recognition site for a nicking endonuclease (NE). Upon contacting the target polynucleotide with the hairpin, the hairpin A’ sequence hybridizes to the A sequence of the target polynucleotide to serve as a toehold for the hairpin as the B sequence of the secondary target sequence invades the hairpin’s stem structure, thereby resulting in the hairpin’s B’ sequence hybridizing to the secondary target, which, in turn, causes the hairpin’s stem-loopPCT Patent Application Attorney Docket No: 181.0004-WO00 structure to open and expose a single-stranded sequence comprising the hairpin C sequence and the hairpin B sequence. Subsequently, an oligonucleotide probe of the invention, which contains a C’ sequence that is complementary to the C sequence of the hairpin, hybridizes to the C sequence, thereby forming a complex of the opened hairpin and the probe that is double-stranded where the hairpin’s C sequence and the probe’s C’ hybridize. The probe is labeled with a quenchable fluorescent dye moiety linked to the probe at or near its 3' end or 5’ end, depending on the orientation of the 5’ or 3’ order of the A’, B’, C, and B sequences of the hairpin, or the fluorescent dye moiety is positioned at an internal position in the probe. The probe is also labeled with a fluorescence quenching molecule (the quencher), which is also linked to the probe at or near its 3' end or 5’ end, depending on the 5’ or 3’ orientation of the hairpin, or the quencher is positioned at an internal position in the probe, so long as the NE site is located between the fluorescent and quencher moieties. Upon a NE contacting a double stranded hairpin-probe complex, a nicking reaction generates two probe fragments, one labeled with the fluorescence dye moiety, and the other with the quencher moiety, thereby, permitting the fluorescence dye moiety to emit a fluorescence signal. Following the denaturation of the two probe cleavage fragments from the C sequence of the open hairpin, a new labeled probe hybridizes to the C sequence of the hairpin, which, in turn, is followed by repeating cycles of probe hybridization, cleavage, denaturation, and signal emission.

[0102] Some methods of the invention use pluralities of pairs of hairpin oligonucleotides, single universal probes, and nicking endonucleases to detect a target single-stranded nucleotide sequence in a sample and then linearly amplify the number of detectable fluorescence signaling events over the course of multiple cycles of the reaction. In such methods of the invention, generally referred to herein as a Method 1.0 methods, the use of dual hairpins enables the presentation of additional universal probe binding sites. In Method 1.0 methods, the method detects target nucleotide sequences in a sample by contacting a target polynucleotide in a sample, wherein the target polynucleotide contains the target nucleotide sequence, with an first oligonucleotide comprising a hairpin structure (“the HP1”) that is characterized by having at least one single-stranded region that is adjacently positioned either 5’ or 3’ to a double-stranded stem and single-stranded loop region. The single-stranded sequence contains an “A’ sequence”, which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is a portion of the target sequence designated as the primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is contiguous with the A sequence and is described herein as secondary (or second) target sequence. The B’ sequence of a HP1 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing to a complementary B sequence. The loop structure of the hairpin contains a “C sequence”, which is flanked by the B’ and B sequences and contains the sequence of a recognition site for a nicking endonuclease (NE). Upon contacting the targetPCT Patent Application Attorney Docket No: 181.0004-WO00 polynucleotide with the HP1, the HP1 A’ sequence hybridizes to the A sequence of the target polynucleotide to serve as a toehold for the hairpin as the B sequence of the secondary target sequence invades the HP1’s stem structure, thereby resulting in the HP1’s B’ sequence hybridizing to the secondary target, which, in turn, causes the HP1’s stem-loop structure to open and expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence.

[0103] Method 1.0 methods also use a plurality of second hairpin oligonucleotides (HP2s), that are specific for HP1, and that are characterized by having at least one single-stranded region that is adjacently positioned either 5’ or 3’ to a double-stranded stem and single-stranded loop region. The single-stranded sequence contains an “B’S sequence” (i.e., a shortened B’ sequence), which is complementary to at least a contiguous portion of the HP1 B sequence. The B’S sequence is adjacent to the C’ sequence of the HP2. The C’ sequence is complementary to the C sequence of HP1. The C’ sequence is contiguous with the B’S sequence. The C’ sequence of a HP2 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing to a complementary C sequence. The loop structure of the HP2 contains a “B sequence”, which is contiguous with the C’ sequence, and a “AS sequence” that is complementary to at least a portion of the HP A’ sequence (i.e., a shortened A sequence), which is flanked by the HP2 C sequence. Upon contacting the HP1 complexed with target polynucleotide, the HP2 B’S sequence hybridizes to the B sequence of the HP1 to serve as a toehold for HP2 as the C sequence of HP1 invades the HP2’s stem structure, thereby resulting in the HP2’s C’ sequence hybridizing to the HP1 C sequence, which, in turn, causes the HP2’s stem-loop structure to hybridize with the complementary sequences on HP1, and expose singe-stranded sequence comprising the HP2 C sequence.

[0104] Subsequently, an oligonucleotide probe of the invention, which contains a C’ sequence that is complementary to the C sequence of both HP1 and HP2 and the sequence of a recognition site for a nicking endonuclease, hybridizes to the HP1 and HP2 C sequences, thereby forming a complex of the opened hairpins and the probe that is double-stranded where the hairpin’s C sequence and the probe’s C’ hybridize. The probe is labeled with a quenchable fluorescent dye moiety linked to the probe at or near its 3' end or 5’ end, depending on the orientation of the 5’ or 3’ order of the A’, B’, C, and B sequences of the hairpin, or the fluorescent dye moiety is positioned at an internal position in the probe. The probe is also labeled with a fluorescence quenching molecule (the quencher), which is also linked to the probe at or near its 3' end or 5’ end, depending on the 5’ or 3’ orientation of the hairpin, or the quencher is positioned at an internal position in the probe, so long as the NE site is located between the fluorescent and quencher moieties. Upon a NE contacting a double stranded hairpin-probe complex, a nicking reaction generates two probe fragments, one labeled with the fluorescence dye moiety, and the other with the quencher moiety, thereby, permitting the fluorescence dye moiety to emit a fluorescence signal. Following the denaturation of the two probe cleavage fragments from the C sequence of the open hairpin, a new labeled probePCT Patent Application Attorney Docket No: 181.0004-WO00 hybridizes to the C sequence of the hairpin, which, in turn, is followed by repeating cycles of probe hybridization, cleavage, denaturation, and signal emission.

[0105] In Method 1.0 methods, the HP2 C’ sequence may contain a modified phosphorothioate residue to prevent unwanted endonuclease nicking of the hairpin.

[0106] In preferred Method 1.0 methods, both HP1 and HP2 contain single-stranded regions at the 5’ end (5’ end overhangs).

[0107] Some methods of the invention use pluralities of pairs of hairpin oligonucleotides, single universal probes, and two different nicking endonucleases to detect a target single-stranded nucleotide sequence in a sample and then exponentially amplify the number of detectable fluorescence signaling events over the course of multiple cycles of the reaction. In such methods of the invention, generally referred to herein as a Method 4.2 or Method 4.1 methods, the use of dual hairpins and two different nicking endonucleases enables the formation of new target polynucleotide sites for exponential signal generation by a single universal probe.

[0108] In Method 4.2 methods, the methods detect target nucleotide sequences in a sample by contacting a target polynucleotide in a sample, wherein the target polynucleotide contains the target nucleotide sequence, with an oligonucleotide comprising a hairpin structure (“the HP1”) that is characterized by having at least one single-stranded region that is adjacently positioned either 5’ to a double stranded stem and single-stranded loop region. The single-stranded sequence contains an “A’ sequence”, which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is a portion of the target sequence designated as the primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is contiguous with the A sequence and is described herein as secondary (or second) target sequence, and also contains the sequence of a recognition site for a first nicking endonuclease (NE1). The B’ sequence of HP1 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing to a complementary B sequence. The loop structure of the hairpin contains a “C sequence”, which is flanked by the B’ and B sequences. Upon contacting the target polynucleotide with the hairpin, the hairpin A’ sequence hybridizes to the A sequence of the target polynucleotide to serve as a toehold for the hairpin as the B sequence of the secondary target sequence invades the hairpin’s stem structure, thereby resulting in the hairpin’s B’ sequence hybridizing to the secondary target, which, in turn, causes the hairpin’s stem-loop structure to open and expose a single-stranded sequence comprising the hairpin C sequence and the hairpin B sequence.

