Reagents and methods

Abstract

A method of detecting a target nucleic acid sequence in a test solution comprising: A. providing a test solution comprising: a. RPA single-stranded DNA-binding protein (RPA-SSB) molecule; b. cleavage

Description

Abstract A method for detecting a target nucleic acid sequence in a test solution includes: A. providing a test solution comprising: a. an RPA single-stranded DNA binding protein (RPA-SSB) molecule; b. a cleavage molecule; c. a detection probe comprising: A. one or two single-stranded oligonucleotides complementary to the target nucleic acid sequence; and B. a suppressable cleavage-dependent signaling system; and d. a sample suspected of containing the target nucleic acid sequence, and B. a signal-facilitating molecule of the detection probe generating a detectable signal when: a. one or two oligonucleotides of the probe hybridize to the target nucleic acid sequence; and b. the cleavage molecule cleaves the probe at a cleavage site. Protection is also claimed for single-stranded oligonucleotide probes comprising a suppressable cleavage-dependent signaling system. The method can be used for various detection applications, real-time monitoring of isothermal nucleic acid amplification reactions, particularly recombinase polymerase amplification (RPA) reactions.

Claims

1. A method of detecting a target nucleic acid sequence of interest in a test solution, the method comprising:1) providing a test solution comprising:A) a RPA single-stranded DNA-binding protein (RPA-SSB) molecule;B) a cleavage molecule;C) a detection probe comprising a signal-promoting molecule; andD) a sample suspected of comprising a target nucleic acid sequence of interest; and2) detecting a detectible signal produced by the signal-promoting molecule of the probe, upon:A) hybridisation of the one or two oligonucleotides of the probe with the target nucleic acid sequence of interest; andB) cleavage at the cleavage site of the probe by the cleavage molecule, wherein cleavage occurs by the action of phosphatase or exonuclease activity, and not by apurinic / apyrimidinic (AP) endonuclease activity;wherein the probe comprises a signal-promoting molecule which is:I. a polypeptide, preferably which:A) has enzymatic activity;B) is a component of an enzyme complex, is a domain of an enzyme, is a fragment of an enzyme or is an enzyme cofactor; orC) is an activator of an enzyme or an enzyme complex;and wherein the probe is a probe according to any one of claims 25 to 44; orII. a small molecule, preferably which:A) promotes the formation of a molecular complex, such as a protein:protein complex;B) promotes the activation of an enzyme or an enzyme complex; or09 12 24C) is a cofactor of an enzyme or an enzyme complex;and wherein the probe is a probe according to any one of claims 25 to 37 and 45 to 47;2. A method according to claim 1, wherein the test solution is a RPA reaction mixture comprising RPA reaction components, wherein in addition to the RPA-SSB, the RPA reaction components comprise:1) a recombinase agent;2) a recombinase loading protein;3) a polymerase; and4) forward and reverse nucleic acid primers for amplification.

3. A method according to claim 2, wherein after the providing step (A), the method further comprises performing a RPA reaction comprising one or more cycles of amplification, and detecting the detectible signal during the one or more cycles of amplification in real time, or at the end of the one or more cycles of amplification.

4. A method according to any one of claims 1 to 3, wherein the RPA-SSB is selected from the group consisting of Gp32, E. coli SSB protein, phage T4 Gp32 protein, phage Rb69 Gp32, phage vB EcoM NBGl Gp32, or any functional analog, homolog or derivative thereof, and any combination thereof, preferably the RPA-SSB is Gp32 or phage vBEco\1_\ Gp32, optionally wherein the RPA-SSB is tagged with one or more functional intrinsically disordered regions (IDRs), preferably as a genetically engineered fusion protein comprising the RPA-SSB and an amino acid sequence comprising or consisting of the one or more functional IDRs.09 12 245. A method according to any one of claims 2 to 4, wherein the recombinase agent is selected from the group consisting of UvsX, T4 UvsX, T6 UvsX, RBI8 UvsX, E.coli phage wV7 UvsX, Shigella phage CB8 UvsX, Shigella phage Shfl2 UvsX, E.coli phage ARI UvsX, phage xBEcoM^G UvsX, Shigella phage SHFML-11 UvsX, Escherichia phage vBEcoXID UvsX, E. coli RecA, E. coli RadA, E. coli RadB, E. coli Rad 51 or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the recombinase agent is UvsX, more preferably Escherichia phage vBEcoMDalCa UvsX.

