Kits and reaction mixtures containing modified probe molecules

• US 7,399,852 B2
• Filed: 03/14/2001
• Issued: 07/15/2008
• Est. Priority Date: 07/16/1996
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Majlessi, Mehrdad, Brentano, Steven T., Becker, Michael M.
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): CALAMITA, HEATHER
• Application Number:
  US09/808,558
• Publication Number:
  US 20030036058A1
• Time in Patent Office: 2,680 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/24.31, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 536/24.3
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The present invention concerns oligonucleotides containing one or more modified nucleotides which increase the binding affinity of the oligonucleotides to target nucleic acids having a complementary nucleotide base sequence. These modified oligonucleotides hybridize to the target sequence at a faster rate than unmodified oligonucleotides having an identical nucleotide base sequence. Such modified oligonucleotides include oligonucleotides containing at least one 2′-O-methylribofuranosyl moiety joined to a nitrogenous base. Oligonucleotides can be modified in accordance with the present invention to preferentially bind RNA targets. The present invention also concerns methods of using these modified oligonucleotides and kits containing the same.

Claims

1. A kit comprising:

a probe molecule for use in determining the presence of an RNA target in a sample, the probe comprising complementary first and second base regions that form a hybrid containing at least one 2′

-O-alkyl ribonucleotide, wherein the probe forms a stable complex with the RNA target but not with a non-targeted nucleic acid under nucleic acid assay conditions, such that the RNA target can be detected, and wherein the complex comprises a single-stranded form of the probe; and

a solid support for immobilizing the RNA target so that unbound nucleic acids and other components of the sample can be removed from the RNA target.

2. The kit of claim 1, wherein the solid support has a magnetic charge.

3. The kit of claim 1 further comprising:

one or more nucleic acid polymerases;

nucleotide triphosphates; and

one or more amplification oligonucleotides, wherein each of said amplification oligonucleotides is, in the presence of a nucleic acid analyte and under amplification conditions, extended to form part of a nucleic acid extension product containing the RNA target or directs the synthesis of a nucleic acid transcription product containing the RNA target.

4. The kit of claim 3, wherein the nucleic acid polymerases and amplification oligonucleotides are sufficient to perform a transcription-based amplification reaction.

5. The kit of claim 1, wherein the first base region contains at least one 2′

-O-alkyl ribonucleotide, and wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.

6. The kit of claim 1, wherein that portion of the first base region which hybridizes to the second base region includes a cluster of at least 4 2′

-O-alkyl ribonucleotides.

7. The kit of claim 6, wherein the probe includes a conjugate molecule joined to the probe at a site located within the cluster of the first base region.

8. The kit of claim 6, wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.

9. The kit of claim 1, wherein that portion of the first base region which hybridizes to the second base region includes at least one nucleotide which is not a 2′

-O-alkyl ribonucleotide.

10. The kit of claim 9, wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.

11. The kit of claim 1, wherein each nucleotide of that portion of the first base region which hybridizes to the second base region is a 2′

-O-alkyl ribonucleotide.

12. The kit of claim 11, wherein the first base region complexes with the RNA target under the nucleic acid assay conditions.

13. The kit of claim 1, wherein each nucleotide of the probe is a 2′

-O-alkyl ribonucleotide.

14. The kit of claim 1, wherein the first and second base regions form a hybrid that is more stable than a hybrid formed between unmodified forms of the first and second base regions consisting of RNA and/or DNA.

15. The kit of claim 1, wherein the probe includes a conjugate molecule.

16. The kit of claim 1, wherein the first and second base regions are contained within an oligonucleotide that is between 10 and 100 bases in length.

17. The kit of claim 1, wherein the probe comprises a detectable label.

18. The kit of claim 17, wherein the detectable label comprises a fluorescent molecule.

19. The kit of claim 1, wherein the RNA target is ribosomal RNA.

20. The kit of claim 1, wherein the probe forms a stable complex with a region of the RNA target that is folded under the nucleic acid conditions.

21. The kit of claim 20, wherein the probe includes at least 5 contiguous 2′

-O-alkyl ribonucleotides.

22. The kit of claim 20, wherein the RNA target is ribosomal RNA.

23. The kit of claim 20, wherein the kit does not include helper probes.

24. The kit of any one of claims 1 to 23, wherein each 2′

-O-alkyl ribonucleotide is a 2′

-O-methyl ribonucleotide.

25. A reaction mixture comprising:

one or more amplification oligonucleotides in the presence of at least one nucleic acid polymerase and nucleotide triphosphates sufficient to form an RNA amplification product; and

a probe molecule comprising first and second base regions hybridized to each other and having at least one 2′

-O-alkyl ribonucleotide, wherein the probe forms a stable and detectable complex with the amplification product but not with non-target nucleic acid, and wherein the complex comprises a single-stranded form of the probe.

26. The reaction mixture of claim 25, wherein the one or more amplification oligonucleotides and the probe are present in the reaction mixture when the amplification reaction is initiated.

27. The reaction mixture of claim 25, wherein the first base region contains at least one 2′

-O-alkyl ribonucleotide, and wherein the first base region forms a complex with the amplification product.

28. The reaction mixture of claim 25, wherein that portion of the first base region which hybridizes to the second base region includes a cluster of at least 4 2′

-O-alkyl ribonucleotides.

29. The reaction mixture of claim 28, wherein the probe includes a conjugate molecule joined to the probe at a site located within the cluster of the first base region.

30. The reaction mixture of claim 28, wherein the first base region forms a complex with the amplification product.

31. The reaction mixture of claim 25, wherein that portion of the first base region which hybridizes to the second base region includes at least one nucleotide which is not a 2′

-O-alkyl ribonucleotide.

32. The reaction mixture of claim 31, wherein the first base region forms a complex with the amplification product.

33. The reaction mixture of claim 25, wherein each nucleotide of that portion of the first base region which hybridizes to the second base region is a 2′

-O-alkyl ribonucleotide.

34. The reaction mixture of claim 33, wherein the first base region forms a complex with the amplification product.

35. The reaction mixture of claim 25, wherein each nucleotide of the probe is a 2′

-O-alkyl ribonucleotide.

36. The reaction mixture of claim 25, wherein the first and second base regions form a hybrid that is more stable than a hybrid formed between unmodified forms of the first and second base regions consisting of RNA and/or DNA.

37. The reaction mixture of claim 25, wherein the probe includes a conjugate molecule.

38. The reaction mixture of claim 25, wherein the first and second base regions are contained within an oligonucleotide that is between 10 and 100 bases in length.

39. The reaction mixture of claim 25, wherein the probe comprises a detectable label.

40. The reaction mixture of claim 39, wherein the detectable label comprises a fluorescent molecule.

41. The reaction mixture of claim 25, wherein the the amplification product is formed from a ribosomal RNA.

42. The reaction mixture of claim 25, wherein the probe forms a stable complex with a folded region of the amplification product.

43. The reaction mixture of claim 42, wherein the reaction mixture does not include helper probes.

44. The reaction mixture of claim 42, wherein the probe includes at least 5 contiguous 2′

-O-alkyl ribonucleotides.

45. The reaction mixture of claim 42, wherein the amplification product is formed from a ribosomal RNA.

46. The reaction mixture of claim 25, wherein the amplification oligonucleotides and the at least one nucleic acid polymerase are sufficient to perform a transcription-based amplification reaction.

47. The reaction mixture of any one of claims 25 to 46, wherein each 2′

-O-alkyl ribonucleotide is a 2′

-O-methyl ribonucleotide.