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WO2024264074A2 - Émission de cycle initiée par coupure et réaction de remplacement - Google Patents

Émission de cycle initiée par coupure et réaction de remplacement Download PDF

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Publication number
WO2024264074A2
WO2024264074A2 PCT/US2024/035320 US2024035320W WO2024264074A2 WO 2024264074 A2 WO2024264074 A2 WO 2024264074A2 US 2024035320 W US2024035320 W US 2024035320W WO 2024264074 A2 WO2024264074 A2 WO 2024264074A2
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Prior art keywords
sequence
probe
stranded
hairpin
target
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WO2024264074A3 (fr
Inventor
Joseph Dunham
Benjamin AKINS
Arnel Arnold Santos GANCENIA
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Seqonce Biosciences Inc
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Seqonce Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • the reagents for this process can be lyophilized to further facilitate accessibility of this technology.
  • 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.
  • the limits of detection are at the single molecule level, which allows for single nucleotide allelic differentiation.
  • 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.
  • NICER is a method, which can be performed directly from specimen collection to analysis.
  • 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
  • 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.
  • 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 nucle
  • the HP2 C’ sequence may contain at least one phosphorothioate modification.
  • 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
  • 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.
  • 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);
  • 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.
  • 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; and PCT Patent Application Attorney Docket No: 181.0004-WO00 the B’ and B sequences are hybridized to each other to form a stem-loop structure
  • the A’ sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the B’ and B sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the C sequence contains 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • 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.
  • 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
  • the A sequence contains 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the B sequences contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the C and the C’ sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • modified nucleic acid of (e) is a phosphorothioate-modified nucleic acid, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a PCT Patent Application Attorney Docket No: 181.0004-WO00 dNTP/ribonucleotide triphosphates (rNTP) hybrid, isoguanosine (isoG); isocytosine (isoC), dUTP, rATP, rCTP, rGTP, or rUTP.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • rNTP PCT Patent Application Attorney Docket No: 181.0004-WO00 dNTP/ribonucleotide triphosphates (rNTP) hybrid, isoguanosine (isoG); isocytosine (isoC), dUTP, rATP, rCTP, rGTP, or rUTP.
  • 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.
  • 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.
  • 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.
  • the A sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the B and B’ sequences contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the C’ sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the 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.
  • 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.
  • 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.
  • the probe contains an X sequence.
  • 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.
  • the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).
  • 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.
  • the quencher moiety is Black Hole Quencher (BHQ).
  • BHQ Black Hole Quencher
  • the probe contains an Y sequence.
  • 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.
  • the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.
  • 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.
  • the quencher moiety is Black Hole Quencher (BHQ).
  • 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.
  • 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.
  • the probe contains an X sequence.
  • 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.
  • the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine (ROX).
  • 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.
  • the quencher moiety is Black Hole Quencher (BHQ).
  • BHQ Black Hole Quencher
  • the probe contains an Y sequence.
  • 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.
  • the dye is 6-Carboxyfluorescein (6-FAM) or carboxy-X-rhodamine.
  • 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.
  • the quencher moiety is Black Hole Quencher (BHQ).
  • 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.
  • B’ hybridizes with its complement, B, to form a stem-loop structure in which the loop includes the C sequence (C).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • Fig.7 depicts the increase in fluorescent signal as the events of nicking and strand displacement are cycled repeatedly until the reaction is terminated.
  • 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 ⁇ L.
  • Fig.8B s is a signal generation plot corresponding to Fig.8A using reactions with no target RNA.
  • Fig.8C shows limit of detection (LoD) triplicate plots for 40 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8D shows LoD triplicate plots for 20 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8E shows LoD triplicate plots for 18 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8F shows LoD triplicate plots for 16 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8G shows LoD triplicate plots for 14 cp/ ⁇ l of SARS-CoV-2N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8H shows LoD triplicate plots for 12 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8I shows LoD triplicate plots for 10 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8J shows LoD triplicate plots for 5 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • Fig.8K shows LoD triplicate plots for 1 cp/ ⁇ l of SARS-CoV-2 N1 (nucleocapsid) target DNA/ ⁇ L.
