Compositions and methods of detecting an analyte by using a nucleic acid hybridization switch probe

• US 20060194240A1
• Filed: 02/28/2006
• Published: 08/31/2006
• Est. Priority Date: 02/28/2005
• Status: Abandoned Application

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Russell, James, Dai, Lizhong, Arnold, Lyle J. Jr., Brentano, Steven T.
• Application Number:
  US11/363,848
• Publication Number:
  US 20060194240A1
• Time in Patent Office: Days
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

Compositions are described for detecting binding of an analyte to a binding partner attached to a nucleic acid hybridization switch probe that includes first and second arm sequences and a support sequence that is at least partially complementary to both arm sequences, allowing the probe under hybridization conditions to form a first conformation in the absence of the analyte and to form a second conformation in the presence of the analyte, and a label associated with the probe that produces a signal that indicates the conformation of the probe. Methods are described for detecting an analyte that forms a specific binding pair with the binding partner attached to the hybridization switch probe, thereby changing the probe from a first to a second conformation that results in a detectable signal that indicates the presence of the analyte in the sample.

Claims

1. A hybridization switch probe (HSP) specific for detection of an analyte, comprising:

a first nucleic acid arm sequence;

a second nucleic acid arm sequence that is different from the first nucleic acid arm sequence;

a nucleic acid support sequence that is at least partially complementary to the first nucleic acid arm sequence and at least partially complementary to the second nucleic acid arm sequence, whereby under hybridization conditions the support sequence forms a hybridization duplex with either

the first nucleic acid arm sequence thereby forming a first HSP conformation, or

the second nucleic acid arm sequence thereby forming a second HSP conformation;

a label that produces a signal that indicates the conformation of the hybridization switch probe,

and a binding pair member that forms a specific binding pair complex with the analyte, wherein the specific binding pair complex produces a conformational change in the hybridization switch probe that results in a detectable signal.

2. The hybridization switch probe of claim 1, wherein the first arm sequence is shorter than the second arm sequence.

3. The hybridization switch probe of claim 1, wherein the label produces a signal that is detectable in a homogeneous assay system.

4. The hybridization switch probe of claim 1, wherein the label is a portion of the HSP nucleic acid.

5. The hybridization switch probe of claim 1, wherein the label is a separate moiety joined directly or indirectly to the HSP.

6. The hybridization switch probe of claim 4, wherein the label is selected from the group consisting of:

a HSP nucleic acid sequence that binds a separate nucleic acid probe sequence,

a HSP nucleic acid sequence that serves as a primer in a nucleic acid amplification reaction,

a HSP nucleic acid sequence that serves as a template in a nucleic acid amplification reaction, and an aptamer.

7. The hybridization switch probe of claim 5, wherein the label is selected from the group consisting of a radionuclide, a ligand, an enzyme, an enzyme substrate, an enzyme cofactor, a reactive group, a chromophore, a particle, a bioluminescent compound, a phosphorescent compound, a chemiluminescent compound, and a fluorophore.

8. The hybridization switch probe of claim 1, wherein the label is a chemiluminescent compound attached to either the first arm sequence or the second arm sequence.

9. The hybridization switch probe of claim 1, wherein

the label is a fluorophore attached to the first arm sequence and the support sequence includes a quencher compound that is in close proximity to the fluorophore when the first arm sequence and the support sequence form a hybridization duplex, or

the label is a fluorophore attached to the second arm sequence and the support sequence includes a quencher compound that is in close proximity to the fluorophore when the second arm sequence and the support sequence form a hybridization duplex, or

the label is a fluorophore attached to the support sequence and the first arm sequence includes a quencher compound that is in close proximity to the fluorophore when the first arm sequence and the support sequence form a hybridization duplex, or

the label is a fluorophore attached to the support sequence and the second arm sequence includes a quencher compound that is in close proximity to the fluorophore when the second arm sequence and the support sequence form a hybridization duplex.

10. The hybridization switch probe of claim 1, wherein the first arm sequence is joined to the support sequence by a linking element and the second arm sequence is joined to the support sequence by a linking element.

11. The hybridization switch probe of claim 1, wherein the binding pair member that forms a specific binding pair complex with the analyte is an aptamer.

12. The hybridization switch probe of claim 1, wherein the detectable signal is an amplified nucleic acid that is produced by use of a portion of the HSP participating in a nucleic acid amplification reaction.

13. A kit comprising a hybridization switch probe that comprises:

a first nucleic acid arm sequence;

a second nucleic acid arm sequence that is different from the first nucleic acid arm sequence;

a nucleic acid support sequence that is at least partially complementary to the first nucleic acid arm sequence and to the second nucleic acid arm sequence, whereby under hybridization conditions the support sequence forms a hybridization duplex with the first nucleic acid arm sequence to form a first conformation of the hybridization switch probe, or with the second nucleic acid arm sequence to form a second conformation of the hybridization switch probe;

a label that produces a signal that indicates the conformation of the hybridization switch probe;

and a binding pair member that forms a specific binding pair complex with an analyte detected by the hybridization switch probe, wherein the specific binding pair complex produces a conformational change in the hybridization switch probe that results in a detectable signal from the label.

