Turn Over Probes and Use Thereof for Nucleic Acid Detection

• US 20090280477A1
• Filed: 05/03/2006
• Published: 11/12/2009
• Est. Priority Date: 05/03/2005
• Status: Active Grant

• Current Assignee: Ensemble Therapeutics
• Original Assignee: Ensemble Discovery
• Inventors: Li, Xiaoyu, Rosenberg, Alexander F., Coull, James M., McLaughlin, Larry W., Bittker, Joshua A., Haff, Lawrence A.
• Granted Patent: US 7,968,289 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2537/155 Cyclic reactions

Abstract

The invention provides turnover probes for use in a variety of detection assays, for example, nucleic acid detection assays. In addition, the invention provides assays, for example, nucleic acid detection assays, using such turnover probes.

Claims

1. A method of disassociating a nucleic acid probe from a target nucleic acid sequence, the method comprising the steps of:

(a) combining a nucleic acid probe with a sample suspected of containing the target nucleic acid sequence under conditions to permit the probe to anneal to the target nucleic acid sequence if the target nucleic acid is present in the sample, wherein the nucleic acid probe comprises a chemical moiety, which if modified or removed, permits the nucleic acid probe or a fragment thereof to form a hairpin structure and disassociate from the complex; and

(b) providing a reagent capable of modifying or removing the chemical moiety so that if a complex is present in the sample the nucleic acid probe or the fragment thereof forms a hairpin structure and disassociates from the complex.

2. The method of claim 1 comprising the additional step of detecting a product produced by step (b) indicative of the existence of the complex so as to determine whether the target nucleic acid is present in the sample.

3. The method of claim 1, wherein the hairpin structure contains a stem.

4. The method of claim 1, wherein in step (b) the reagent removes the chemical moiety.

5. The method of claim 1, wherein in step (b) the reagent modifies the chemical moiety.

6. The method of claim 1, wherein the nucleic acid probe is cleaved in the vicinity of the chemical moiety.

7. The method of claim 1, wherein the nucleic acid probe comprises a masked thiol linkage.

8. The method of claim 7, wherein the nucleic acid probe comprises a thiol linkage masked by a disulfide moiety.

9. The method of claim 1, wherein the reagent comprises a nucleic acid sequence complementary to the target nucleic acid sequence.

10. The method of claim 1, wherein the reagent is linked to a second nucleic acid sequence capable of annealing to the target nucleic acid sequence so that when the second nucleic acid sequence anneals to the target nucleic acid sequence the reagent modifies or removes the chemical moiety contained in the nucleic acid probe.

11. The method of claim 10, wherein the second nucleic acid sequence anneals to a first location of the target nucleic acid sequence and the nucleic acid probe anneals at a second location of the target nucleic acid sequence.

12. The method of claim 11, wherein the first and second locations are adjacent one another.

13. The method of claim 1, wherein the reagent comprises a nucleic acid sequence complementary to the nucleic acid probe.

14. The method of claim 1, wherein the reagent comprises an agent selected from the group consisting of a metal, a reducing agent, a chelating agent, a nucleophile, a nucleophilic compound, an electrophile, an electrophilic compound, a catalytic peptide, a catalytic small molecule, a protease, a nuclease, or a protein that induces a chemical transformation.

15. The method of claim 1, wherein the chemical moiety is selected from the group consisting of a base, a modified base, an oligonucleotide, a modified oligonucleotide, an internucleotide linkage, a modified internucleotide linkage, a protecting group, a peptide, a protein, a polymer, a bead and a nanoparticle.

16. The method of claim 1, wherein the nucleic acid probe further comprises a detectable label.

17. The method of claim 16, wherein in step (b) the modification or removal of the chemical moiety causes the detectable label to generate a detectable signal.