[0109] Subsequently, an oligonucleotide probe of the invention, which contains a C’ sequence that is complementary to the C sequence of the hairpin and the sequence of a recognition site for a second nickingPCT Patent Application Attorney Docket No: 181.0004-WO00 endonuclease (NE2), hybridizes to the C sequence, thereby forming a complex of the opened hairpin and the probe that is double-stranded where the hairpin’s C sequence and the probe’s C’ hybridize. The probe is labeled with a quenchable fluorescent dye moiety linked to the probe at or near its 3' end or 5’ end, depending on the orientation of the 5’ or 3’ order of the A’, B’, C, and B sequences of the hairpin, or the fluorescent dye moiety is positioned at an internal position in the probe. The probe is also labeled with a fluorescence quenching molecule (the quencher), which is also linked to the probe at or near its 3' end or 5’ end, depending on the 5’ or 3’ orientation of the hairpin, or the quencher is positioned at an internal position in the probe, so long as the NE2 site is located between the fluorescent and quencher moieties. Upon a NE2 contacting a double stranded hairpin-probe complex, a nicking reaction generates two probe fragments, one labeled with the fluorescence dye moiety, and the other with the quencher moiety, thereby, permitting the fluorescence dye moiety to emit a fluorescence signal. Following the denaturation of the two probe cleavage fragments from the C sequence of the open hairpin, a new labeled probe hybridizes to the C sequence of the hairpin, which, in turn, is followed by repeating cycles of probe hybridization, cleavage, denaturation, and signal emission.

[0110] Method 4.2 methods also use a plurality of second hairpin oligonucleotides (HP2s), that are specific for HP1, and that are characterized by having at least one single-stranded region that is adjacently positioned either 3’ to a double-stranded stem and single-stranded loop region. The single-stranded sequence contains a “C’ sequence”, which is complementary to the HP1 C sequence and contains a nucleotide sequence recognition site for a second nicking endonuclease (NE2). The C’ sequence is adjacent to the B’ sequence of the HP2. The B’ sequence is complementary to the B sequence of HP1. The B’ sequence is contiguous with the C’ sequence. The B’ sequence of a HP2 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing to a complementary B sequence. The loop structure of the HP2 contains an “A sequence”, which is contiguous with the B’ sequence, that is complementary the HP A’ sequence, which is flanked by the HP2 B sequence. Upon contacting the HP1 complexed with target polynucleotide, the HP2 C’ sequence hybridizes to the C sequence of the HP1 to serve as a toehold for HP2 as the B sequence of HP1 invades the HP2’s stem structure, thereby resulting in the HP2’s B’ sequence hybridizing to the HP1 B sequence, which, in turn, causes the HP2’s stem-loop structure to open and expose singe-stranded sequence comprising the HP2 A and B sequences.

[0111] Subsequently, contacting the complex formed by HP2 and HP1 with the target polynucleotide sequence with the NE1 and NE2 enzymes, nick NE1 and to NE2 sites. Nicking the NE1 site allows a nicked HP1 B fragment to denature from the complex. Nicking the NE2 site NE2 site of the HP2 C’ sequence of the complex of HP2 and HP1 with the target polynucleotide allows a short 3’ HP2 C’ fragment to denature; and a long HP2 fragment comprising a 5’ HP2 C’ sequence and the HP2 B’, A, and B sequences that is still hybridized to HP1. The nicking of both sites on HP1 and HP2 facilitates the denaturation of the long HP2PCT Patent Application Attorney Docket No: 181.0004-WO00 fragment comprising a 5’ HP2 C’ sequence and the HP2 B’, A, and B sequences, which can act as a “free” target polynucleotide to which additional HP1 can hybridize. Furthermore, the long HP2 fragment comprising a 5’ HP2 C’ sequence and the HP2 B’, A, and B sequences, can be displaced by another invading, full-length HP2.

[0112] In Method 4.2 methods, these probe hybridization and HP2 hybridization and nicking processes can recur to generate a fluorescent signal in an exponential fashion, as more target polynucleotide is generated by the process of liberating free HP2 fragments. The cycles may continue until a reaction component is depleted.

[0113] In preferred Method 4.2 methods, HP1 contains single-stranded regions at the 5’ end (5’ end overhangs) and HP2 contains single-stranded regions at the 3’ end (3’ end overhangs).

[0114] In Method 4.1 methods, the methods detect target nucleotide sequences in a sample by contacting a target polynucleotide in a sample, wherein the target polynucleotide contains the target nucleotide sequence, with an oligonucleotide comprising a hairpin structure (“the HP1”) that is characterized by having at least one single-stranded region that is adjacently positioned either 5’ to a double stranded stem and single-stranded loop region. The single-stranded sequence contains an “A’ sequence”, which is complementary to the “A sequence” of the target polynucleotide sequence. The A sequence is a portion of the target sequence designated as the primary (or first) target sequence. The A’ sequence is adjacent to the B’ sequence of the hairpin. The B’ sequence is complementary to at least a portion of the “B sequence” of the target polynucleotide sequence. The B sequence is contiguous with the A sequence and is described herein as secondary (or second) target sequence. The B’ sequence of HP1 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing to a complementary B sequence. The loop structure of the hairpin contains a “C sequence”, which is flanked by the B’ and B sequences. Upon contacting the target polynucleotide with the hairpin, the hairpin A’ sequence hybridizes to the A sequence of the target polynucleotide to serve as a toehold for the hairpin as the B sequence of the secondary target sequence invades the hairpin’s stem structure, thereby resulting in the hairpin’s B’ sequence hybridizing to the secondary target, which, in turn, causes the hairpin’s stem-loop structure to open and expose a single-stranded sequence comprising the hairpin C sequence and the hairpin B sequence.

[0115] Subsequently, an oligonucleotide probe of the invention, which contains a C’ sequence that is complementary to the C sequence of the hairpin and the sequence of a recognition site for a second nicking endonuclease (NE1), hybridizes to the C sequence, thereby forming a complex of the opened hairpin and the probe that is double-stranded where the hairpin’s C sequence and the probe’s C’ hybridize. The probe is labeled with a quenchable fluorescent dye moiety linked to the probe at or near its 3' end or 5’ end, depending on the orientation of the 5’ or 3’ order of the A’, B’, C, and B sequences of the hairpin, or thePCT Patent Application Attorney Docket No: 181.0004-WO00 fluorescent dye moiety is positioned at an internal position in the probe. The probe is also labeled with a fluorescence quenching molecule (the quencher), which is also linked to the probe at or near its 3' end or 5’ end, depending on the 5’ or 3’ orientation of the hairpin, or the quencher is positioned at an internal position in the probe, so long as the NE1 site is located between the fluorescent and quencher moieties. Upon a NE1 contacting a double stranded hairpin-probe complex, a nicking reaction generates two probe fragments, one labeled with the fluorescence dye moiety, and the other with the quencher moiety, thereby, permitting the fluorescence dye moiety to emit a fluorescence signal. Following the denaturation of the two probe cleavage fragments from the C sequence of the open hairpin, a new labeled probe hybridizes to the C sequence of the hairpin, which, in turn, is followed by repeating cycles of probe hybridization, cleavage, denaturation, and signal emission.

[0116] Method 4.1 methods also use a plurality of second hairpin oligonucleotides (HP2s), that are specific for HP1, and that are characterized by having at least one single-stranded region that is adjacently positioned either 5’ to a double-stranded stem and single-stranded loop region. The single-stranded sequence contains a “C’ sequence”, which is complementary to the HP1 C sequence and contains a nucleotide sequence recognition site for a second nicking endonuclease (NE2). The C’ sequence is adjacent to the B sequence of the HP2. The B sequence is complementary to the B’ sequence of HP1. In Method 4.1, HP2 contains a nucleotide sequence recognition site for a second nicking endonuclease (NE2) at the interface of or between the C’ and B sequences. The B sequence of a HP2 oligonucleotide forms the double-stranded stem structure of the hairpin by hybridizing to a complementary B’ sequence. The loop structure of the HP2 contains an “A sequence”, which is contiguous with the B sequence, that is complementary the HP A’ sequence, which is flanked by the HP2 B’ sequence. Upon contacting the HP1 complexed with target polynucleotide, the HP2 C’ sequence hybridizes to the C sequence of the HP1 to serve as a toehold for HP2 as the B’ sequence of HP1 invades the HP2’s stem structure, thereby resulting in the HP2’s B sequence hybridizing to the HP1 B’ sequence, which, in turn, causes the HP2’s stem-loop structure to open and expose singe-stranded sequence comprising the HP2 B’ sequence.

[0117] Subsequently, contacting the complex formed by HP2 and HP1 with the target polynucleotide sequence with the NE1 and NE2 enzymes, nick NE1 and to NE2 sites. Nicking the NE1 site allows a nicked HP2 C’ fragment at the 5’ end to denature from the complex. Nicking the NE2 site of the HP2 at the interface of the HP2 B and C’ sequences allows a short 3’ HP2 C’ fragment to denature. The nicking of both sites on HP2 facilitates the displacement of an HP2 fragment containing contiguous B, A, and B’ sequences by invasion and hybridization of an new, un-nicked HP2. The B’ sequence at the 3’ end of the liberated HP2 fragment may then hybridize to the exposed, single-stranded HP1 B sequence, thereby presenting additional target polynucleotide A and B sequences.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0118] In Method 4.1 methods, these probe and HP2 hybridization and nicking processes can recur to generate a fluorescent signal in an exponential fashion, as more target polynucleotide is generated by the process of hybridizing additional HP1s to HP2 fragments presenting as additional target polynucleotides. The cycles may continue until a reaction component is depleted.

[0119] In preferred Method 4.1 methods, both HP1 and HP2 contain single-stranded regions at the 5’ end (5’ end overhangs).

