Molecular torches

• US 6,534,274 B2
• Filed: 10/31/2001
• Issued: 03/18/2003
• Est. Priority Date: 07/02/1998
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Schroth, Gary P., Becker, Michael M.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/001,344
• Publication Number:
  US 20020090633A1
• Time in Patent Office: 503 Days
• Field of Search: 435/6, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• 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 2563/107 fluorescence

Abstract

The present invention features “molecular torches” and the use of molecular torches for detecting the presence of a target nucleic acid sequence. Molecular torches contain a target binding domain, a target closing domain, and a joining region. The target binding domain is biased towards the target sequence such that the target binding domain forms a more stable hybrid with the target sequence than with the target closing domain under the same hybridization conditions. The joining region facilitates the formation or maintenance of a closed torch.

Claims

1. A method for determining the presence of a target nucleic acid sequence in a sample, said method comprising the steps of:

a) contacting said sample with a molecular torch comprising;

a target binding domain comprising nucleotide base recognition groups;

a target closing domain comprising nucleotide base recognition groups, wherein said target binding domain is biased toward said target sequence, such that said target binding domain forms a more stable hybrid with said target sequence than with said target closing domain under strand displacement conditions; and

a joining region which joins said target binding domain and said target closing domain, wherein said joining region is selected from the group consisting of;

one or more non-nucleotide linkers;

first and second polynucleotides, or derivatives thereof, said first and second polynucleotides, or derivatives thereof, being substantially complementary to each other; and

a combination of said one or more non-nucleotide linkers and said first and second polynucleotides or derivatives thereof,

wherein said joining region facilitates the formation of a target binding domain;

target closing domain hybrid in the absence of said target sequence;

b) exposing said sample to strand displacement conditions; and

c) determining whether a target binding domain;

target sequence hybrid is present in said sample as an indication of the presence or absence of said target sequence in said sample.

2. The method of claim 1, wherein said target binding domain comprises 7 to about 40 of said nucleotide base recognition groups and 0 to about 4 non-nucleotide monomeric groups, each said non-nucleotide monomeric group being opposite one of said nucleotide base recognition groups present in said target closing domain.

3. The method of claim 2, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

4. The method of claim 1, wherein said target closing domain comprises 7 to about 40 of said nucleotide base recognition groups and 0 to about 6 non-nucleotide monomeric groups or mismatches with said target binding domain.

5. The method of claim 4, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

6. The method of claim 1, wherein at least 70% of said nucleotide base recognition groups of said target binding domain bind to said nucleotide base recognition groups of said target closing domain under said strand displacement conditions.

7. The method of claim 1, wherein at least 80% of said nucleotide base recognition groups of said target binding domain bind to said nucleotide base recognition groups of said target closing domain under said strand displacement conditions.

8. The method of claim 1, wherein at least 90% of said nucleotide base recognition groups of said target binding domain bind to said nucleotide base recognition groups of said target closing domain under said strand displacement conditions.

9. The method of claim 1, wherein said target binding domain includes from 1 to about 10 of said nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

10. The method of claim 9, wherein up to 5 of said nucleotide base recognition groups of said target binding domain are accessible to said target sequence under said strand displacement conditions.

11. The method of claim 9, wherein up to 3 of said nucleotide base recognition groups of said target binding domain are accessible to said target sequence under said strand displacement conditions.

12. The method of claim 1, wherein the 5′

end or the 3′

end of said target binding domain includes from 1 to about 10 of said nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

13. The method of claim 1, wherein said target binding domain:

target closing domain hybrid includes from 1 to about 10 nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

14. The method of claim 1, wherein:

said target sequence is RNA;

said target binding domain is substantially comprised of nucleotide base recognition groups which more stably bind to ribonucleotides than to deoxyribonucleotides; and

said target closing domain is substantially comprised of deoxyribonucleotides.

