Molecular torches and their use under denaturing conditions

• US 20050123988A1
• Filed: 01/27/2005
• Published: 06/09/2005
• Est. Priority Date: 07/02/1998
• Status: Active Grant

• Current Assignee: Gary P. Schroth, Michael M. Becker
• Original Assignee: Gary P. Schroth, Michael M. Becker
• Inventors: Schroth, Gary P., Becker, Michael M.
• Granted Patent: US 7,252,947 B2
• 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 2525/161 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; 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 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.

3. The molecular torch of claim 2, 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.

4. The molecular torch of claim 3, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

5. The molecular torch 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 hybridization conditions.

6. The molecular torch of claim 1, 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.

7. The molecular torch of claim 1, 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.

8. The molecular torch of claim 7, wherein said target binding domain comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

9. The molecular torch of claim 1, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

10. The molecular torch of claim 1, 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.

11. The molecular torch of claim 1, 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:

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.

12. The molecular torch of claim 11, 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.

13. The molecular torch of claim 11, 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 Forrester energy transfer pair or a dye dimer pair.

14. The molecular torch of claim 1, 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.

15. The molecular torch of claim 14, 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.

16. 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 said molecular torch of claim 1;

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.

17. The method of claim 16, 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.

18. The method of claim 17, 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.

19. The method of claim 18, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

20. The method of claim 16, 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.

21. The method of claim 16, 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.

22. The method of claim 16, 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.

23. The method of claim 22, wherein said target binding domain substantially comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

24. The method of claim 16, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

25. The molecular torch of claim 16, 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.

26. The molecular torch of claim 16, 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:

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.

27. The method of claim 26, 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.

28. The method of claim 26, 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 Forrester energy transfer pair or a dye dimer pair.

29. The method of claim 16, 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.

30. The method of claim 29, 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. The method of claim 16 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.

32. The method of claim 16, wherein said target sequence is the product of an amplification reaction and said molecular torch is added to said sample prior to initiating said amplification reaction.

33. A reaction mixture comprising, prior to amplification, said molecular torch of claim 1 and a primer for use in generating multiple copies of said target sequence.

34. The reaction mixture of claim 33, 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.

35. The reaction mixture of claim 34, 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.

36. The reaction mixture of claim 35, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

37. The reaction mixture of claim 33, 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.

38. The reaction mixture of claim 33, 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.

39. The reaction mixture of claim 33, 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.

40. The reaction mixture of claim 39, wherein said target binding domain comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

41. The reaction mixture of claim 33, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

42. The reaction mixture of claim 33, 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.

43. The reaction mixture of claim 33, 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:

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.

44. The reaction mixture of claim 43, 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.

45. The reaction mixture of claim 43, 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 Forrester energy transfer pair or a dye dimer pair.

46. The reaction mixture of claim 33, 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.

47. The reaction mixture of claim 46, 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.

48. The reaction mixture of claim 33 comprising a set of primers for use in generating multiple copies of said target sequence.

49. The reaction mixture of claim 33, wherein an amplification procedure carried out under essentially constant conditions is used to generate multiple copies of said target sequence.

50. The reaction mixture of claim 33, wherein a transcription-associated amplification procedure is used to generate multiple copies of said target sequence.

51. The reaction mixture of claim 50, wherein said amplification procedure is performed without the addition of an exogenous RNAse H activity.