6. A method according to any one of claims 2 to 5, wherein the recombinase loading protein is selected from the group consisting of UvsY, E. coli RecO, E. coli RecR or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the recombinase loading protein is UvsY, more preferably Escherichia phage STO UvsY.

7. A method according to any one of claims 2 to 6, wherein the polymerase is:1) a eukaryotic polymerase selected from the group consisting of pol-a, pol-P, pol-5, pol-s or any functional analog, homolog or derivative thereof, and any combination thereof;2) a prokaryotic polymerase selected from the group consisting of Bacillus stearothermophilus polymerase I large fragment, Bacillus subtilis Pol I large fragment (Bsu polymerase), Listeria monocytogenes DNA polymerase I, S. aureus DNA polymerase I (Sau polymerase), E. coli DNA polymerase I Klenow fragment, E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V, or any functional analog, homolog or derivative thereof, and any combination thereof, preferably wherein the polymerase is S. aureus DNA polymerase I (Sau polymerase) or Bacillus subtilis Pol I large fragment (Bsu polymerase);09 12 243) a bacteriophage polymerase selected from the group consisting of bacteriophage T4 gp43 DNA polymerase, T7 DNA polymerase and Phi-29 DNA polymerase, or any functional analog, homolog or derivative thereof, and any combination thereof.

8. A method according to any one of claims 1 to 7, wherein the cleavage molecule is Escherichia coli Exonuclease III, preferably having the amino acid sequence set forth in SEQ ID NO: 1; wherein the cleavage molecule is Neisseria gonorrhoeae Exonuclease III, preferably having the amino acid sequence set forth in SEQ ID NO: 2; or wherein the cleavage molecule is tyrosyl-DNA phosphodiesterase 1 (TDP-1), preferably having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

9. A method according to any one of claims 1 to 8, wherein the probe is a probe according to claim 38, and wherein the detectible signal is produced in a reaction which is dependent upon the polypeptide of the probe.

10. A method according to claim 9, wherein the probe is a probe according to claim 39, and wherein the detectible signal is produced in a reaction which is dependent upon the enzymatic activity of the polypeptide of the probe.

11. A method according to claim 10, wherein the probe is a probe according to claim 40 or 41, and wherein the detectible signal is produced in a reaction in a solution which is dependent upon the enzymatic activity of HRP of the probe, the method comprising detecting the activity of the HRP enzyme (HRP holoenzyme) in the solution, optionally wherein the cleavage molecule is tyrosyl-DNA phosphodiesterase 1 (TDP-1), optionally wherein TDP-1 has the amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 4.09 12 2412. A method according to claim 9, wherein the probe is a probe according to claim 42, and wherein the detectible signal is produced in a reaction which is dependent upon the enzymatic activity of the enzyme complex comprising the component, the enzymatic activity of the enzyme comprising the domain, the enzymatic activity of the enzyme comprising the fragment, or the enzymatic activity of the enzyme comprising the cofactor.

13. A method according to claim 12, wherein the probe is a probe according to claim 43 or 44, and wherein the detectible signal is produced in a reaction which is dependent upon the enzymatic activity of beta-galactosidase mediated by the betagalactosidase alpha peptide of the probe, optionally wherein the cleavage molecule is Escherichia coli Exonuclease III, preferably having the amino acid sequence set forth in SEQIDNO: 1.