  • 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.
  • 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.
  • 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.
  • 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.
  • Fig.10B is a signal generation plot showing the negative control results corresponding to Fig.10A using reactions with no target RNA.
  • Fig.10C is a consolidated signal generation plot of the data in Figs.8A, 8B, 10A, and 10B.
  • 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.
  • 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.
  • NS nicking nuclease site
  • 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.
  • 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.
  • 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.
  • 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 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 contains a shortened B’ sequence (B’ S ), C, C’, and B sequences, and a shortened A sequence (A S ).
  • C and C’ flank B and A s , 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.
  • 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).
  • A’ of hairpin 1 (HP1) hybridizes to target template sequence A;
  • HP1 performs strand displacement;
  • HP1 B’ hybridizes to target sequence B;
  • C and B of HP1 become single stranded;
  • probe C hybridizes to the exposed C sequence of HP1;
  • new un-nicked probe hybridizes to the exposed C sequence of HP1 and (v)-(vii) repeat and cycle;
  • C’ of hairpin 2 (HP2) hybridizes to the exposed C sequence of HP1;
  • HP2 performs strand displacement, B’ of HP2 hybridizes to B of HP1;
  • 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.
  • 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 hybridize
  • 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.
  • Fig.20 illustrates the signal generation between Method 0 (linear method, hairpin specific probe), and Method 0.5 (linear, universal probe).
  • 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.
  • 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.
  • Fig.23 shows multiplexing NICER signal amplification data for two different targets, including a mismatched hairpin and probe combination to demonstrate specificity.
  • Fig.24 shows data obtained from comparing NICER 0.5 methods in which the reaction mix contained different amounts of 6000 MW PEG.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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 PCT 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.
  • 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.
  • the quencher a fluorescence quenching molecule
  • a nicking reaction 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.
  • 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.
  • Method 1.0 methods the use of dual hairpins enables the presentation of additional universal probe binding sites.
  • 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 HP1 hairpin structure
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a nicking reaction 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.
  • a new labeled probe PCT 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.
  • the HP2 C’ sequence may contain a modified phosphorothioate residue to prevent unwanted endonuclease nicking of the hairpin.
  • both HP1 and HP2 contain single-stranded regions at the 5’ end (5’ end overhangs).
  • 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.
  • Method 4.2 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.
  • 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.
  • 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.
  • 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 PCT 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.
  • a nicking reaction 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.
  • 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.
  • 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.
  • 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.
  • 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 HP2 PCT 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • NE1 second nicking endonuclease
  • 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 PCT 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.
  • a nicking reaction 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Steps (C)(i-iii) are repeated until a desired level of fluorescence signal is detected.
  • 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.
  • 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).
  • 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.
  • the method detects and/or distinguishes a specific species of mature small RNA from among other species of small RNAs in the sample.
  • 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/ ⁇ l, 5 to 15 copies/ ⁇ l, 15-25 copies/ ⁇ l, 25-100 copies/ ⁇ l, 100-200 copies/ ⁇ l, 200-300 copies/ ⁇ l, 300- 400 copies/ ⁇ l, 400-500 copies/ ⁇ l, 500-1000 copies/ ⁇ l, 1000-1500 copies/ ⁇ l, or 1500-2000 copies/ ⁇ l of target polynucleotide.
  • LiD limit of detection
  • 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.
  • 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.
  • 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.
  • extraction-free methods are preferably RNA extraction methods.
  • PCT Patent Application Attorney Docket No: 181.0004-WO00 [00125] Methods of the invention may also be utilized for analyzing non-nucleic acid targets.
  • 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.
  • the plurality of target agents are protein target agents, such as, for example, antibodies.
  • 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.
  • 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.
  • 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.
  • the A’ sequence contains a minimum number of nucleotides required for strand displacement mediated hairpin opening and subsequent nicking reactions.
  • 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.
  • 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.
