Turn over probes and use thereof for nucleic acid detection

• US 7,968,289 B2
• Filed: 05/03/2006
• Issued: 06/28/2011
• Est. Priority Date: 05/03/2005
• Status: Expired due to Fees

• Current Assignee: Ensemble Therapeutics
• Original Assignee: Ensemble Therapeutics
• Inventors: Li, Xiaoyu, McLaughlin, Larry W., Bittker, Joshua A., Haff, Lawrence A., Coull, James M.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/913,521
• Publication Number:
  US 20090280477A1
• Time in Patent Office: 1,882 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• 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 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, 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;

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

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

2. 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.

3. The method of claim 2, wherein the signal generating moiety is covalently attached to the target nucleic acid.

4. The method of claim 2, wherein the signal generating moiety is a catalyst.

5. The method of claim 2, wherein the signal generating moiety is a light emitting moiety.

6. The method of claim 2, wherein the detectable event is an optical event.

7. The method of claim 6, wherein the optical event comprises a fluorescent event.

8. The method of claim 2, 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.

9. The method of claim 2, 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.

10. The method of claim 9, wherein the detectable event occurs after hairpin formation.

14. The method of claim 2, wherein the chemical moiety comprises a masked thiol linkage produced using the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group.

11. 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 to form a complex 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, wherein the chemical moiety comprises a thiol linkage masked by a disulfide moiety produced using the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group; and

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

12. 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, wherein the chemical moiety comprises a thiol linkage masked by a disulfide moiety produced using the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group;

,

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

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

13. 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, wherein the chemical moiety comprises a thiol masked by a disulfide linkage produced using the phosphoramidite as shown in FIG. 8 (compound I) wherein R is a linear or branched C_1-6 alkyl group.