[0120] In some methods of the invention, a target polynucleotide contains a first (first) target nucleotide sequence (an A sequence) and a second (secondary) target nucleotide sequence (a B sequence), which flanks the first target nucleotide sequence. In such methods, which are generally referred to herein as Method 0.0 methods, one or more target polynucleotides is contacted with a plurality of oligonucleotides, each having a hairpin structure, wherein each hairpin oligonucleotide has: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region, wherein: the stem includes a nucleotide sequence (hairpin B’ sequence) that is complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (hairpin B sequence); and the single-stranded loop region comprises (i) a nucleotide sequence of a nicking endonuclease (NE) site (the hairpin C sequence); and (ii) a single-stranded, primary target-specific nucleotide sequence (hairpin A’ sequence), located 3’ or 5’ of the hairpin stem region, comprising a nucleotide sequence complementary to the A sequence of the target polynucleotide; hybridizing the hairpin A’ sequence and the hairpin B’ sequence of one or more of the hairpin oligonucleotides in the plurality of hairpin oligonucleotides to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of the hairpin oligonucleotide with the target polynucleotide, wherein hybridization of the hairpin B sequence to the B’ sequence of the target polynucleotide opens the stem-loop structure of the hairpin to expose a single-stranded sequence comprising the hairpin C sequence and the hairpin B sequence; hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the hairpin C sequence (C’ sequence), and a nucleotide sequence complementary to the hairpin B sequence of the hairpin structure, to the exposed single- stranded C and B’ sequences of the hairpin structure to form one or more double stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3’ end, an internal nucleotide position, or the 5’ end of the probe; and (ii) a fluorescent moiety-quenching molecule (the quencher) linked to the 3’ end, an internal position, or the 5’ end of the probe, wherein the probe nicking site is located between the fluorescent and quencher moieties; contacting the one or more double stranded hybridized probe complexes with one or more nicking endonucleases (NE)s under reaction conditions that allow the one or more NEs to nick the double stranded hybridized probe complex at the one or more respective NE sites, wherein the nicking reactions release one or more of a single-strandedPCT Patent Application Attorney Docket No: 181.0004-WO00 fragment of the probe comprising the fluorescent moiety-linked end of the probe, from each of the one or more hybridized probe complex comprising the quencher, thereby allowing: (i) the fluorescent moiety to emit a fluorescent signal; and (ii) the probe fragment, comprising the fluorescent moiety, to denature from the loop region, thereby exposing the loop site comprising the C sequence; and detecting the fluorescent signal of the one or more released probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide. Steps (C)(i-iii) are repeated until a desired level of fluorescence signal is detected.

[0121] Methods of the invention can be used to detect target nucleotide sequences in essentially any single-stranded polynucleotide, including, for example, RNA, microRNA (miRNA), single-stranded DNA, or circulating free DNA (cfDNA). Single-stranded DNA targets include single-stranded DNA prepared from double-stranded DNA by using heat, random nicking, a helicase, or strand displacement by a replicative or strand displacing polymerase. In one method of the invention, the target polynucleotide is RNA extracted from a virus, such as a variant of a SARS-CoV-2, including but not limited to any one of the following: the N501Y, variant, U.K. (alpha, B.1.1.7); South African (beta, B.1.351); Brazil (gamma, P.1); India (delta, B.1.617.2); variant B.1.617.2 (Delta), and California (epsilon, B.1.429 / 427).

[0122] In another method of the invention, the target polynucleotide is used to detect a locus in a tail sequence of a target-specific oligonucleotide primer. One or more target polynucleotides to be detected in a method of the invention may, in some invention, be bound to an antibody or to a target protein. In a method of the invention used for the multiplex detection of more than one target sequence, the method detects and / or distinguishes a specific species of mature small RNA from among other species of small RNAs in the sample.

[0123] In some methods of the invention, the methods detect very low quantities of target polynucleotide, for example, in some methods of the invention has a limit of detection (LoD) of 1 to 5 copies / µl, 5 to 15 copies / µl, 15-25 copies / µl, 25-100 copies / µl, 100-200 copies / µl, 200-300 copies / µl, 300- 400 copies / µl, 400-500 copies / µl, 500-1000 copies / µl, 1000-1500 copies / µl, or 1500-2000 copies / µl of target polynucleotide.

[0124] Methods of the invention are used to target nucleotide sequences in a sample obtained from any source known to contain, suspected of containing, or suspected of possible containing a target polynucleotide. In one method of the invention, the sample is derived from a biological sample, including, for example, a sample derived from a nasal swab, an oral swab, a throat swab, an ear swab, blood or a blood fraction, saliva, urine, or feces. Preparation of a sample may include a nucleic acid extraction step, or it could be prepared by following an extraction-free method. In some methods of the invention, the target polynucleotide is prepared using an extraction-free method, such as by heat inactivation or lysis, whereas extraction-based methods are preferably RNA extraction methods.PCT Patent Application Attorney Docket No: 181.0004-WO00

[0125] Methods of the invention may also be utilized for analyzing non-nucleic acid targets. For example, a method of the invention may be used to detect interactions between a binding agent and a target analyte that is specifically bound by the binding agent by: (i) contacting a sample comprising a plurality of target analytes with a plurality of binding agents conjugated to one or more target polynucleotides under conditions to allow the binding agents to bind specifically to the binding agents; (ii) removing unbound binding agents, typically by including a washing step; and (ii) performing the method of detecting target polynucleotides, wherein detecting the fluorescence signal of the one or more released probe fragments is indicative of specific binding of the binding agent to target protein. In some methods of the invention the plurality of target agents are protein target agents, such as, for example, antibodies.

[0126] An A’ sequence of a hairpin of the invention is typically, but not necessarily limited to 6-24 nucleotides in length. In some hairpin oligonucleotides of the invention, the A’ sequence contains a minimum number of nucleotides required for strand displacement mediated hairpin opening and subsequent nicking reactions. For example, an A’ sequence may be 2 nucleotides.3 nucleotides.4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides in length.

[0127] A B’ sequence and its complement sequence, B, of a hairpin of the invention are typically, but not limited to 6-24 nucleotides in length. In some hairpin oligonucleotides of the invention, the A’ sequence contains a minimum number of nucleotides required for strand displacement mediated hairpin opening and subsequent nicking reactions. For example, a B’ sequence may be 2 nucleotides.3 nucleotides.4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides in length.

[0128] The minimum number of nucleotides in a C sequence of a hairpin of the invention is the minimum number of nucleotides required to function as a nicking endonuclease recognition site recognized by the particular nicking endonuclease (NE) used in a method of the invention when the C sequence of the hairpin hybridizes to the C’ sequence of a probe of the invention. In general, a NE used in a method of the invention is 3-10 nucleotides in length. In one method of the invention, the C sequence of a hairpin contains an NE site recognized by Nt.BspQI, while in another method the site is recognized by Nt.BsmAI,PCT Patent Application Attorney Docket No: 181.0004-WO00 while in other methods of the invention , it is Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, or Nb.BssSI.

[0129] Hairpin structures of the invention can contain modifications. For example, the hairpin structure in some methods of the invention contains one or more phosphorothioate molecules at or near their 5’ or 3’ ends, or at an internal nucleotide position. In the same or other methods of the invention, the hairpin structure contains one or more Locked Nucleic Acids (LNA)s at the 5’ end, an internal nucleotide position, or at the 3’end of one or more of the sequence regions of the hairpin. In such methods of the invention the A’ sequence of the hairpin structure, for example, may contain (LNA)s or other exotic nucleic acids to increase Tm at specific sites. In the same or other methods of the invention, the hairpin structure contains one or more Bridged Nucleic Acids (BNA)s at the 5’ end, an internal nucleotide position, or at the 3’end of one or more of the sequence regions of the hairpin.

[0130] In some methods of the invention, one or more of the sequences of the hairpin or probe can include one or more nonnatural nucleic acids, such as, for example, a peptide nucleic acid (PNA), , a dNTP / ribonucleotide triphosphates (rNTP) hybrid, isoguanosine (isoG); isocytosine (isoC), dUTP, rATP, rCTP, rGTP, or rUTP. Furthermore, one or more nonnatural nucleic acids are incorporated into the hairpin C sequence of the single-stranded loop region or the hairpin A’ sequence of the single-stranded nucleotide sequence.

[0131] In some methods of the invention, a hairpin structure contains internal spacer sequences. For example, in one method of the invention, the hairpin structure contains a spacer in its A’ domain that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide positions in length. In another method of the invention, the hairpin structure contains a spacer in its B’ domain that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide positions in length. In another method of the invention, the hairpin structure contains a spacer in its B domain that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide positions in length. In another method of the invention, the hairpin structure contains a spacer in its C’ domain that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide positions in length. In another method of the invention, the hairpin structure contains a spacer in its C domain that is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide positions in length.