14. A method according to claim 12 or 13, wherein the step of detecting the detectible signal comprises:1) contacting the beta-galactosidase alpha peptide of the probe in solution with beta-galactosidase omega fragment (omega peptide) to form beta-galactosidase holoenzyme; and2) detecting the presence of beta-galactosidase holoenzyme in solution.

15. A method according to claim 14, wherein the step of detecting the presence of beta-galactosidase holoenzyme in solution comprises: a colorimetric assay, a fluorescence assay, a chemiluminescence assay, a bioluminescence assay or an electrochemical assay.09 12 2416. A method according to claim 15, wherein the step of detecting the presence of beta-galactosidase holoenzyme in solution comprises:1) reacting beta-galactosidase holoenzyme with chlorophenol red-P-D-galactopyranoside (CPRG) in solution, thereby forming a chromophore reaction product chlorophenol red; and2) detecting the presence of chlorophenol red, optionally by measuring the absorbance of the solution at 570 to 595 nm, e.g. 575 nm.

17. A method according to any one of claims 1 to 8, wherein the probe is a probe according to claim 46, and wherein the detectible signal is produced in a reaction which is dependent upon the conversion of an apoenzyme or apoenzyme complex into a holoenzyme or holoenzyme complex mediated by the small molecule cofactor of the probe.

18. A method according to claim 17, wherein the probe is a probe according to claim 47, and wherein the detectible signal is produced in a reaction which is dependent upon the conversion of an apoenzyme or apoenzyme complex into a holoenzyme or holoenzyme complex mediated by the hemin small molecule of the probe.

19. A method according to claim 18, wherein the detectible signal is produced in a method comprising:1) contacting in a solution the hemin of the probe with horseradish peroxidase (HRP) apoenzyme, thereby forming HRP holoenzyme; and2) detecting the activity of HRP holoenzyme in the solution,09 12 24optionally wherein the step of detecting the activity of HRP holoenzyme in the solution comprises reacting HRP holoenzyme in the solution with an oxidase and a substrate for the oxidase, and detecting the presence of a product produced by the reaction.

20. A method according to claim 11 or 19, wherein the step of detecting the activity of HRP holoenzyme in the solution comprises:1) (A) reacting HRP holoenzyme in the solution with:i) glucose, e.g. P-d-glucose, glucose oxidase, p-hydroxybenzoic acid and 4-aminoantipyrine; orii) p-hydroxybenzoic acid, 4-aminoantipyrine and H2O2,thereby forming a chromophore reaction product; and(B) detecting the presence of the chromophore reaction product, optionally by measuring the absorbance of the solution at 504 nm; or2) (A) reacting HRP holoenzyme in the solution with:i) glucose, e.g. P-d-glucose, glucose oxidase and 3,3',5,5'-tetramethybenzidine (TMB); orii) 3,3',5,5'-tetramethybenzidine (TMB) and H2O2,thereby forming a chromophore reaction product; and(B) detecting the presence of the chromophore reaction product, optionally by measuring the absorbance of the solution at 450 nm or 650 nm; or3) (A) reacting HRP holoenzyme in the solution with:i) glucose, e.g. P-d-glucose, glucose oxidase and 2,2’-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS); orii) 2,2’-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) and H2O2,09 12 24thereby forming a chromophore reaction product; and(B) detecting the presence of the chromophore reaction product, optionally by measuring the absorbance of the solution at 405 nm.