18. The method of claim 1, wherein the nucleic acid probe or the target nucleic acid is coupled to a ligand.

19. The method of claim 1, wherein the chemical moiety is a sulfur-containing internucleotide linkage.

20. The method of claim 1, further comprising the step of:

c) detecting a product produced by step (b) indicative of the existence of the complex so as to determine whether the target nucleic acid is present in the sample.

53. The method of claim 8, wherein the masked disulfide-containing linkage comprises the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group.

21-33. -33. (canceled)

34. A composition comprising a nucleic acid probe capable of annealing to a target nucleic acid to form a complex, wherein the probe comprises

(i) a nucleic acid sequence complementary to the target nucleic acid;

(ii) a chemical moiety associated with the nucleic acid sequence, which if modified or removed by a chemical reagent permits the probe to form a hairpin structure and to disassociate from the complex;

(iii) a signal generating moiety associated with the nucleic acid sequence; and

(iv) optionally, a signal quencher associated with the nucleic acid sequence.

35. The composition of claim 34, wherein the chemical moiety is disposed within the nucleic acid sequence.

36. The composition of claim 35, wherein the chemical moiety is a chemical bond.

37. The composition of claim 36, wherein the chemical moiety is defined by at least one atom.

38. The composition of claim 34, wherein the chemical moiety is attached to the nucleic acid sequence.

39. The composition of claim 38, wherein the chemical moiety is covalently bonded to the nucleic acid sequence.

40. The composition of claim 34, wherein the chemical moiety is selected from the group consisting of a base, a modified base, an oligonucleotide, a modified oligonucleotide, a modified internucleotide linkage, a protecting group, a peptide, a protein, a polymer, a bead and a nanoparticle.

54. The composition of claim 34, wherein the chemical moiety comprises the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group.

41. A composition comprising a target nucleic acid sequence and a nucleic acid probe capable of annealing to the target nucleic acid sequence to form a complex, wherein the nucleic acid probe comprises a chemical moiety, which if modified or removed permits the nucleic acid probe to form a hairpin structure and disassociate from the complex.

42. The composition of claim 41, wherein the nucleic acid probe further comprises a signal generating moiety.

43. The composition of claim 41, wherein the nucleic acid probe further comprises a quencher capable of quenching a signal from detectable label.

55. The composition of claim 41, wherein the chemical moiety comprises the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group.

44. A method of amplifying a signal indicative of the presence of a target nucleic acid in a sample, the method comprising the steps of:

(a) incubating a sample suspected of containing the target nucleic acid with a first nucleic acid probe under conditions to permit the first nucleic acid probe to anneal to the target nucleic acid if present in the sample, wherein the first nucleic acid probe comprises (i) a signal generating moiety capable of producing a detectable event and (ii) a nucleotide sequence that anneals to the target nucleic acid;

(b) providing a non-enzymatic turnover inducing reagent that reacts with the first nucleic acid probe to promote separation of the first nucleic acid probe from the target nucleic acid and create a detectable event; and

(c) allowing a second nucleic acid probe to bind to the target nucleic acid after the first nucleic acid probe has been separated from the target nucleic acid and produce another detectable event.

45. The method of claim 44, wherein the signal generating moiety is covalently attached to the target nucleic acid.

46. The method of claim 44, wherein the signal generating moiety is a catalyst.

47. The method of claim 44, wherein the signal generating moiety is a light emitting moiety.

48. The method of claim 44, wherein the detectable event is an optical event.

49. The method of claim 48, wherein the optical event comprises a fluorescent event.

50. The method of claim 44, wherein the turnover inducing reagent is attached to a different nucleotide sequence that anneals to the target nucleic acid at a location adjacent to where the probe anneals to the target nucleic acid.

51. The method of claim 44, wherein the turnover inducing reagent reacts with the first nucleic acid probe to cause the first nucleic acid probe or a fragment thereof produce a hairpin structure.

52. The method of claim 51, wherein the detectable event occurs after hairpin formation.

56. The composition of claim 44, wherein the chemical moiety comprises the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group.