[0132] Like a C sequence of a hairpin used in a method of the invention, the minimum number of nucleotides in a C’ sequence of a probe of the invention, is the minimum number of nucleotides required to function as a nicking endonuclease recognition site recognized by the particular nicking endonuclease (NE) used in a method of the invention when the C’ sequence of the hairpin hybridizes to the C sequence of a hairpin of the invention. In general, a NE used in a method of the invention is 3-10 nucleotides in length. In one method of the invention, the C’ sequence of a probe contains an NE site recognized by Nt.BspQI, while in another method the site is recognized by Nt.BsmAI, while in other methods of the invention , it isPCT Patent Application Attorney Docket No: 181.0004-WO00 Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, or Nb.BssSI. The following table describes a nonlimiting summary of Nes that can be used in method of the invention. Table 1: Nicking Endonuclease Sites Nicking Optimal Reaction Endonuclease Temp. (°C) Cleavage Site SEQ ID NO Nt.BspQI 50 GCTCTTCN (SEQ ID NO.1) NGAAGAGC (SEQ ID NO.2) Nt.CviPII 37 CCD (SEQ ID NO.3) HGG (SEQ ID NO.4) Nt.BstNBI 55 GAGTCNNNNN (SEQ ID NO.5) NNNNNGACTC (SEQ ID NO.6) Nb.BsrDI 65 GCAATGNN (SEQ ID NO.7) NNCATTGC (SEQ ID NO.8) Nb.BtsI 37 GCAGTGNN (SEQ ID NO.9) NNCACTGC (SEQ ID NO.10) Nt.AlwI 37 GGATCNNNNN (SEQ ID NO.11) NNNNNGATCC (SEQ ID NO.12) Nb.BbvCI 37 CCTCAGC (SEQ ID NO.13) GCTGAGG (SEQ ID NO.14) Nt.BbvCI 37 CCTCAGC (SEQ ID NO.15) GCTGAGG (SEQ ID NO.16) Nb.BsmI 65 GAATGCN (SEQ ID NO.17) NGCATTC (SEQ ID NO.18) Nb.BssSI 37 CACGAG (SEQ ID NO.19) CTCGTG (SEQ ID NO.20) Nt.BsmAI 37 GTCTCNN (SEQ ID NO.21) NNGAGAC (SEQ ID NO.22)

[0133] In some methods on the invention, the probe has at least two different NE recognition sites. Such probes are used in multiplex applications of a method of the invention, wherein each different NE corresponds to a respective hairpin that is specific for only one of the at least two target sequences of the multiplex analysis. In such methods, the probes, which may be referred to as double-enzyme or double nicking endonuclease probes, can contain optional X’, Y’, and Z’ sequences that are used to differentially target the probes to the different hairpins in a reaction, or serve as spacers.

[0134] In some methods of the invention, the quenchable fluorescent moiety and the quenching molecule are separated by no more than 30 nucleotide positions. Accordingly, in a method of the invention, the quenchable fluorescent moiety and the quenching molecule are separated by 1 nucleotide position, 2 nucleotide positions, 3 nucleotide positions, 4 nucleotide positions, 5 nucleotide positions, 6 nucleotide positions, 7 nucleotide positions, 8 nucleotide positions, 9 nucleotide positions, 10 nucleotide positions, 11 nucleotide positions, 12 nucleotide positions, 13 nucleotide positions,PCT Patent Application Attorney Docket No: 181.0004-WO00 14 nucleotide positions, 15 nucleotide positions, 16 nucleotide positions, 17 nucleotide positions, 18 nucleotide positions, 19 nucleotide positions, 20 nucleotide positions, 21 nucleotide positions, 22 nucleotide positions, 23 nucleotide positions, 24 nucleotide positions, 25 nucleotide positions, 26 nucleotide positions, 27 nucleotide positions, 28 nucleotide positions, 29 nucleotide positions, or 30 nucleotide positions.

[0135] Probes used in methods of the invention can be linked to any fluorescent dye moiety that one of ordinary skill in the art would know is appropriate for an application of the invention, including, for example, fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT (Hydroxy Tryptamine); 6-HAT; 6-JOE; 6-carboxyfluorescein (6-FAM); FITC); Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY™ fluorophores (e.g, 492 / 515, 493 / 503, 500 / 510, 505 / 515, 530 / 550, 542 / 563, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650-X, 650 / 665-X, 665 / 676, FL, FL ATP, Fl-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR- X SE), coumarins (e.g, 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S, AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin, CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes (e.g, calcium crimson, calcium green, calcium orange, calcofluor white), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g, 3, 3.18, 3.5, 5, 5.18, 5.5, 7), cyanGFP, cyclic AMP Fluorosensor (FiCRhR), fluorescent proteins (e.g, green fluorescent protein (e.g, P. EGFP), blue fluorescent protein (e.g, BFP, EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent protein (e.g, ECFP, Cerulean, CyPet), yellow fluorescent protein (e.g YFP, Citrine, Venus, YPet), FRET donor / acceptor pairs (e.g., fluorescein / tetramethylrhodamine, lAEDANS / fluorescein, EDANS / dabcyl, fluorescein / fluorescein, BODIPY™ FL / BODIPY™ FL, Fluorescein / QSY7 and QSY9), LysoTracker™ and LysoSensor™ (e.g., LysoTracker™ Blue DND-22, LysoTracker™ Blue-White DPX, LysoTracker™ Yellow HCK-123, LysoTracker™ Green DND-26, LysoTracker™ Red DND-99, LysoSensor™ Blue DND-167, LysoSensor™ Green DND-189, LysoSensor™ Green DND-153, LysoSensor™ Yellow / Blue DND-160, LysoSensor Yellow / Blue 10,000 MW dextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g, 110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, Red, Rhod-2, 5-ROX (carboxy-X-rhodamine), Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetramethylrhodamine (TRITC), WT), Texas Red, Texas Red-X, VIC and other labels described in, e.g, US Pub. No.2009 / 0197254), among others as would be known to those of skill in the art. Other detectable labels can also be used (see, e.g, US Pub. No.2009 / 0197254), as would be known to those of skill in the art.

[0136] In some embodiments of the invention, the quencher moiety is 5IABkFQ having broad absorbance spectra ranging from 420 to 620 nm with peak absorbance at 531 nm. This quencher can be used with fluorescein and other fluorescent dyes that emit in the green to pink spectral range. In somePCT Patent Application Attorney Docket No: 181.0004-WO00 embodiments, the quencher is any of the Black Hole Quenchers® (available from Biosearch Technologies), either of the Iowa Black® quenchers (available from Integrated DNA technologies), Zen® quencher (available from Integrated DNA Technologies), any of the Onyx® quenchers (available from Millipore- Sigma), or any of the ATTO® quenchers (available from ATTO-TEC GmbH).

[0137] The reaction of a method of the invention is typically conducted under isothermal conditions, at, for example, temperatures from about 20 to 40 °C. In one method of the invention, the reaction is conducted at about 25 °C. In another method of the invention, the reaction is conducted at about 30 °C, while in yet another method of the invention, the reaction is conducted at about 35 °C. While methods of the invention are described herein as repeating “cycles” to amplify signal, but they are not thermal cycles. For example, a method of the invention may be repeated for 1-500 cycles, 1-450 cycles, 1-400 cycles, 1-350 cycles, 1-300 cycles, 1-250, 1-200 cycles, 1-100 cycles, 1-50 cycles, 1-45 cycles, 1-40 cycles, 1-35 cycles, 1-30 cycles, 1-25 cycles, 1-20 cycles, 1-15 cycles, 1-14 cycles, 1-13 cycles, 1-12 cycles, 1- 11 cycles, 1-10 cycles, 1-9 cycles, 1-9 cycles, 1-8 cycles, 1-7 cycles, 1-6 cycles, 1-5 cycles, 1-4 cycles, 1-3 cycles, or 2 cycles.

[0138] In some methods of the invention, one or more of the hairpins and / or the probe is conjugated to one or more nanoparticles. For example, a nanoparticle that is conjugated to a hairpin oligonucleotide of the invention is a polystyrene microsphere or lanthanide nanoparticle, while in another method of the invention, one or more of the hairpin oligonucleotides is conjugated to a metal chelating polymer, such as, for example, a lanthanide metal-chelating polymer. EXAMPLES

[0139] Example 1: Detection of SARS-CoV-2 N1 Locus A linear NICER process was used to detect SARS-CoV-2 in nasal swab specimens contrived with chemically inactivated and SARS-CoV-2 virus particles (heat lysed for 5 minutes at 95°C). The specimens were prepared for analysis by placing nasal swabs in saline and adding the inactivated virus particles directly to swab samples and diluting the samples to 40, 20, 18, 16, 14, 12, 10, 5, and 1 genome copies / copy per µL. Hairpins and probe were tested at 0.25 µM final concentrations.

[0140] The hairpin nucleotide sequences used in the analysis included a CDC-approved forward primer sequence, specific for the N1 region of the gene coding for nucleocapsid protein (N) in SARS-CoV-2 (SEQ ID NO.23), which is complementary to the A’ and B domains of the hairpin structure. Nt.BbvCI was added to the reactions to serve as a nicking endonuclease (NE). The fluorophore linked to the hairpin hybridizing probe in these studies was carboxyfluorescein (FAM).