21. A detection probe for detecting a target nucleic acid sequence of interest in a test solution, the test solution comprising a recombinase polymerase amplification (RPA) single-stranded DNA-binding protein (RPA-SSB), a cleavage molecule having phosphatase or exonuclease activity and a target nucleic acid sequence of interest; wherein the probe comprises:1) one or two single-stranded oligonucleotides each comprising a sequence which is complementary to a target nucleic acid sequence of interest; and2) a repressible cleavage-dependent signalling system which:A) comprises a signal-promoting molecule tethered to the oligonucleotide(s) via at least one linker, wherein the at least one linker is attached to the signalpromoting molecule and to a chemical group of the oligonucleotide(s) and thereby defines a cleavage site on the oligonucleotide(s);B) provides a detectible signal which:i) is dependent upon cleavage at the cleavage site by the cleavage molecule by the action of phosphatase or exonuclease activity, andii) following hybridisation of the oligonucleotide(s) with the target sequence, is not dependent upon:a) cleavage of the oligonucleotide(s) at an apurinic / apyrimidinic site (abasic site) of the oligonucleotide(s), e.g. at a 3'-side of an abasic site and / or at a 5'-side of the abasic site, or at a site comprising an abasic furan; orb) cleavage of the oligonucleotide(s) at a mismatch site; andC) is configured so that when the probe is present in the test solution:i) when the oligonucleotide(s) is not hybridised with the target sequence, cleavage at the cleavage site by the cleavage molecule09 12 24is inhibited and production of a detectible signal is thereby repressed; andii) upon hybridisation of the oligonucleotide(s) with the target sequence, the cleavage molecule cleaves at the cleavage site, thereby producing a detectible signal;wherein the signal-promoting molecule of the probe is:I. a polypeptide, and wherein the probe is configured such that when the probe is present in the test solution, upon hybridisation of the oligonucleotide(s) with the target sequence the cleavage molecule cleaves at the cleavage site, whereupon a detectible signal is produced in a reaction which is dependent upon the polypeptide; orII. a small molecule, and wherein the probe is configured such that when the probe is present in the test solution, upon hybridisation of the oligonucleotide(s) with the target sequence the cleavage molecule cleaves at the cleavage site, whereupon a detectible signal may be produced in a reaction which is dependent upon the small molecule.

22. A detection probe according to claim 21, wherein the linker(s):1) is not attached to an abasic nucleotide or an abasic residue of the oligonucleotide(s), e.g. the sugar molecule of an abasic nucleotide; and / or2) is not attached to the oligonucleotide(s) at a nucleotide or residue position adjacent to a position in the oligonucleotide which is occupied by an abasic nucleotide or an abasic residue.

23. A detection probe according to claim 21 or claim 22, wherein each of the one or two oligonucleotides is about 20 to 70 nucleotide positions in length, preferably between about 30 to 60 nucleotide positions in length.09 12 2424. A detection probe according to any one of claims 21 to 23, further comprising a polymerase extension blocking group attached to the 3’ end of the oligonucleotide(s), optionally wherein the blocking group is a spacer, such as a C3-spacer.

25. A detection probe according to any one of claims 21 to 24, wherein:1) the target nucleic acid sequence of interest is DNA and the detection probe is a detection probe for detecting a target DNA sequence of interest; or2) the target nucleic acid sequence of interest is RNA and the detection probe is a detection probe for detecting a target RNA sequence of interest.

26. A detection probe according to any one of claims 21 to 25, wherein when present in the test solution the recombinase polymerase amplification (RPA) singlestranded DNA-binding protein (RPA-SSB) is selected from the group consisting of Gp32, E. coli SSB protein, phage T4 Gp32 protein, phage Rb69 Gp32, phage vBEcoMNBGl Gp32, or any functional analog, homolog or derivative thereof, and any combination thereof, preferably the RPA-SSB is Gp32 or phage vB_EcoM_NBGl Gp32.

27. A detection probe according to claim 26, wherein the RPA-SSB is tagged with one or more functional intrinsically disordered regions (IDRs), preferably as a genetically engineered fusion protein comprising the RPA-SSB and an amino acid sequence comprising or consisting of the one or more functional IDRs.

