Molecular torches

• US 20030165957A1
• Filed: 01/27/2003
• Published: 09/04/2003
• 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 6,835,542 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 consisting essentially of:

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

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

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

first and second labels, wherein said first label is attached to said target binding domain and said second label is attached to said target closing domain, and 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 from each other.

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 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.

11. The molecular torch of claim 1, 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.

12. 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.

13. The molecular torch of claim 12, 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.

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 consisting essentially of;

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 hybridize under hybridization conditions to said target sequence when said target binding domain is hybridized to said target closing domain;

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

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

first and second labels, wherein said first label is attached to said target binding domain and said second label is attached to said target closing domain, and 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 from each other;

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 sequence hybrid is more stable than said target binding domain;

target closing domain hybrid under said hybridization conditions; and

d) determining whether there has been a change in the interaction between said first and second labels after step c) as an indication of the presence or absence of a hybrid formed between said target binding domain and said target sequence in said sample.

16. The method of claim 15, 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.

17. The method of claim 16, 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.

18. The method of claim 17, wherein each said non-nucleotide monomeric group is an abasic nucleotide.

19. The method of claim 15, 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.

20. The method of claim 15, 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.

21. The method of claim 15, 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.

22. The method of claim 21, wherein said target binding domain substantially comprises 2′

-methoxy or 2′

-fluoro substituted ribonucleotides.

23. The method of claim 15, wherein at least one of said non-nucleotide linkers is a polysaccharide or a polypeptide.

24. The method of claim 15, 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 15, 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.

26. The method of claim 15, wherein said molecular torch further comprises a blocking group which can inhibit primer extension by a nucleic acid polymerase.

27. The method of claim 25, wherein said blocking group is located at or near a 3′

end of said molecular torch.

28. The method of claim 26, 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.

29. The method of claim 15, wherein the temperature of said sample is raised in step b) and lowered in step c).

30. The method of claim 15 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.

31. The method of claim 15, 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.