[0141] Two reaction buffer conditions were assessed for performing NICER: (i) ( 50 mM Potassium acetate, 20 mM tris-acetate, 10 mM Magnesium acetate, 100 µg / ml recombinant albumin (pH 7.9 @ 25 °C); and (ii) (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 100 µg / ml recombinant albumin (pH 7.9 @ 25 °C).PCT Patent Application Attorney Docket No: 181.0004-WO00 Higher salt conditions could have been used to promote hybridization stability while allowing for enzymatic activity. The reactions were carried out, without using a heated lid, in 20 µL of buffer at 25 °C for the entirety of the reaction time. Each imaging cycle was approximately 40 seconds.

[0142] Unlike for PCR, each cycle of the NICER process represents an imaging event, not a series of thermal cycles coupled with imaging. Detection of the fluorescence signal generated when the fluorophore of the nicked portion of the probe denatured from the loop sequence was performed using the optics of a standard qPCR instrument.

[0143] Amplification of signal was observed across all viral dilutions by cycle 10 (approximately 6.7 min.; Fig.8A). A few samples showed later cycle signal generation as well as random drop out (Table 2). One of the three negative controls had an amplification product (Fig.8B). The cycle number data corresponding to the Cq is found in Table 2. Replicate plots for each limit of detection (LoD) are presented in Figs.8C-8K for samples 9, 8, 7, 6, 5, 4, 3, 2, and 1, respectively, in Table 2. Table 2: N1 Cq Replicate LoD Data Sample Dye Copies / µL Cq - Replicate 1 Cq - Replicate 2 Cq – Replicate 3 1 FAM 1 8.13 9.71 8.65 2 FAM 5 10.24 9.13 10.86 3 FAM 10 8.81 -- 37.19 4 FAM 12 12.62 9.83 9.55 5 FAM 14 9.49 12.8 11.76 6 FAM 16 8.33 10.52 -- 7 FAM 18 10.39 12.65 -- 8 FAM 20 9.55 12.22 11.43 9 FAM 40 9.49 13.95 13.5

[0144] Given the detection of fluorescence signal in a few negative controls, tests were performed to determine the proper concentration of hairpin to minimize the potential for oligonucleotide impurities that could cause background noise. A temperature series from 20-30 °C was tested across 50, 25, 10, and 5 nM final concentrations of hairpin in negative control samples (Fig.9A-9D). NICER 0.0 control signal amplification was only detected when the reaction was performed at elevated temperatures while using a 5 nM concentration of hairpin, suggesting that using a lower concentration of N1 hairpin would be favored when performing NICER 0.0 at 25 °C, which is a standard reaction temperature. However, identifying an optimal hairpin concentration to use at a standard temperature is likely to be dependent upon variables like the sequence, structure, and size of the specific target hairpin.

[0145] Example 2: Limited signal amplification when a hairpin’s target recognition sequence contains a single nucleotide variation To assess whether a single nucleotide variant of a target recognition sequence of a NICER hairpin would inhibit detection of the target, a universal probe NICER method (NICER 0.5) was performed using a SARS- CoV-2 B.1.1.7 - N501Y specific hairpin (“N501 hairpin”) and a SARS-CoV-2 wild-type target. The N501YPCT Patent Application Attorney Docket No: 181.0004-WO00 encoding variant contains a single nucleotide A / T change in the gene coding for surface glycoprotein (S). The N501 hairpin was designed to contain a single nucleotide mismatch at the tail base immediately flanking the stem. The reaction conditions and FAM fluorophore were the same as described in Example 1 but the hairpin was shorter. The same LoD samples described in Example 1 were used. The test target was the wild-type genome of SARS-CoV-2. It was hypothesized that a single nucleotide change would be sufficient to disrupt the binding / invasion process of hairpin opening, which would result in little to no signal generation. Although signal was initially observed, the signal amplification pattern was atypical and signal generation collapsed over multiple cycles, as indicated by the dome shaped plot that was generated (Figs.10A, 10C). Two out of three negative controls showed domed shaped signal generation plots (Fig.10B, 10C). A composite plot of N1 versus the N501 variant hairpin highlighted the atypical signal amplification of the N501 hairpin and suggested that a NICER method could be used to detect single nucleotide mutations.

[0146] Example 3: Linear NICER using Universal Probe (“Method 0.5”) NICER signal amplification was tested using a probe designed to be capable of non-specific hairpin binding. FIG.14 depicts the universal probe structure. In NICER methods using a universal probe (“Method 0.5”); FIG.13), (i) A’ of a genomic-target-specific hairpin hybridizes to A of the target template sequence and (ii) the hairpin performs strand displacement, and B’ of the hairpin hybridizes to B of target template sequence; (iii) C and B of the hairpin become single stranded, allowing (iv) C’ of the probe to bind to the exposed C portion of the hairpin; (v) the nicking endonuclease nicks at the nicking site on probe to generate two fragments, each with similar Tm, below 25 °C, which (vi) denature off of the hairpin; and (vii) new un- nicked probe binds to exposed C portion of the hairpin and steps (iv)-(vi) repeat as a cycle. Method 0.5 makes use of multiple hairpins specific for different target sequences on the same genomic target along with a single, universal (i.e., hairpin non-specific) probe, allowing for increased signal amplification.

[0147] NICER amplification using a universal probe was compared to linear NICER (“Method 0”) methods as described above in Example 1. Reactions were conducted on CFX96 rt-PCR platform at 25 °C, 1s cycle x 250 cycles (not including imaging); with 50nM template, 50nM hairpin, and 50nM probe. Results are shown in Fig.33.

[0148] Example 4: Method 0.5 Using Internal Dye and Quencher The effects of placing the quencher and / or fluorescent dye internally on the probe was investigated. Fig.19 depicts the signal generation between a probe with an internal quencher and a probe with an internal dye. Reactions were conducted on CFX96 rt-PCR platform at 25 °C, 1s cycle x 250 cycles (not including imaging); conducted with 50nM template, 50nM hairpin, and 50nM probe.

[0149] Use of an internal quencher was essential in significantly decreasing the background signal generation produced by probe binding directly to template. Fig.27 depicts signal amplification comparingPCT Patent Application Attorney Docket No: 181.0004-WO00 background signals between probe with terminal quencher and dye and probe with internal quencher and terminal dye.

[0150] Example 5: Effect of Probe and Hairpin Concentration in Method 0.5 The effect of probe (FIG.21, upper panel) and hairpin concentration (FIG.21, lower panel) in Method 0.5 was also investigated. Reactions were conducted on CFX96 rt-PCR platform at 25 °C; using 5nM template, 50nM hairpin, and 1s cycle x 250 cycles (not including imaging). Experimental conditions investigating variable hairpin concentrations were 25 °C; and 5nM template, 250nM probe at 1s cycle x 250 cycles (not including imaging).

[0151] Example 6: Multiplexing Using Method 0.5 Multiplexing with two different targets generated distinguishable signals using FAM and FOX fluorescent probes (FIG.23, upper panel, 1-4). Reactions were conducted on CFX96 rt-PCR platform, 50nM template, 50nM hairpin, 50nM probe at 25 °C, ~1s cycle x 125 cycles (not including imaging). Control experiments are shown in FIG.23 (bottom panel, 5-8).

[0152] Example 7: Effect of PEG on Method 0.5 and Method 1.0 (see Example 10 below) Method 0.5 reactions were supplemented with 6000 MW polyethylene glycol (PEG) (FIG.39; upper panel). PEG percentages reflect the final proportion of reaction mixture. Reactions were conducted on CFX96 rt- PCR platform, 25 °C; 50nM hairpin, 50nM probe, 1s cycles x 250 cycles. Varying PEG concentrations were also tested at different template concentrations (FIG.39, lower panel).

[0153] 6000 MW PEG and 8000 MW PEG were also assessed in Method 1.0 reactions (FIG.25), upper panel). Reactions were conducted on CFX96 rt-PCR platform, 25 °C, cycles 1s x 250 cycles; 50nM template, 50nM hairpin, 50nM probe. The effects of 12% 8000 MW PEG on Method 0.5 and Method 1.0 are shown in FIG 40, lower panel. Reactions were conducted on CFX96 rt-PCR platform, 25 °C, cycles 1s x 250 cycles; 5 nM template, 50 nM / 150 nM hairpin, 50nM probe.

