Small nucleic acid detection probes and uses thereof

• US 20070015176A1
• Filed: 02/14/2006
• Published: 01/18/2007
• Est. Priority Date: 02/18/2005
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Straus, Neil, Lao, Kai
• Application Number:
  US11/354,618
• Publication Number:
  US 20070015176A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide

Abstract

The present teachings are directed to compositions, methods, and kits for detecting and quantitating small nucleic acid molecules, including small DNA molecules and small RNA molecules. The detector probes of the current teachings, including single-loop detector probes, double-loop detector probes, and bimolecular detector probes, are designed to selectively hybridize with a corresponding small nucleic acid target and to produce, under appropriate conditions, a detectable signal or a detectably different signal. The detector complexes of the current teachings comprise a detector probe comprising a first reporter group and a displaceable sequence comprising a second reporter group, wherein the displaceable sequence is hybridized to the detector probe. According to certain methods, detecting a small nucleic acid target comprises the target displacing the displaceable sequence of a detector complex to form a detector probe-small nucleic acid target duplex, illuminating the duplex with light of an appropriate wavelength, and determining the presence of a detectable fluorescent signal or the change in a detectable signal.

Claims

1. A detector probe comprising (a) a target-complementary portion, (b) a reporter group, and (c) a first stem-loop structure, wherein (i) the first stem-loop structure comprises a first segment, a second segment, and a third segment, (ii) the second segment is located between the first segment and the third segment, and (iii) the first segment and the third segment are complementary with each other.

2. The detector probe of claim 1, further comprising (d) a second stem-loop structure, wherein (i) the second stem-loop structure comprises a fourth segment, a fifth segment, and a sixth segment, (ii) the fifth segment is located between the fourth segment and the sixth segment, and (iii) the fourth segment and the sixth segment are complementary with each other.

3. The detector probe of claim 2, wherein the reporter group comprises a tether and an intercalating dye molecule.

4. The detector probe of claim 2, wherein the target-complementary portion further comprises a minor groove binder.

5. The detector probe of claim 1, wherein the reporter group comprises a tether and an intercalating dye molecule.

6. The detector probe of claim 1, wherein the target-complementary portion further comprises a minor groove binder.

7. A bimolecular detector probe comprising, (a) a first component comprising (i) a first target-complementary portion and (ii) a first reporter group and (b) a second component comprising (i) a second target-complementary portion and (ii) a second reporter group.

8. A detector complex comprising:

(1) a detector probe comprising (a) a target-complementary portion, (b) a reporter group, and (c) a first stem-loop structure, wherein (i) the first stem-loop structure comprises a first segment, a second segment, and a third segment, (ii) the second segment is located between the first segment and the third segment, and (iii) the first segment and the third segment are complementary with each other; and

(2) a displaceable sequence comprising a second reporter group, wherein the displaceable sequence is annealed to the detector probe.

9. The detector complex of claim 8, wherein the detector probe further comprises:

(d) a second stem-loop structure, wherein (i) the second stem-loop structure comprises a fourth segment, a fifth segment, and a sixth segment, (ii) the fifth segment is located between the fourth segment and the sixth segment, and (iii) the fourth segment and the sixth segment are complementary with each other.

10. A method for detecting a small nucleic acid sequence comprising:

combining a detector complex with a sample, wherein the detector complex comprises (a) a detector probe comprising (i) a target-complementary portion and (ii) a first reporter group and (b) a displaceable sequence comprising a second reporter group, and wherein the detector probe is annealed with the displaceable sequence;

displacing the displaceable sequence of the detector complex and annealing a small nucleic acid sequence with the detector probe to form a duplex; and

detecting the detector probe-small nucleic acid sequence duplex.

11. The method of claim 10, wherein the first reporter group is a fluorophore and the second reporter group is a fluorescent quencher or a dark quencher.

12. The method of claim 10, wherein the detecting comprises quantitating the detector probe-small nucleic acid sequence duplex.

13. The method of claim 10, wherein the detecting comprises fluorescence microscopy.

14. The method of claim 10, wherein the detecting comprises single molecule detection.

15. The method of claim 10, wherein the sample comprises a cell or a tissue section.

16. The method of claim 10, wherein the sample comprises a cell lysate.

17. A method for detecting a small nucleic acid sequence comprising:

combining a detector probe with a sample, wherein the detector probe comprises (a) a target-complementary portion, (b) a reporter group, and (c) a first stem-loop structure, wherein (i) the first stem-loop structure comprises a first segment, a second segment, and a third segment, (ii) the second segment is located between the first segment and the third segment, and (iii) the first segment and the third segment are complementary, and wherein the reporter group comprises a tether and an intercalating dye molecule;

annealing the small nucleic acid sequence with the detector probe to form a detector probe-small nucleic acid sequence duplex; and

detecting the detector probe-small nucleic acid sequence duplex.