28. A detection probe according to any one of claims 21 to 27, wherein the cleavage molecule is Escherichia coli Exonuclease III, preferably having the amino acid sequence set forth in SEQ ID NO: 1; wherein the cleavage molecule is Neisseria gonorrhoeae Exonuclease III, preferably having the amino acid sequence set forth in09 12 24SEQ ID NO: 2; or wherein the cleavage molecule is tyrosyl-DNA phosphodiesterase 1 (TDP-1), preferably having the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

29. A detection probe according to any one of claims 21 to 28, wherein in each one of the one or two oligonucleotides the signal-promoting molecule is tethered to the oligonucleotide(s) by a linker which is attached to the signal-promoting molecule and to a chemical group of the terminal nucleotide at the 3’ end of the oligonucleotide(s), preferably to the 3’ terminal phosphate group of the oligonucleotide(s), and wherein:1) the chemical group to which the linker is attached, preferably the 3’ terminal phosphate group, comprises the cleavage site; and2) when the probe is present in the test solution, upon hybridisation of the oligonucleotide(s) with the target sequence in the presence of a cleavage molecule having phosphatase or exonuclease activity, the cleavage molecule cleaves at the cleavage site thereby producing a detectible signal.

30. A detection probe according to any one of claims 21 to 28, wherein in each one of the one or two oligonucleotides the signal-promoting molecule is tethered to the oligonucleotide(s) by two linkers, wherein: (i) a first linker is attached to a first position on the signal-promoting molecule and to a chemical group at nucleotide position n in the oligonucleotide(s), and (ii) a second linker is attached to a second position on the signal-promoting molecule and to a chemical group at nucleotide position n+x in the oligonucleotide(s) wherein:1) the chemical group on the oligonucleotide(s) to which the first linker is attached comprises the cleavage site, preferably the 3’ terminal phosphate group; and2) when the probe is present in the test solution, upon hybridisation of the oligonucleotide(s) with the target sequence in the presence of a cleavage molecule having phosphatase or exonuclease activity, the cleavage09 12 24molecule cleaves at the cleavage site thereby producing a detectible signal; andwherein the terminal nucleotide position at the 3’ end of the oligonucleotide(s) is defined as position n, and subsequent nucleotide positions relative to position n in the direction proximal to the 5’ end of the oligonucleotide(s) are defined as n+x, wherein x is an integer of 0 or more.

31. A detection probe according to claim 30, wherein the chemical group of the oligonucleotide(s) to which the first linker is attached is the 3’ terminal phosphate group at position n of the oligonucleotide(s).

32. A detection probe according to claim 31, wherein:1) the second linker is attached to a chemical group at nucleotide position n in the oligonucleotide(s); or2) the second linker is attached to a chemical group at nucleotide position n+1 in the oligonucleotide(s); or3) the second linker is attached to a chemical group at nucleotide position n+x in the oligonucleotide(s), wherein x is an integer of 2 or more.

33. A detection probe according to claim 30, wherein the chemical group of the oligonucleotide(s) to which the first linker is attached is the nucleobase of a nucleotide at position n, preferably a thymine nucleobase.

34. A detection probe according to claim 33, wherein:1) the second linker is attached to a chemical group at nucleotide position n+1 in the oligonucleotide(s); or09 12 242) the second linker is attached to a chemical group at nucleotide position n+x in the oligonucleotide(s), wherein x is an integer of 2 or more.

35. A detection probe according to any one of claims 32 to 34, wherein the chemical group of the oligonucleotide(s) to which the second linker is attached is the nucleobase of a nucleotide, preferably a thymine nucleobase.

36. A detection probe according to claim 30, wherein the second linker is attached to a chemical group which is attached to the terminal nucleotide position at the 5’ end of the oligonucleotide(s), preferably wherein the chemical group is a phosphate group.