14. A kit of claim 13, further comprising one or more reagents for preparation of a sample containing the analyte, one or more reagents that promote binding of the analyte and the binding pair member, one or more reagents that treat the label to produce a detectable signal, or one or more reagents used in a nucleic acid amplification reaction that amplifies a nucleic acid sequence by using a portion of the HSP sequence.

15. A method of detecting an analyte in a sample, comprising:

forming a reaction mixture comprising a sample containing an analyte and a hybridization switch probe specific for the analyte,

wherein the hybridization switch probe is made up of a first nucleic acid arm sequence, a second nucleic acid arm sequence that is different from the first nucleic acid arm sequence, a nucleic acid support sequence that is at least partially complementary to the first nucleic acid arm sequence and to the second nucleic acid arm sequence, a label that produces a detectable signal, and a binding pair member that binds the analyte to form a specific binding pair complex that produces a conformational change in the hybridization switch probe, and

wherein the hybridization switch probe is in a first HSP conformation in which one arm sequence is in a hybridization duplex with the support sequence;

binding the analyte to the binding pair member, thereby forming a specific binding pair complex on the hybridization switch probe;

producing a conformational change from the first HSP conformation to a second HSP conformation resulting from formation of the specific binding pair complex; and

detecting a signal change from the label that indicates the conformational change, thereby indicating the presence of the analyte in the sample.

16. The method of claim 15, wherein the first arm sequence of the hybridization switch probe has an attached label, the second arm sequence has an attached binding pair member, and the first HSP conformation includes a hybridization duplex made up of the second arm sequence and the support sequence which is destabilized when the specific binding pair complex is formed, thereby changing the hybridization switch probe to the second HSP conformation that includes a hybridization duplex made up of the first arm sequence and the support sequence.

17. The method of claim 15, wherein the second arm sequence of the hybridization switch probe has an attached label, the first arm sequence has an attached binding pair member, and the first HSP conformation includes a hybridization duplex made up of the first arm sequence and the support sequence which is destabilized when the specific binding pair complex is formed, thereby changing the hybridization switch probe to the second HSP conformation that includes a hybridization duplex made up of the second arm sequence and the support sequence.

18. The method of claim 15, wherein the one arm sequence of the hybridization switch probe is a labeled arm sequence that has both an attached label and an attached binding pair member, and the first HSP conformation includes a hybridization duplex made up of the labeled arm sequence and the support sequence which is destabilized when the specific binding pair complex is formed, thereby changing the hybridization switch probe to the second HSP conformation in which the labeled arm sequence is not hybridized to the support sequence.

19. The method of claim 15, wherein the analyte is a ligand that binds specifically to the binding pair member and both the binding pair member and the analyte are known chemical or biochemical structures.

20. The method of claim 15, wherein the analyte is a ligand that binds specifically to the binding pair member and either the ligand or the binding pair member has an unknown chemical or biochemical structure.

21. The method of claim 15, wherein the binding pair member is a portion of a nucleic acid sequence in the hybridization switch probe.

22. The method of claim 15, wherein the binding pair member is an aptamer.

23. The method of claim 15, wherein the detecting step detects an increase in a detectable signal to indicate the presence of the analyte in the sample.

24. The method of claim 15, wherein the detecting step detects a decrease in a detectable signal to indicate the presence of the analyte in the sample.

25. The method of claim 15, wherein the detecting step detects a signal resulting from in vitro amplification of a nucleic acid sequence present in the hybridization switch probe.

26. The method of claim 15, wherein the detecting step detects a signal resulting from using a portion of the hybridization switch probe in the second HSP conformation as a primer in an in vitro nucleic acid amplification reaction.

27. The method of claim 15, wherein the detecting step detects a signal resulting from using a portion of the hybridization switch probe in the second HSP conformation as a template in an in vitro nucleic acid amplification reaction.

28. The method of claim 15, wherein the detecting step detects a signal resulting from using a portion of the hybridization switch probe in the first HSP conformation as a primer in an in vitro nucleic acid amplification reaction.

29. The method of claim 15, wherein the detecting step detects a signal resulting from using a portion of the hybridization switch probe in the first HSP conformation as a template in an in vitro nucleic acid amplification reaction.

30. The method of claim 15, wherein the detecting step detects a signal resulting from in vitro amplification of a sequence that is only amplified when the hybridization switch probe is in the second HSP conformation.

31. The method of claim 15, wherein the detecting step detects a signal resulting from in vitro amplification of a sequence that is only amplified when the hybridization switch probe is in the first HSP conformation.

32. The method of claim 15, wherein the detecting step is performed in a homogeneous format.