Nucleic acid hairpin probes and uses thereof

• US 20020142309A1
• Filed: 03/30/2001
• Published: 10/03/2002
• Est. Priority Date: 07/14/2000
• Status: Active Grant

• Current Assignee: Applied Gene Technologies Incorporated
• Original Assignee: Applied Gene Technologies Incorporated
• Inventors: Dattagupta, Nanibhushan
• Granted Patent: US 6,596,490 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates generally to nucleic acid hybridization analysis. More specifically, an oligonucleotide probe for hybridization analysis is provided, which probe comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with. Arrays comprising the hairpin probes immobilized on a solid support and methods for nucleic acid hybridization analysis using the probes or array of immobilized probes are also provided. Methods for transcribing and/or amplifying a probe DNA sequence using a hairpin probe are further provided.

Claims

1. An oligonucleotide probe for hybridization analysis, which probe comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with.

2. The probe of claim 1, which comprises DNA, RNA, PNA or a derivative thereof.

3. The probe of claim 1, which comprises both DNA and RNA or derivatives thereof.

4. The probe of claim 1, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is substantially complementary to its corresponding nucleotide sequence in the target nucleotide sequence.

5. The probe of claim 4, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is a perfect match to its corresponding nucleotide sequence in the target nucleotide sequence.

6. The probe of claim 1, wherein the single stranded loop of the probe comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or 15 nucleotides.

7. The probe of claim 1, wherein the double stranded segment of the hairpin structure is formed between two perfectly matched nucleotide sequences or two substantially matched nucleotide sequences.

8. The probe of claim 1, further comprising an element or a modification that facilitates intramolecular crosslinking of the probe upon suitable treatment.

9. The probe of claim 8, wherein the element is a chemically or photoactively activatable crosslinking agent.

10. The probe of claim 9, wherein the photoactively activatable crosslinking agent is a furocoumarin.

11. The probe of claim 8, wherein the element is a macromolecule having multiple ligand binding sites.

12. The probe of claim 11, wherein the macromolecule is a component of biotin-avidin binding system.

13. The probe of claim 1, further comprising an element or a modification that renders the probe sensitive or resistant to nuclease digestion.

14. The probe of claim 13, wherein the element is a restriction enzyme cleavage site.

15. The probe of claim 13, wherein at least a portion of the double stranded segment of the probe is a duplex between a DNA strand and a RNA strand, the DNA strand containing methylphosphonates, and wherein at least a portion of the RNA strand is complementary to the target nucleotide sequence.

17. The array of claim 16, wherein the plurality of probes comprise DNA, RNA, PNA or a derivative thereof.

18. The array of claim 16, wherein at least one of the probes comprises both DNA and RNA or derivatives thereof.

19. The array of claim 16, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop of at least one of the probes is substantially complementary to its corresponding nucleotide sequence in the target nucleotide sequence.

20. The array of claim 19, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop of at least one of the probes is a perfect match to its corresponding nucleotide sequence in the target nucleotide sequence.

21. The array of claim 16, wherein the single stranded loop of at least one of the probes comprises at least 3, 4, 5, 6, 7, 8, 9, 10 or 15 nucleotides.

22. The array of claim 16, wherein the double stranded segment of the hairpin structure of at least one of the probes is formed between two perfectly matched nucleotide sequences or two substantially matched nucleotide sequences.

23. The array of claim 16, wherein at least one of the probes further comprises an element or modification that facilitates intramolecular crosslinking of the probes upon suitable treatment.

24. The array of claim 23, wherein the element is a chemically or photoactively activatable crosslinking agent.

25. The array of claim 24, wherein the photoactively activatable crosslinking agent is a furocoumarin.

26. The array of claim 23, wherein the element is a macromolecule having multiple ligand binding sites.

27. The array of claim 26, wherein the macromolecule is a component of biotin-avidin binding system.

28. The array of claim 16, wherein at least one of the probes further comprises an element or modification that renders the probe sensitive or resistant to nuclease digestion.

29. The array of claim 28, wherein the element is a restriction enzyme cleavage site.

30. The array of claim 28, wherein at least a portion of the double stranded segment of at least one of the probes is a duplex between a DNA strand and a RNA strand, said DNA strand contains methylphosphonates and at least a portion of said RNA strand is complementary to a target nucleotide sequence to be detected.

31. The array of claim 16, wherein each of the oligonucleotide probe is capable of forming a target nucleotide sequence /oligonucleotide probe duplex with a different target nucleotide sequence.

16. An array of oligonucleotide probes immobilized on a solid support for hybridization analysis, which array comprises a solid support suitable for use in nucleic acid hybridization having immobilized thereon a plurality of oligonucleotide probes, at least one of the probes comprises a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be hybridized with.

32. A method for detecting a target nucleotide sequence in a sample, which method comprises the steps of:

a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions, and wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to a target nucleotide sequence to be detected;

b) contacting the probe provided in step a) with a sample containing or suspected of containing the target nucleotide sequence under conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself; and

c) assessing the intermolecular hybrid formed in step b).

33. The method of claim 32, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is substantially complementary to its corresponding nucleotide sequence in the target nucleotide sequence.

34. The method of claim 32, wherein the portion of the nucleotide sequences located within the double stranded segment and the single stranded loop is a perfect match to its corresponding nucleotide sequence in the target nucleotide sequence.

35. The method of claim 32, wherein the single stranded loop of the probe comprises at least 3, 4, 5, 6, 7, 8, 9 or 10,15 nucleotides.