[0154] Example 8: Accelerated Linear NICER (“Method 1.0”) A dual hairpin system using the universal probe was also developed, termed Accelerated Linear NICER (“Method 1.0”; FIG.16). A modified phosphorothioate residue in the second hairpin prevents unwanted endonuclease nicking. In a first phase of Method 1.0 reactions, (i) A’ of hairpin 1 (HP1) hybridizes to A of target template sequence; (ii) HP1 performs strand displacement and B’ of HP1 hybridizes to B of target template sequence; (iii) C and B of HP1 become single stranded; (iv) C’ probe can bind to exposed C portion of HP1; (v) nicking endonuclease nicks at recognition site on probe to generate two fragments, each with a similar Tm below 25 °C, which (vi) denature off of HP1; and (vii) new un-nicked probe binds to exposed C portion of HP1 and (iv)-(vi) repeat in cycles. In a second phase of Method 1.0 reactions, (viii) B’s of hairpin 2 (HP2) hybridize to B of HP1; (ix) both HP1 and HP2 experience strand displacement and HP1 denatures from template sequence and HP2 hybridizes to HP1, and C’, B, and A sequences of HP2 hybridize to C, B’, and A’PCT Patent Application Attorney Docket No: 181.0004-WO00 of HP1; (x) original template sequence is available for new HP1 to hybridize; (xi) C’ probe can bind to exposed C portion of HP2; (xii) nicking endonuclease nicks at nicking site on probe to generate two fragments, each with a similar Tm below 25 °C, which (xiii) denature off HP2; and (xiv) new un-nicked probe binds to exposed C portion of HP2 and (xi)-(xiii) repeat in cycles.

[0155] NICER signal amplification using Method 1.0 was compared to Method 0.5 (FIG.26). Reactions were conducted on a CFX96 rt-PCR platform at 25 °C, 1s cycle x 250 cycles (not including imaging); with 50nM template, 50nM hairpin, and 50nM probe.

[0156] The presence of the modified phosphorothioate residue in the C’ region of the second hairpin for reducing background signal was determined to be important (FIG.28). Reactions were conducted on a CFX96 rt-PCR platform at 25 °C, 1s cycle x 250 cycles (not including imaging). Representative of Method 1 without modified hairpin 2 protecting the nicking site in the C’ region is shown in the upper panel (FIG.28). The background signal in the sample without template (gray) is indistinguishable from the sample with template (black). Representative of Method 1 with modified hairpin 2 protecting the nicking site in the C’ region is shown in the lower panel (FIG.28). There was a noticeable distinction between background signal in sample without template (gray) and sample with template (black).

[0157] Example 9: Exponential NICER (“Method 4.2”) NICER methodologies using dual hairpins, a universal probe, and two endonuclease sites / enzymes were also developed for exponential signal generation. A first iteration / embodiment of exponential NICER reactions use a first hairpin with a 5’ overhang a second hairpin with a 3’ overhang (“Method 4.2”; FIG.17). In Method 4.2 reactions, (i) A’ of hairpin 1 (HP1) hybridizes to A of target template sequence; (ii) HP1 performs strand displacement, B’ of HP1 hybridizes to B of target template sequence; (iii) C and B of HP1 become single stranded; (iv) C’ probe can bind to exposed C portion of HP1; (v) a first nicking endonuclease nicks at a first recognition site on the probe to generate two fragments, each with a similar Tm below 25 °C, which (vi) denature off of HP1; and (vii) new un-nicked probe binds to exposed C portion of HP1 and (iv)-(vi) repeat and cycle; then (viii) C’ of hairpin 2 (HP2) hybridizes to exposed C of HP1; (ix) HP2 performs strand displacement, B’ of HP2 hybridizes to B of HP1 (x) A and B of HP2 single stranded and exposed, acting as template for a non-hybridized HP1; (xi) the first endonuclease may nick at recognition site C’ on HP2 and make it possible for a new un-nicked HP2 to displace nicked HP2; and (xii) a second endonuclease may nick HP1 at the second recognition site in B’, allowing a short, double-stranded sequence resulting from nicking in C’ of HP1 and B’ of HP2 to denature, resulting in a free target template sequence on the resulting large HP2 fragment (right-hand side of FIG.17).

[0158] Example 10: Exponential NICER (“Method 4.1”) A second iteration / embodiment of exponential NICER reactions were also developed, using a first and a second hairpin, both with 5’ overhangs (FIG.18; “Method 4.1”). In Method 4.1 reactions, (i) A’ of HP1PCT Patent Application Attorney Docket No: 181.0004-WO00 hybridizes to A of target template sequence; (ii) HP1 performs strand displacement, B’ of HP1 hybridizes to B of target template sequence; (iii) C and B of HP1 becomes single stranded; (iv) C’ probe can bind to exposed C portion of HP1; (v) a first nicking endonuclease nicks at recognition site on probe to generate two fragments, each with a similar Tm below 25 °C, which (vi) denature off of HP1; and (vii) new un-nicked probe binds to exposed C portion of HP1 and (iv)-(vi) repeat and cycle; then (viii) C’ of HP2 hybridizes to B of HP1; (ix) both HP1 and HP2 experience strand displacement and HP1 denatures from template sequence and HP2 hybridizes to HP1, while B and A of HP2 hybridize to B’ and A’ of HP1; (x) original template sequence presented on HP2 becomes available for a new HP1 to hybridize; (xi) C’ probe can bind to exposed C portion of HP2; (xii) the first endonuclease nicks at the first recognition site on the probe to generate two fragments, each with a similar Tm below 25 °C, which (xiii) denature off of HP1; (xiv) new un- nicked probe binds to exposed C portion of HP2 and (xi)-(xiii) repeat and cycle; and then (xv) C’ of HP2 may be nicked by both the first and second endonuclease, creating small fragments, each with a similar Tm below 25 °C that may denature off of HP1; (xvi) a new, un-nicked HP2 may invade and hybridize to HP1, displacing the nicked HP2; and (xvii) the displaced large HP2 fragment may either form a hairpin, or B’ of HP2 may hybridize to an exposed B of HP1, keeping the nicked HP2 open and providing a new target template sequence for unhybridized HP1 to invade.

[0159] The viability of Method 4.2 reactions was demonstrated by developing reaction products on agarose gel electrophoresis (FIG.29). Samples were run at 37 °C, 1s cycle x 250 cycles not including imaging, then loaded onto a 3% agarose gel and developed for 30 min. at 250V on ice. Template [5nM], hairpin [50nM] or [50nM] / [5x10-4nM], probe [150nM]. Agarose gel lanes contain samples ran in different reaction buffer formulations to observe the effect of different salt and enzyme compositions and concentrations.

Claims

PCT Patent Application Attorney Docket No: 181.0004-WO00 What is claimed:

1. A method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method comprising: (A) contacting the one or more target polynucleotides with a plurality of hairpin oligonucleotides, wherein each hairpin oligonucleotide comprises: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the hairpin C sequence), wherein the double-stranded stem region comprises a nucleotide sequence (the hairpin B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the hairpin B sequence); and (ii) a single-stranded nucleotide sequence (the hairpin A’ sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the hairpin A’ sequence and the hairpin B’ sequence of one or more of the hairpin oligonucleotides to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of the hairpin oligonucleotide with the target polynucleotide, wherein hybridization of the hairpin B’ sequence to the B sequence of the target polynucleotide opens the stem-loop structure of the hairpin to expose a single-stranded sequence comprising the hairpin C sequence and the hairpin B sequence; (C) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the hairpin C sequence (the C' sequence) and a nucleotide sequence of a nicking endonuclease (NE) site, to the exposed single-stranded C sequence of the hairpin structure to form one or more double- stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; and (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE site is located between the fluorescent and quencher moieties; (D) contacting the one or more double stranded hybridized probe complexes with one or more NEs under reaction conditions that allow the one or more NEs to nick the double stranded hybridizedPCT Patent Application Attorney Docket No: 181.0004-WO00 probe complex at the one or more respective NE sites, wherein the nicking reactions generate two probe fragments, each of which denature from the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (i) the fluorescent moiety to emit a fluorescent signal; and (ii) the harpin C sequence to hybridize another probe in the plurality of probes; and (E) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

2. The method of claim 1 further comprising repeating steps (C) and (D) until a desired level of signal amplification is reached or a until a reaction component is exhausted.

3. A method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method comprising: (A) contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1s), wherein each HP1 comprises: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP1 C sequence), wherein the double-stranded stem region comprises a nucleotide sequence (the HP1 B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the HP1 B sequence); and (ii) a single-stranded nucleotide sequence (the HP1 A’ sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of HP1 with the target polynucleotide, wherein hybridization of the HP1 B’ sequence to the B sequence of the target polynucleotide opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence; (C) contacting the complex of HP1 with the target polynucleotide with a plurality of second hairpin oligonucleotides (HP2s), wherein each HP2 comprises: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region, wherein:PCT Patent Application Attorney Docket No: 181.0004-WO00 (a) the double-stranded stem region comprises a nucleotide sequence (the HP2 C’ sequence) complementary to the HP1 C sequence in a double strand with a complementary sequence (the HP2 C sequence); and (b) the single-stranded loop region comprises a nucleotide sequence complementary to the HP1 B’ sequence (the HP2 B sequence) flanked by a nucleotide sequence that is complementary to at least a contiguous portion of the HP1 A’ sequence (the HP2 AS sequence) (ii) a single-stranded nucleotide sequence (the HP2 B’S sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence that is complementary to at least a contiguous portion of the HP1 B sequence; (D) hybridizing the HP2 B’S, HP2 C’, the HP2 B, and HP2 AS sequences of one or more of the HP2s to the HP1 B, HP1 C, HP1 B’, and HP1 A’ sequences, respectively, of the complex of HP1 with the target polynucleotide, to form a complex of HP2 and HP1 with the target polynucleotide, wherein hybridization of the HP2 to HP1 opens the stem-loop structure of the HP2 to expose a single- stranded sequence comprising the HP2 C sequence; (E) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the HP1 C sequence (the C' sequence) and a nucleotide sequence of a nicking endonuclease (NE) site, to the exposed HP1 C sequence and exposed single-stranded HP2 sequence of the complex of HP2 and HP1 with the target polynucleotide, to form one or more double-stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; and (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE site is located between the fluorescent and quencher moieties; (F) contacting the one or more double stranded hybridized probe complexes with one or more NEs under reaction conditions that allow the one or more NEs to nick the double stranded hybridized probe complex at the one or more respective NE sites, wherein the nicking reactions generate two probe fragments, each of which denature from the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (i) the fluorescent moiety to emit a fluorescent signal; andPCT Patent Application Attorney Docket No: 181.0004-WO00 (ii) the exposed HP1 and exposed single-stranded HP2 C sequences to hybridize another probe in the plurality of probes; and (G) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