15. The method of claim 14, wherein said target binding domain substantially comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

16. The method of claim 1, wherein each of said first and second polynucleotides substantially comprises nucleotide base recognition groups which more stably bind to ribonucleotides than to deoxyribonucleotides.

17. The method of claim 16, wherein said first and second polynucleotides comprise 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

18. The method of claim 1, wherein said joining region consists of said one or more non-nucleotide linkers.

19. The method of claim 18, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

20. The method of claim 1, wherein said joining region consists of said first and second polynucleotides or derivatives thereof.

21. The method of claim 1, wherein said joining region consists of two of said non-nucleotide linkers and said first and second polynucleotides or derivatives thereof.

22. The method of claim 1, wherein:

said target binding domain or said target closing domain comprises a label; and

said label produces a first signal when said target binding domain;

target closing domain hybrid is formed and a second signal when said target binding domain;

target closing domain hybrid is not formed, said first and second signals being distinguishable.

23. The method of claim 1, wherein:

said target binding domain comprises a first label and said target closing domain comprises a second label;

said first and second labels interact to produce a first signal when said target binding domain and said target closing domain form a target binding domain;

target closing domain hybrid and a second signal when said target binding domain and said target closing domain do not form said target binding domain;

target closing domain hybrid, said first and second signals being distinguishable; and

an interaction change between said first and second labels is detected in step c) as an indication of the presence of a target binding domain;

target sequence hybrid.

24. The method of claim 23, wherein said first label is attached to the end of said target binding domain which is not joined to said joining region and said second label is attached to the end of said target closing domain which is not joined to said joining region.

25. The method of claim 23, wherein said first and second labels comprise a luminescent/quencher pair.

26. The method of claim 23, wherein said first and second labels comprise a fluorophore/quencher pair.

27. The method of claim 23, wherein said first and second labels comprise a luminescent/adduct pair.

28. The method of claim 23, wherein said first and second labels comprise a Fö

rrester energy transfer pair.

29. The method of claim 23, wherein said first and second labels comprise a dye dimer pair.

30. The method of claim 1, wherein said molecular torch further comprises a blocking group which can inhibit primer extension by a nucleic acid polymerase.

31. The method of claim 30, wherein said blocking group is located at or near a 3′

end of said molecular torch.

32. The method of claim 31, wherein said blocking group is selected from the group consisting of an alkyl group, a non-nucleotide linker, an alkane-diol dideoxynucleotide residue and cordycepin.

33. The method of claim 1, wherein said strand displacement conditions constitute essentially constant environmental conditions.

34. The method of claim 1 further comprising separating said molecular torch which has formed said target binding domain:

target sequence hybrid from molecular torches present in said sample which have not formed a target binding domain;

target sequence hybrid.

35. The method of claim 1, wherein said target sequence is the product of a transcription-associated amplification and said molecular torch is added to said sample prior to said amplification.

36. A molecular torch comprising:

a target binding domain comprising nucleotide base recognition groups;

a target closing domain comprising nucleotide base recognition groups, wherein said target binding domain is biased toward a target nucleic acid sequence, such that said target binding domain forms a more stable hybrid with said target sequence than with said target closing domain under strand displacement conditions; and

a joining region which joins said target binding domain and said target closing domain, wherein said joining region is selected from the group consisting of;

one or more non-nucleotide linkers;

first and second polynucleotides, or derivatives thereof, said first and second polynucleotides, or derivatives thereof, being substantially complementary to each other; and

a combination of said one or more non-nucleotide linkers and said first and second polynucleotides or derivatives thereof,

wherein said joining region facilitates the hybridization of said target binding domain to said target closing domain.

37. The molecular torch of claim 36, wherein said target binding domain comprises 7 to about 40 of said nucleotide base recognition groups and 0 to about 4 non-nucleotide monomeric groups, each said non-nucleotide monomeric group being opposite one of said nucleotide base recognition groups present in said target closing domain.