37. A detection probe according to any one of claims 21 to 28, wherein the probe comprises two oligonucleotides, and wherein:1) the signal-promoting molecule is tethered to a first oligonucleotide by a first linker which is attached to a first position on the signal-promoting molecule and to a chemical group of the terminal nucleotide at the 3’ end of the first oligonucleotide, preferably to the 3’ terminal phosphate group of the first oligonucleotide; and2) the signal-promoting molecule is tethered to a second oligonucleotide by a second linker which is attached to a second position on the signalpromoting molecule and to a chemical group of the terminal nucleotide at the 3’ end of the second oligonucleotide, preferably to the 3’ terminal phosphate group of the second oligonucleotide;and further wherein:A) the chemical group of the first oligonucleotide to which the first linker is attached, preferably the 3' terminal phosphate group, comprises a first cleavage site, and the chemical group of the second oligonucleotide to09 12 24which the second linker is attached, preferably the 3’ terminal phosphate group, comprises a second cleavage site; andB) when the probe is present in the test solution, upon hybridisation of the oligonucleotide with the target sequence in the presence of a cleavage molecule having phosphatase or exonuclease activity, the cleavage molecule cleaves at the cleavage sites thereby producing a detectible signal.

38. A detection probe according to any one of claims 21 to 37 and wherein the signal-promoting molecule of the probe is according to claim 21(1); wherein when the probe is present in the test solution, upon hybridisation of the oligonucleotide(s) with the target sequence the cleavage molecule cleaves at the cleavage site, whereupon a detectible signal is produced in a reaction which is dependent upon the polypeptide.

39. A detection probe according to claim 38, wherein the polypeptide has enzymatic activity, and the detectible signal is produced in a reaction which is dependent upon the enzymatic activity of the polypeptide.

40. A detection probe according to claim 39, wherein the polypeptide is a horseradish peroxidase (HRP) enzyme, preferably wherein the HRP enzyme has the amino acid sequence set forth in SEQ ID NO: 5.

41. A detection probe according to claim 40, wherein the probe has the structure:wherein * is the terminal phosphate group at the 3’ end of the oligonucleotide, L is the linker, and HRP is an HRP enzyme, preferably which has the amino acid sequence set forth in SEQ ID NO: 5.09 12 2442. A detection probe according to claim 38, wherein the polypeptide is a component of an enzyme complex, a domain of an enzyme, a fragment of an enzyme or an enzyme cofactor, and the detectible signal is produced in a reaction which is dependent upon the polypeptide.

43. A detection probe according to claim 42, wherein the polypeptide is a betagalactosidase alpha peptide, preferably having an amino acid sequence as set forth in SEQ ID NOS: 6 to 56.

44. A detection probe according to claim 43, wherein the probe has the structure:09 12 24wherein * is the terminal phosphate group at the 3’ end of the oligonucleotide, LI is the first linker, and L2 is the second linker.

45. A detection probe according to any one of claims 21 to 37 and wherein the signal-promoting molecule of the probe is according to claim 21(11); wherein when the probe is present in the test solution, upon hybridisation of the oligonucleotide(s) with the target sequence the cleavage molecule cleaves at the cleavage site, whereupon a detectible signal may be produced in a reaction which is dependent upon the small molecule.

46. A detection probe according to claim 45, wherein the small molecule is a cofactor of an enzyme or an enzyme complex, and the detectible signal is produced in a reaction which is dependent upon the activation of the enzyme or enzyme complex.

47. A detection probe according to claim 46, wherein the signal-promoting molecule is hemin, which has the structure:optionally, wherein the probe has the structure:09 12 24rS'-oligonudeotide—o / Lo.wherein * is the terminal phosphate group at the 3’ end of the oligonucleotide, and L is the linker.

48. A composition comprising composition components:1) a RPA single-stranded DNA-binding protein (RPA-SSB) molecule;2) a cleavage molecule; and3) a detection probe according to any one of claims 21 to 47.CM49. A recombinase polymerase amplification (RPA) reaction composition comprising composition components:1) RPA reaction components, including a RPA single-stranded DNA-binding protein (RPA-SSB) molecule,2) a cleavage molecule; and3) a detection probe according to any one of claims 21 to 47.

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