18. The method of claim 17, wherein the detecting comprises quantitating the detector probe-small nucleic acid sequence duplex.

19. The method of claim 18, wherein the quantitating comprises single molecule detection.

20. The method of claim 17, wherein the detecting comprises fluorescence microscopy.

21. The method of claim 17, wherein the sample comprises a cell or a tissue section.

22. The method of claim 17, wherein the sample comprises a cell lysate.

23. The method of claim 17, wherein the detector probe further comprises:

(d) a second stem-loop structure, wherein (i) the second stem-loop structure comprises a fourth segment, a fifth segment, and a sixth segment, (ii) the fifth segment is located between the fourth segment and the sixth segment, and (iii) the fourth segment and the sixth segment are complementary with each other.

24. The method of claim 23, wherein the detecting comprises quantitating the detector probe-small nucleic acid sequence duplex.

25. The method of claim 24, wherein the quantitating comprises single molecule detection.

26. The method of claim 23, wherein the detecting comprises fluorescence microscopy.

27. The method of claim 23, wherein the sample comprises a cell or a tissue section.

28. The method of claim 23, wherein the sample comprises a cell lysate.

29. A method for detecting a small nucleic acid sequence comprising:

combining a bimolecular detector probe with a sample, wherein the bimolecular detector probe comprises, (a) a first component comprising (i) a first target-complementary portion and (ii) a first reporter group and (b) a second component comprising (i) a second target-complementary portion and (ii) a second reporter group;

annealing the small nucleic acid sequence with the two components of the bimolecular probe to form a trimolecular complex; and

detecting the trimolecular complex.

30. The method of claim 29, wherein the detecting comprises quantitating the trimolecular complex.

31. The method of claim 30, wherein the quantitating comprises single molecule detection.

32. The method of claim 29, wherein the detecting comprises fluorescence microscopy.

33. The method of claim 29, wherein the sample comprises a cell or a tissue section.

34. The method of claim 29, wherein the sample comprises a cell lysate.

35. A kit comprising a detector complex comprising a detector probe and a displaceable sequence.

36. The kit of claim 35, wherein the detector probe comprises:

(a) a target-complementary portion, (b) a reporter group, and (c) a first stem-loop structure, wherein (i) the first stem-loop structure comprises a first segment, a second segment, and a third segment, (ii) the second segment is located between the first segment and the third segment, and (iii) the first segment and the third segment are complementary with each other.

37. The kit of claim 35, wherein the detector probe comprises:

(a) a target-complementary portion, (b) a reporter group, (c) a first stem-loop structure, wherein (i) the first stem-loop structure comprises a first segment, a second segment, and a third segment, (ii) the second segment is located between the first segment and the third segment, and (iii) the first segment and the third segment are complementary with each other, and (d) a second stem-loop structure, wherein (i) the second stem-loop structure comprises a fourth segment, a fifth segment, and a sixth segment, (ii) the fifth segment is located between the fourth segment and the sixth segment, and (iii) the fourth segment and the sixth segment are complementary with each other.

38. A kit comprising a detector probe that comprises:

(a) a target-complementary portion, (b) a reporter group, and (c) a first stem-loop structure, wherein (i) the first stem-loop structure comprises a first segment, a second segment, and a third segment, (ii) the second segment is located between the first segment and the third segment, and (iii) the first segment and the third segment are complementary with each other, wherein the reporter group comprises a tether and an intercalating dye molecule.

39. The kit of claim 38, wherein the detector probe further comprises:

(d) a second stem-loop structure, wherein (i) the second stem-loop structure comprises a fourth segment, a fifth segment, and a sixth segment, (ii) the fifth segment is located between the fourth segment and the sixth segment, and (iii) the fourth segment and the sixth segment are complementary with each other

40. A kit comprising a bimolecular detector probe comprising:

(a) a first component comprising (i) a first target-complementary portion and (ii) a first reporter group; and

(b) a second component comprising (i) a second target-complementary portion and (ii) a second reporter group.

41. The kit of claim 40, wherein the first reporter group comprises a fluorescent reporter group and the second reporter group comprises a fluorescent quencher.