36. The method of claim 32, wherein the conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself is achieved by controlling compositions of the probe and the target nucleotide sequence so that the Tm of the intermolecular hybrid is higher than the Tm of the intramolecular hybrid.

37. The method of claim 36, wherein the Tm of the intermolecular hybrid is at least 2°

C. higher than the Tm of the intramolecular hybrid.

38. The method of claim 36, wherein the intermolecular hybrid is a RNA:

DNA, RNA;

RNA or a DNA;

DNA hybrid or a derivative thereof and the intramolecular hybrid is a RNA;

DNA, RNA;

RNA or a DNA;

DNA hybrid or a derivative thereof.

39. The method of claim 36, wherein the intermolecular hybrid is a RNA:

DNA hybrid whereas the intramolecular hybrid is a DNA;

DNA hybrid.

40. The method of claim 32, wherein the target nucleotide sequence is detectably labeled and the formation of the intermolecular hybrid is assessed by detecting the label of the target nucleotide sequence in the intermolecular hybrid.

41. The method of claim 40, wherein the label is selected from the group consisting of a chemical, an enzymatic, a radioactive, a fluorescent, a luminescent and a FRET label.

42. The method of claim 32, wherein the formation of the intermolecular hybrid is assessed by addition of a detectably labeled secondary probe that specifically hybridizes with at least a portion of the intermolecular hybrid and the detection of a secondary intermolecular hybrid formed between the secondary probe and the original intermolecular hybrid indicates the presence of the target nucleotide sequence in the sample.

43. The method of claim 42, further comprising a step of crosslinking the intermolecular hybrid and the intramolecular hybrid after the formation of the intermolecular hybrid but before the addition of the detectably labeled secondary probe.

44. The method of claim 43, wherein the crosslinking step is effected via addition of a crosslinking agent subsequent to hybridization of the original probe with the target nucleotide sequence.

45. The method of claim 43, wherein the original hairpin probe is synthesized with the crosslinking agent attached and crosslinking is achieved by addition of an appropriate agent or treatment.

46. The method of claim 42, wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is not involved in the hybridization of the target nucleotide sequence and the original probe.

47. The method of claim 42, wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is involved in the hybridization of the target nucleotide sequence and the original probe and the nucleotide sequence in the original probe that is complementary to the same portion of the target nucleotide sequence is removed prior to or concurrently with the addition of the secondary probe.

48. The method of claim 47, wherein the hairpin structure in the original probe is formed between a DNA strand that contains methylphosphonates and a RNA strand that is complementary to the target nucleotide sequence and wherein the RNA strand, after forming a hybrid with the target nucleotide sequence but before the addition of the secondary probe, is removed by a RNase H treatment.

49. The method of claim 42, wherein the secondary probe specifically hybridizes with a portion of the original probe that is involved in the formation of the intramolecular hybrid but is not involved in the formation of the intermolecular hybrid with the target nucleotide sequence and wherein the same portion of the original probe that remains within the unhybridized original probe is removed prior to or concurrently with the addition of the secondary probe.

50. The method of claim 49, wherein the portion of the original probe that remains within the unhybridized original probe contains a restriction enzyme cleavage site and is removed by cleavage with said restriction enzyme.

51. The method of claim 42, wherein the detectably labeled secondary probe is a specific probe.

52. The method of claim 42, wherein the detectably labeled probe is a degenerate probe.

53. The method of claim 32, wherein the probe is immobilized on a solid support.

54. The method of claim 32, wherein a plurality of the probes immobilized on a solid support is used.

55. The method of claim 32, wherein the sample is a biosample.

56. The method of claim 32, wherein a plurality of samples is assayed simultaneously.

57. The method of claim 32, wherein the oligonucleotide probe and the target nucleotide sequence is contacted at a temperature from about 4°

C. to about 90°

C.

58. The method of claim 32, wherein the oligonucleotide probe and the target nucleotide sequence is contacted for a time from about 1 minute to about 60 minutes.

60. The method of claim 59, wherein at least a portion of said nucleotide sequences located within said double stranded segment and a portion of said nucleotide sequence located within said single stranded loop collectively form a region that is complementary to the DNA sequence.

61. The method of claim 59, further comprising a step of reverse transcribing the synthesized RNA sequence into a DNA sequence.

62. The method of claim 61, wherein the RNA synthesis and the reverse transcription are conducted for a plurality of cycles to amplify the probe sequence.

59. A method for transcribing and/or amplifying an oligonucleotide probe sequence, which method comprises the steps of:

a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a double stranded segment and a single stranded loop, wherein said loop contains at least 3 nucleotides, said double stranded segment is formed between two complementary nucleotide sequences under suitable conditions and contains a promoter sequence, and wherein at least a portion of said nucleotide sequence located within said single stranded loop is complementary to a DNA sequence and said portion of said nucleotide sequence comprises both ribonucleotide sequence and deoxyribonucleotide sequence;

b) contacting said probe provided in step a) with said DNA sequence under suitable conditions to form a probe/DNA duplex;

c) cleaving said ribonucleotide sequence within said portion of said nucleotide sequence complementary to said DNA sequence by RNase H treatment to open said single stranded loop; and

d) synthesizing a RNA sequence using a RNA polymerase that is compatible with said promoter contained within said double stranded segment of said probe,

whereby at least a portion of said single stranded loop is transcribed.