Molecular torches and their use under denaturing conditions

• US 7,252,947 B2
• Filed: 01/27/2005
• Issued: 08/07/2007
• Est. Priority Date: 07/02/1998
• Status: Expired due to Fees

• 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:
  US11/044,090
• Publication Number:
  US 20050123988A1
• Time in Patent Office: 922 Days
• Field of Search: 435/6, 435/91.2, 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 molecular torch for use in detecting the presence of a target nucleic acid sequence in a sample, said 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 the same assay conditions, and wherein said molecular torch does not detectably hybridize under hybridization conditions to said target sequence when said target binding domain is hybridized to said target closing domain;

a label associated with said target binding domain or said target closing domain, 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; and

an unlabeled joining region comprising one or more non-nucleotide linkers, wherein said joining region joins said target binding domain and said target closing domain, and wherein said joining region facilitates the formation of a target binding domain;

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

2. The molecular torch of claim 1, wherein a first label is associated with said target binding domain and a second label is associated with said target closing domain, wherein said first and second labels interact to produce said first signal when said target binding domain:

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

target closing domain hybrid is not formed.

3. The molecular torch of claim 2, wherein said target binding domain comprises 7 to 40 of said nucleotide base recognition groups and 0 to 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.

4. The molecular torch of claim 3, wherein said target closing domain comprises 7 to 40 of said nucleotide base recognition groups and 0 to 6 non-nucleotide monomeric groups or mismatches with said target binding domain.

5. The molecular torch of claim 4, wherein each said non-nucleotide monomeric group is an a basic nucleotide.

6. The molecular torch of claim 2, 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 hybridization conditions.

7. The molecular torch of claim 2, wherein said target binding domain and said target closing domain each comprise a sugar-phosphodiester type linkage and nucleotide base recognition groups able to hydrogen bond to adenine, guanine, cytosine, thymine or uracil joined to said backbone.

8. The molecular torch of claim 2, 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.

9. The molecular torch of claim 8, wherein said target binding domain comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

10. The molecular torch of claim 2, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

11. The molecular torch of claim 2, 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.

12. The molecular torch of claim 2, wherein said first and second labels comprise an enzyme/substrate pair, an enzyme/cofactor pair, a luminescent/quencher pair, a fluorophore/quencher pair, a luminescent/adduct pair, a Fö

rrester energy transfer pair or a dye dimer pair.

13. The molecular torch of claim 2, wherein said molecular torch further comprises a blocking group which can inhibit primer extension by a nucleic acid polymerase, and wherein said blocking group is located at or near a 3′

end of said molecular torch.

14. The molecular torch of claim 13, 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.

31. A method for determining the presence or amount of a target nucleic acid in a sample, said method comprising the steps of:

a) performing an amplification reaction in a sample containing said molecular torch of claim 1; and

b) detecting a product of said amplification reaction as an indication of the presence or amount of said target nucleic acid in said sample.

32. The method of claim 31, wherein a first label is associated with said target binding domain and a second label is associated with said target closing domain, wherein said first and second labels interact to produce said first signal when said target binding domain:

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

target closing domain hybrid is not formed.

33. The method of claim 32, wherein said target binding domain comprises 7 to 40 of said nucleotide base recognition groups and 0 to 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.

34. The method of claim 33, wherein said target closing domain comprises 7 to 40 of said nucleotide base recognition groups and 0 to 6 non-nucleotide monomeric groups or mismatches with said target binding domain.

35. The method of claim 34, wherein each said non-nucleotide monomeric group is an a basic nucleotide.

36. The method of claim 32, 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 hybridization conditions.

37. The method of claim 32, wherein said target binding domain and said target closing domain each comprise a sugar-phosphodiester type linkage and nucleotide base recognition groups able to hydrogen bond to adenine, guanine, cytosine, thymine or uracil joined to said backbone.

38. The method of claim 32, 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.

39. The method of claim 38, wherein said target binding domain comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

40. The method of claim 32, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

41. The method of claim 32, 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.

42. The method of claim 32, wherein said first and second labels comprise an enzyme/substrate pair, an enzyme/cofactor pair, a luminescent/quencher pair, a fluorophore/quencher pair, a luminescent/adduct pair, a Fö

rrester energy transfer pair or a dye dimer pair.

43. The method of claim 32, wherein said molecular torch further comprises a blocking group which can inhibit primer extension by a nucleic acid polymerase, and wherein said blocking group is located at or near a 3′

end of said molecular torch.

44. The method of claim 43, 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.

45. The method of claim 32, wherein said amplification reaction is carried out under essentially constant conditions.

46. The method of claim 32, wherein said amplification reaction is a transcription-associated amplification procedure.

47. The method of claim 46, wherein said amplification reaction is performed without the addition of an exogenous RNAse H activity.

15. 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 the same assay conditions, and wherein said molecular torch does not detectably hybridize under hybridization conditions to said target sequence when said target binding domain is hybridized to said target closing domain; and

an unlabeled joining region comprising one or more non-nucleotide linkers, wherein said joining region joins said target binding domain and said target closing domain, and 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 denaturing conditions, such that said target binding domain and said target closing domain do not form a stable target binding domain;

target closing domain hybrid

c) exposing said sample to hybridization conditions, such that a target binding domain;

target closing domain hybrid is formed in the absence of said target sequence and a target binding domain;

target sequence hybrid is formed in the presence of said target sequence, wherein said target binding domain;

target closing domain hybrid is more stable than said target binding domain;

target closing domain hybrid under said hybridization conditions; and

d) 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.

16. The method of claim 15, 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.

17. The method of claim 16, wherein a first label is associated with said target binding domain and a second label is associated with said target closing domain, wherein said first and second labels interact to produce said first signal when said target binding domain:

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

target closing domain hybrid is not formed.

18. The method of claim 17, wherein said target binding domain comprises 7 to 40 of said nucleotide base recognition groups and 0 to 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.

19. The method of claim 18, wherein said target closing domain comprises 7 to 40 of said nucleotide base recognition groups and 0 to 6 non-nucleotide monomeric groups or mismatches with said target binding domain.

20. The method of claim 19, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

21. The method of claim 17, 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 hybridization conditions.

22. The method of claim 17, wherein said target binding domain and said target closing domain each comprise a sugar-phosphodiester type linkage and nucleotide base recognition groups able to hydrogen bond to adenine, guanine, cytosine, thymine or uracil joined to said backbone.

23. The method of claim 17, 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.

24. The method of claim 23, wherein said target binding domain substantially comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

25. The method of claim 17, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

26. The method of claim 17, 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.

27. The method of claim 17, wherein said first and second labels comprise an enzyme/substrate pair, an enzyme/cofactor pair, a luminescent/quencher pair, a fluorophore/quencher pair, a luminescent/adduct pair, a Fö

rrester energy transfer pair or a dye dimer pair.

28. The method of claim 17, wherein said molecular torch further comprises a blocking group which can inhibit primer extension by a nucleic acid polymerase, and wherein said blocking group is located at or near a 3′

end of said molecular torch.

29. The method of claim 28, 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.

30. The method of claim 17 further comprising separating said molecular torch which has formed a hybrid with said target sequence from molecular torches present in said sample which have not formed a hybrid with said target sequence.