4. The method of claim 3, wherein the HP2 C’ sequence comprises at least one phosphorothioate modification.

5. A method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method comprising: (A) contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1s), wherein each HP1 comprises: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP1 C sequence), wherein the double-stranded stem region comprises a nucleotide sequence (the HP1 B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the HP1 B sequence), wherein the HP1 B sequence comprises a nucleotide sequence of a first nicking endonuclease (NE1) site; and (ii) a single-stranded nucleotide sequence (the HP1 A’ sequence), either 5’ to the double stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of HP1 with the target polynucleotide, wherein hybridization of the HP1 B’ sequence to the B sequence of the target polynucleotide opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence; (C) contacting the complex of HP1 with the target polynucleotide with a plurality of second hairpin oligonucleotides (HP2s), wherein each HP2 comprises: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP2 A sequence), wherein the double-stranded stem region comprises a nucleotide sequence (the HP2 B’ sequence) complementary to the HP1 B sequence in a double strand with a complementary sequence (the HP2 B sequence); andPCT Patent Application Attorney Docket No: 181.0004-WO00 (ii) a single-stranded nucleotide sequence (the HP2 C’ sequence), 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence that is complementary to the HP1 C sequence and a nucleotide sequence of a second NE (NE2) site; (D) hybridizing the HP2 C’ sequence and HP2 B’ sequence of one or more of the HP2s to the exposed, single-stranded HP1 C sequence and HP1 B sequence, respectively, of the complex of HP1 with the target polynucleotide, to form a complex of HP2 and HP1 with the target polynucleotide, wherein hybridization of the HP2 to HP1 opens the stem-loop structure of the HP2 to expose a single-stranded sequence comprising the HP2 A sequence and the HP2 B sequence; (E) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the HP1 C sequence (the C' sequence) and the nucleotide sequence of the NE2 site, to the exposed HP1 C sequence, to form one or more double-stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; and (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE2 site is located between the fluorescent and quencher moieties; (F) contacting the one or more double stranded hybridized probe complexes with one or more NE2s under reaction conditions that allow the one or more NE2s to: (i) nick the double stranded hybridized probe complex at the one or more respective NE2 sites, wherein the nicking reactions generate two probe fragments, each of which denature from the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (a) the fluorescent moiety to emit a fluorescent signal; and (b) the exposed HP1 and exposed single-stranded HP2 C sequences to hybridize another probe in the plurality of probes; and; (ii) nick the NE2 site of the HP2 C’ sequence of the complex of HP2 and HP1 with the target polynucleotide, thereby allowing: (a) a short 3’ HP2 C’ fragment to denature; and (b) a long HP2 fragment comprising a 5’ HP2 C’ sequence and the HP2 B’, A, and B sequences to be displaced by another HP2PCT Patent Application Attorney Docket No: 181.0004-WO00 (G) contacting the complex of HP2 and HP1 with the target polynucleotide with one or more NE1s under reaction conditions that allow the one or more NE1s to nick the NE1 site of the HP1 B sequence of the complex of HP2 and HP1 with the target polynucleotide, thereby allowing a nicked HP1 B fragment to denature; (H) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

6. The method of claim 5, wherein the method further comprises: (I) contacting the long HP2 fragment comprising a 5’ HP2 C’ sequence and the HP2 B’, A, and B sequences with another HP1 from the plurality of HP1s, thereby hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the HP2 A sequence and the HP2 B sequence, respectively, to form a complex of HP1 with HP2, wherein hybridization of the HP1 B’ sequence to the HP2 B sequence opens the stem-loop structure of the HP1 to expose a single- stranded sequence comprising the HP1 C sequence and the HP1 B sequence; and (J) Repeating steps (D)-(H) until a desired level of signal amplification is reached or a until a reaction component is exhausted.

7. A method for detecting one or more single-stranded target polynucleotides (the target polynucleotide) in a sample, wherein each target polynucleotide comprises a first target nucleotide sequence (the A sequence) and a second target nucleotide sequence (the B sequence) that flanks the first target nucleotide sequence, the method comprising: (A) contacting the one or more target polynucleotides with a plurality of first hairpin oligonucleotides (HP1s), wherein each HP1 comprises: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP1 C sequence), wherein the double-stranded stem region comprises a nucleotide sequence (the HP1 B’ sequence) complementary to the B sequence of the target polynucleotide in a double strand with a complementary sequence (the HP1 B sequence); and (ii) a single-stranded nucleotide sequence (the HP1 A’ sequence), either 5’ or 3’ to the double stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence that is complementary to the A sequence of the target polynucleotide; (B) hybridizing the HP1 A’ sequence and the HP1 B’ sequence of one or more of the HP1s to the A sequence and the B sequence of the one or more target polynucleotides, respectively, to form a complex of HP1 with the target polynucleotide, wherein hybridization of the HP1 B’ sequence toPCT Patent Application Attorney Docket No: 181.0004-WO00 the B sequence of the target polynucleotide opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence; (C) contacting the complex of HP1 with the target polynucleotide with a plurality of second hairpin oligonucleotides (HP2s), wherein each HP2 comprises: (i) a hairpin structure comprising a double-stranded stem region and a single-stranded loop region (the HP2 A sequence), wherein the double-stranded stem region comprises a nucleotide sequence (the HP2 B sequence) complementary to the HP1 B sequence in a double strand with a complementary sequence (the HP2 B’ sequence); (ii) a single-stranded nucleotide sequence (the HP2 C’ sequence), 5’ to the double stranded stem region, wherein the single-stranded nucleotide sequence comprises a nucleotide sequence that is complementary to the HP1 C sequence and a nucleotide sequence of a first nicking endonuclease (NE1) site; and (iii) a nucleotide sequence of second NE (NE2) site between the HP2 B sequence and the HP2 C’ sequence; (D) hybridizing the HP2 C’ sequence,HP2 B’ sequence, and HP2 A sequence of one or more of the HP2s to the exposed, single-stranded HP1 C sequence, HP1 B’ sequence, and HP1 A’ sequence respectively, of the complex of HP1 with the target polynucleotide, to denature HP1 from the target polynucleotide and form a complex of HP2 and HP1, wherein hybridization of the HP2 to HP1 opens the stem-loop structure of the HP2 to expose a single-stranded sequence comprising the HP2 B’ sequence; (E) hybridizing one or more single-stranded oligonucleotide probes (the probe or probes) in a plurality of probes, each comprising a nucleic acid sequence complementary to the HP1 C sequence (the C' sequence) and the nucleotide sequence of the NE1 site, to the exposed HP1 C sequence, to form one or more double-stranded, hybridized probe complexes, wherein each probe is linked to: (i) a quenchable fluorescent moiety linked to the 3' end, an internal position, or the 5' end of the probe; and (ii) a quenching molecule (the quencher) linked to the 3' end, an internal nucleotide position, or the 5' end of the probe, wherein the NE1 site is located between the fluorescent and quencher moieties; (F) contacting the one or more double stranded hybridized probe complexes with one or more NE1s under reaction conditions that allow the one or more NE1s to: (i) nick the double stranded hybridized probe complex at the one or more respective NE1 sites, wherein the nicking reactions generate two probe fragments, each of which denature fromPCT Patent Application Attorney Docket No: 181.0004-WO00 the hairpin C sequence, thereby liberating the quenchable fluorescent moiety from the quencher, and allowing: (a) the fluorescent moiety to emit a fluorescent signal; and (b) the exposed HP1 and exposed single-stranded HP2 C sequences to hybridize another probe in the plurality of probes; and; (ii) nick the NE1 site of the HP2 C’ sequence of the complex of HP2 and HP1, thereby allowing a short 5’ HP2 C’ fragment to denature; and (G) contacting the complex of HP2 and HP1 with one or more NE2s under reaction conditions that allow the one or more NE2s to nick the NE2 site between the HP2 B sequence and the HP2 C’ sequence, thereby allowing a nicked 3’ HP2 C’ fragment to denature and forming a complex nicked HP2 and HP1; (H) contacting another HP2 from the plurality of HP2s with the HP1 of the complex of nicked HP2 and HP1, thereby hybridizing the HP2 C’ sequence, HP2 B’ sequence, and HP2 A sequence of the one or more HP2s to the exposed, single-stranded HP1 C sequence, HP1 B’ sequence, and HP1 A’ sequence respectively the displacing the nicked HP2 comprising the HP2 B, HP2 A, and HPB’ sequence; (I) detecting the florescent signal of the one or more liberated probe fragments, wherein the fluorescent signal correlates with detection of the target polynucleotide.