38. The molecular torch of claim 37, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

39. The molecular torch of claim 36, wherein said target closing domain comprises 7 to about 40 of said nucleotide base recognition groups and 0 to about 6 non-nucleotide monomeric groups or mismatches with said target binding domain.

40. The molecular torch of claim 39, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

41. The molecular torch of claim 36, wherein at least 70% of said nucleotide base recognition groups of said target binding domain bind to said nucleotide base recognition groups of said target closing domain under strand displacement conditions.

42. The molecular torch of claim 36, wherein at least 80% of said nucleotide base recognition groups of said target binding domain bind to said nucleotide base recognition groups of said target closing domain under strand displacement conditions.

43. The molecular torch of claim 36, wherein at least 90% of said nucleotide base recognition groups of said target binding domain bind to said nucleotide base recognition groups of said target closing domain under strand displacement conditions.

44. The molecular torch of claim 36, wherein said target binding domain includes from 1 to about 10 of said nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

45. The molecular torch of claim 44, wherein said target binding domain includes up to 5 of said nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

46. The molecular torch of claim 44, wherein said target binding domain includes up to 3 of said nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

47. The molecular torch of claim 36, wherein the 5′

end or the 3′

end of said target binding domain includes from 1 to about 10 of said nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

48. The molecular torch of claim 36, wherein said target binding domain:

target closing domain hybrid includes from 1 to about 10 of said nucleotide base recognition groups which are accessible to said target sequence under said strand displacement conditions.

49. The molecular torch of claim 36, wherein said target binding domain is substantially comprised of nucleotide base recognition groups which more stably bind to ribonucleotides than to deoxyribonucleotides, and wherein said target closing domain is substantially comprised of deoxyribonucleotides.

50. The molecular torch of claim 49, wherein said target binding domain comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

51. The molecular torch of claim 36, wherein said joining region consists of said one or more non-nucleotide linkers.

52. The molecular torch of claim 51, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

53. The molecular torch of claim 36, wherein said joining region consists of said first and second polynucleotides or derivatives thereof.

54. The molecular torch of claim 36, wherein said joining region consists of two of said non-nucleotide linkers and said first and second polynucleotides or derivatives thereof.

55. The molecular torch of claim 36, wherein said target binding domain or said target closing domain comprises a label, and wherein said label produces a first signal when said target binding domain:

target closing domain hybrid is formed and a second signal when said target binding domain;

target closing domain hybrid is not formed, said first and second signals being distinguishable.

56. The molecular torch of claim 36, wherein said target binding domain comprises a first label and said target closing domain comprises a second label, wherein said first and second labels interact to produce a first signal when said target binding domain and said target closing domain form a target binding domain:

target closing domain hybrid and a second signal when said target binding domain and said target closing domain do not form said target binding domain;

target closing domain hybrid, said first and second signals being distinguishable.

57. The molecular torch of claim 56, wherein said first label is attached to the end of said target binding domain which is not joined to said joining region and said second label is attached to the end of said target closing domain which is not joined to said joining region.

58. The molecular torch of claim 56, wherein said first and second labels comprise a luminescent/quencher pair.

59. The molecular torch of claim 56, wherein said first and second labels comprise a fluorophore/quencher pair.

60. The molecular torch of claim 56, wherein said first and second labels comprise a luminescent/adduct pair.

61. The molecular torch of claim 56, wherein said first and second labels comprise a Fö

rrester energy transfer pair.

62. The molecular torch of claim 56, wherein said first and second labels comprise a dye dimer pair.

63. The molecular torch of claim 36, wherein said molecular torch further comprises a blocking group which can inhibit primer extension by a nucleic acid polymerase.

64. The molecular torch of claim 63, wherein said blocking group is located at or near a 3′

end of said molecular torch.

65. The molecular torch of claim 63, wherein said blocking group is selected from the group consisting of an alkyl group, a non-nucleotide linker, an alkane-diol dideoxynucleotide residue and cordycepin.