  • a NE used in a method of the invention is 3-10 nucleotides in length.
  • 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.
  • Hairpin structures of the invention can contain modifications.
  • 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.
  • 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.
  • LNA Locked Nucleic Acids
  • the A’ sequence of the hairpin structure may contain (LNA)s or other exotic nucleic acids to increase Tm at specific sites.
  • 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.
  • BNA Bridged Nucleic Acids
  • 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.
  • PNA peptide nucleic acid
  • rNTP dNTP/ribonucleotide triphosphates
  • a hairpin structure contains internal spacer sequences.
  • 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.
  • 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.
  • 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.
  • 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.
  • a NE used in a method of the invention is 3-10 nucleotides in length.
  • 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 is PCT 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.
  • 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
  • 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.
  • 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.
  • the quenchable fluorescent moiety and the quenching molecule are separated by no more than 30 nucleotide positions.
  • 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 nucle
  • 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); BODIPYTM 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
  • EGFP blue fluorescent protein
  • BFP blue fluorescent protein
  • 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, BODIPYTM FL/BODIPYTM FL, Fluorescein/QSY7 and QSY9
  • LysoTrackerTM and LysoSensorTM e.g., LysoTrackerTM Blue DND-22, LysoTrackerTM Blue-White DPX, LysoTrackerTM Yellow HCK-123, LysoTrackerTM Green DND-26, LysoTrackerTM Red DND-99, Ly
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • one or more of the hairpins and/or the probe is conjugated to one or more nanoparticles.
  • 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.
  • a metal chelating polymer such as, for example, a lanthanide metal-chelating polymer.
  • 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 ⁇ L. Hairpins and probe were tested at 0.25 ⁇ M final concentrations.
  • 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).
  • FAM carboxyfluorescein
  • Two reaction buffer conditions were assessed for performing NICER: (i) ( 50 mM Potassium acetate, 20 mM tris-acetate, 10 mM Magnesium acetate, 100 ⁇ g/ml recombinant albumin (pH 7.9 @ 25 °C); and (ii) (10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 100 ⁇ g/ml recombinant albumin (pH 7.9 @ 25 °C).
  • 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.
  • 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.
  • Example 2 Limited signal amplification when a hairpin’s target recognition sequence contains a single nucleotide variation
  • NICER 0.5 a universal probe NICER method was performed using a SARS- CoV-2 B.1.1.7 - N501Y specific hairpin (“N501 hairpin”) and a SARS-CoV-2 wild-type target.
  • N501 hairpin SARS-CoV-2 B.1.1.7 - N501Y specific hairpin
  • SARS-CoV-2 wild-type target SARS-CoV-2 wild-type target.
  • the N501Y PCT 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).
  • 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.
  • 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.
  • 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.
  • 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. [00149] 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 comparing PCT 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.
  • 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). [00151] 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).
  • 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). [00153] 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.
  • 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.
  • A’ of hairpin 1 hybridizes to A of target template sequence;
  • HP1 performs strand displacement and B’ of HP1 hybridizes to B of target template sequence;
  • C and B of HP1 become single stranded;
  • C’ probe can bind to exposed C portion of HP1;
  • 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
  • new un-nicked probe binds to exposed C portion of HP1 and (iv)-(vi) repeat in cycles.
  • 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. [00156] 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).
  • 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).
  • 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
  • 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”).

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Abstract

La présente divulgation concerne des détails de procédés isothermes sans polymérase pour détecter des séquences nucléotidiques. Des procédés utilisent des oligonucléotides en épingle à cheveux spécifiques à une cible, qui interagissent également particulièrement avec des sondes marquées par des fractions de colorant fluorescent et un extincteur de fluorescence, pour amplifier la détection de polynucléotides cibles par répétition de cycles de coupure à médiation par endonucléase des sondes pour provoquer la dénaturation de fragments de sonde marquée par fluorophore à l'opposé des complexes en épingle à cheveux-sonde pour dissocier les marqueurs fluorescents des extincteurs de fluorescence.
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