8. The method of claim 7, wherein the HP2 B’ sequence of the displaced, nicked HP2 from step (H) hybridizes to the exposed, single-stranded HP1 sequence formed in step (B), thereby allowing for hybridization of the HP1 A’ sequence and HP1 B’ sequence of another HP1 from the plurality of HP1s, wherein hybridization of the HP1 B’ sequence to the HP2 B sequence of the HP2 fragment B sequence opens the stem-loop structure of the HP1 to expose a single-stranded sequence comprising the HP1 C sequence and the HP1 B sequence, the method further comprising repeating steps (D)-(H) until a desired level of signal amplification is reached or a until a reaction component is exhausted.

9. The method of any one of claims 1-8, wherein the method is conducted isothermally.

10. The method of any one of claims 1-8, wherein the method is conducted at room temperature.

11. A polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ or 3’ to 5’, an A’ sequence, a B’ sequence, a C sequence, and a B sequence, wherein: the A’ sequence comprises a primary target-specific nucleotide sequence; the B’ sequence comprises a secondary target-specific nucleotide sequence; the C sequence comprises a nicking endonuclease recognition site nucleotide sequence;PCT Patent Application Attorney Docket No: 181.0004-WO00 the B sequence comprises a nucleotide sequence complementary to the B’ sequence, and optionally, comprises a nicking endonuclease recognition site nucleotide sequence; and the B’ and B sequences are hybridized to each other to form a stem-loop structure, wherein the loop comprises the C sequence.

12. The polynucleotide of claim 11, wherein the A’ sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

13. The polynucleotide of claim 11 or 12, wherein the B’ and B sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

14. The polynucleotide of any one of claims 11-13, wherein the C sequence comprises 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

15. The polynucleotide of any one of claims 11-14, wherein the nicking endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

16. A polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ or 3’ to 5’, an B’S sequence, a C’ sequence, a B sequence, an AS sequence, and a C sequence, wherein: (a) the B sequence comprises a nucleotide sequence that is identical to a secondary target-specific nucleotide sequence; (b) the B’Ssequence comprises a sequence complementary to, but comprises fewer nucleotides than, the B sequence; (c) the C’ sequence comprises a nicking endonuclease recognition site nucleotide sequence and at least one modified nucleotide; (d) the ASsequence comprises a nucleotide sequence that is identical to at least a contiguous portion of a primary target-specific sequence; (e) the C’ and C sequences are hybridized to each other to form a stem-loop structure, wherein the loop comprises the B and AS sequences; and (f) the C’ sequence comprises at least one modified nucleotide that prevents nicking endonuclease activity at the nicking endonuclease recognition site nucleotide.

17. The polynucleotide of claim 16, wherein the A sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.PCT Patent Application Attorney Docket No: 181.0004-WO00 18. The polynucleotide of claim 17 or 18, wherein the B sequences comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

19. The polynucleotide of any one of claims 16-18, wherein the C and the C’ sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

20. The polynucleotide comprising a hairpin structure of claim 16, wherein modified nucleic acid of (e) is a phosphorothioate-modified nucleic acid, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a dNTP / ribonucleotide triphosphates (rNTP) hybrid, isoguanosine (isoG); isocytosine (isoC), dUTP, rATP, rCTP, rGTP, or rUTP.

21. The polynucleotide hairpin structure of claim 16, wherein the at least one modified nucleic acid is positioned: 1-4 nucleotides from the 5’ end; at an internal nucleotide position; or at or near the 3’end.

22. A polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ a C’ sequence, a B sequence, an A sequence, and a B’ sequence, wherein: (a) the A sequence comprises a primary target-specific nucleotide sequence; (b) the B sequence comprises a secondary target-specific nucleotide sequence and a first nicking endonuclease recognition site nucleotide sequence; (c) the B’ sequence comprises a nucleotide sequence that is complementary to the B sequence; (d) the B and B’ sequences are hybridized to each other to form a stem-loop structure, wherein the loop comprises the A sequence; and (e) the C’ sequence comprises a second nicking endonuclease recognition site nucleotide sequence.

23. A polynucleotide comprising a hairpin structure and, in order from 5’ to 3’ a B sequence, an A sequence, a B’ sequence, and a C’ sequence, wherein: (a) the A sequence comprises a primary target-specific nucleotide sequence; (b) the B sequence comprises a secondary target-specific nucleotide sequence; (c) the B’ sequence comprises a nucleotide sequence that is complementary to the B sequence; (d) the B and B’ sequences are hybridized to each other to form a stem-loop structure, wherein the loop comprises the A sequence; (e) the interface of the C’ and B sequences comprises a second nicking endonuclease recognition site nucleotide sequence; and (f) the C’ sequence comprises a second nicking endonuclease recognition site nucleotide sequence.PCT Patent Application Attorney Docket No: 181.0004-WO00 24. The polynucleotide of claim 22 or 23, wherein the A sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

25. The polynucleotide of claim 22 or 23, wherein the B and B’ sequences comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

26. The polynucleotide of claim 22 or 23, wherein the C’ sequence comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

27. The polynucleotide hairpin structure of any one of claims 16-26, wherein the nicking endonuclease recognition sites, or the first and the second nicking endonuclease recognition sites, are independently a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

28. A linear single-stranded polynucleotide probe comprising a C’ sequence, a dye molecule, and a quencher molecule, wherein: the C’ sequence comprises, in order from 5’ to 3’ or 3’ to 5’: an optional X sequence comprising 1-8 nucleotides; a nicking endonuclease recognition site nucleotide sequence; and an optional Y sequence comprising 1-8 nucleotides.

29. The probe of claim 28, wherein the nicking endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

30. The probe of claim 28 or claim 29, wherein the probe comprises an X sequence.

31. The probe of claim 30, wherein the X sequence is labeled with a dye, wherein the dye is: internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

32. The probe of claim 31, wherein the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

33. The probe of any one of claims 29-32, wherein the X sequence is labeled with a quencher moiety, wherein the quencher moiety is:PCT Patent Application Attorney Docket No: 181.0004-WO00 internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

34. The probe of 33, wherein the quencher moiety is Black Hole Quencher (BHQ).

35. The probe of anyone of claims 28-29, wherein the probe comprises an Y sequence.

36. The probe of claim 30, wherein the Y sequence is labeled with a dye, wherein the dye is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence.

37. The probe of claim 35, wherein the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.

38. The probe of any one of claims 35-37, wherein the Y sequence is labeled with a quencher moiety, wherein the quencher moiety is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence.

39. The probe of 38, wherein the quencher moiety is Black Hole Quencher (BHQ).

40. A linear single-stranded polynucleotide probe comprising a C’ sequence, a dye molecule, and a quencher molecule, wherein: the C’ sequence comprises, in order from 5’ to 3’ or 3’ to 5’: an optional X sequence comprising 1-4 nucleotides; a first nicking endonuclease recognition site nucleotide sequence; an optional Y sequence comprising 1-4 nucleotides; a second nicking endonuclease recognition site nucleotide sequence; and an optional Z sequence comprising 1-4 nucleotides.

41. The probe of claim 29, wherein the nicking endonuclease recognition site is a site required for nicking by Nt.BspQI, Nt.CviPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.BsmI, Nb.BssSI, or Nt.BsmAI.

42. The probe of claim 40 or claim 41, wherein the probe comprises an X sequence.PCT Patent Application Attorney Docket No: 181.0004-WO00 43. The probe of claim 42, wherein the X sequence is labeled with a dye, wherein the dye is: internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

44. The probe of claim 43, wherein the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).

45. The probe of any one of claims 41-44, wherein the X sequence is labeled with a quencher moiety, wherein the quencher moiety is: internally positioned in the X sequence; positioned at or near the 5’ end of the probe if the X sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the X sequence is 3’ of the C’ sequence.

46. The probe of 45, wherein the quencher moiety is Black Hole Quencher (BHQ).

47. The probe of anyone of claims 40-41, wherein the probe comprises an Y sequence.

48. The probe of claim 42, wherein the Y sequence is labeled with a dye, wherein the dye is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence.

49. The probe of claim 47, wherein the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.

50. The probe of any one of claims 47-49, wherein the Y sequence is labeled with a quencher moiety, wherein the quencher moiety is: internally positioned in the Y sequence; positioned at or near the 5’ end of the probe if the Y sequence is 5’ of the C’ sequence; or positioned at or near the 3’ end of the probe if the Y sequence is 3’ of the C’ sequence.

51. The probe of 50, wherein the quencher moiety is Black Hole Quencher (BHQ).

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