Inversion probes

• US 20040005595A1
• Filed: 03/14/2003
• Published: 01/08/2004
• Est. Priority Date: 09/28/2001
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Browne, Kenneth A.
• Granted Patent: US 7,070,933 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Unitary hybridization probes having stem-and-loop structures, wherein the stem portion of the structure comprises a pair of interactive arms that are substantially prevented from interacting with target polynucleotides. One arm of the invented parallel-stem hybridization probe has a backbone polarity opposite that of the target-complementary loop sequence of the probe. Rather than interacting in an antiparallel fashion, the arms of parallel-stem hybridization probes interact in a parallel fashion. The arms of the invented dual inversion probes interact in a conventional antiparallel fashion, but have backbone polarities opposite that of the target-complementary loop portion of the probe. Arm portions of the inversion probes do not substantially contribute to sequence-dependent stabilization of probe:target hybrids. Incorporating inversion linkages into the structures of these probes dramatically simplifies the process of designing stem-and-loop hybridization probes.

Claims

1. A hybridization probe for detecting a target polynucleotide, comprising:

(a) a loop comprising a target-complementary sequence of bases joined to a loop backbone, said target-complementary sequence of bases extending from a first boundary thereof to a second boundary thereof;

(b) a first arm joined to said target-complementary sequence of bases at said first boundary thereof through a first arm linkage, said first arm comprising a first arm sequence of bases joined to a first arm backbone;

(c) a second arm joined to said target-complementary sequence of bases at said second boundary thereof through a second arm linkage, said second arm comprising a second arm sequence of bases joined to a second arm backbone;

wherein at least one of said first arm linkage and said second arm linkage comprises an inversion linkage, said inversion linkage optionally including a non-nucleotide linker, and

(d) at least one detectable label joined to any of said loop, said first arm, said second arm or, if present, said non-nucleotide linker,

wherein said first arm and said second arm interact with each other in the absence of said target polynucleotide to form a stem duplex.

2. The hybridization probe of claim 1, wherein only one of said first arm linkage and said second arm linkage is an inversion linkage, said hybridization probe being a parallel-stem hybridization probe.

3. The hybridization probe of claim 2, wherein said at least one detectable label comprises a pair of interactive labels comprising a first label and a second label, said first label being joined to said first arm and said second label being joined to said second arm.

4. The hybridization probe of claim 3, wherein said first arm linkage is an inversion linkage, wherein said first arm is an inversion arm, and wherein said second arm is an extension arm.

5. The hybridization probe of claim 4, wherein at least one of said loop, said inversion arm or said extension arm comprise at least one nucleotide analog.

6. The hybridization probe of claim 5, wherein said at least one nucleotide analog is selected from the group consisting of a 2′

-methoxy nucleotide analog, an isocytosine nucleotide analog and an isoguanine nucleotide analog.

7. The hybridization probe of claim 4, wherein said inversion arm and said extension arm comprise deoxyribonucleotides.

8. The hybridization probe of claim 5, wherein said loop comprises 2′

-methoxy nucleotide analogs.

9. The hybridization probe of claim 4, wherein the inversion linkage is either a 5′

-5′

inversion linkage or a 3′

-3′

inversion linkage.

10. The hybridization probe of claim 9, wherein said inversion linkage is a 5′

-5′

inversion linkage, and wherein said inversion arm and said extension arm both comprise 3′

termini.

11. The hybridization probe of claim 9, wherein the inversion linkage is a 3′

-3′

inversion linkage, and wherein said inversion arm and said extension arm both comprise 5′

termini.

12. The hybridization probe of claim 1, wherein the target-complementary sequence of bases has a length in the range of from 10-25 bases.

13. The hybridization probe of claim 12, wherein the target-complementary sequence of bases has a length in the range of from 16-22 bases.

14. The hybridization probe of claim 3, wherein the extension arm has a length of from 5-12 bases.

15. The hybridization probe of claim 14, wherein the inversion arm has a length of from 5-12 bases.

16. The hybridization probe of claim 3, wherein both the extension arm and the inversion arm have lengths in the range of from 6-8 bases.

17. The hybridization probe of claim 3, wherein said pair of interactive labels is a pair of FRET interactive labels.

18. The hybridization probe of claim 3, wherein said pair of interactive labels is a pair of non-FRET interactive labels.

19. The hybridization probe of claim 18, wherein one member of said pair of non-FRET interactive labels comprises fluorescein.

20. The hybridization probe of claim 5, wherein the extension arm comprises at least one nucleotide analog.

21. The hybridization probe of claim 5, wherein the inversion arm comprises at least one nucleotide analog.

22. The hybridization probe of claim 20, wherein said at least one nucleotide analog is selected from the group consisting of isocytosine and isoguanine.

23. The hybridization probe of claim 1, wherein both of said first arm linkage and said second arm linkage are inversion linkages different from each other, said hybridization probe being a dual inversion probe.

24. The hybridization probe of claim 23, wherein said at least one detectable label comprises a pair of interactive labels comprising a first label and a second label, said first label being joined to said first arm and said second label being joined to said second arm.

25. The hybridization probe of claim 23, wherein said first arm linkage is a 3′

-3′

inversion linkage, and wherein said second arm linkage is a 5′

-5′

inversion linkage.

26. The hybridization probe of claim 23, wherein said first arm linkage is a 5′

-5′

inversion linkage, and wherein said second arm linkage is a 3′

-3′

inversion linkage.

27. The hybridization probe of claim 24, wherein at least one of said loop, said first arm or said second arm comprise at least one nucleotide analog.

28. The hybridization probe of claim 27, wherein said loop comprises 2′

-methoxy nucleotide analogs.

29. The hybridization probe of claim 24, wherein the target-complementary sequence of bases has a length in the range of from 10-25 bases.

30. The hybridization probe of claim 29, wherein the target-complementary sequence of bases has a length in the range of from 16-22 bases.

31. The hybridization probe of claim 29, wherein the first arm has a length of from 5-12 bases.

32. The hybridization probe of claim 31, wherein the second arm has a length of from 5-12 bases.

33. The hybridization probe of claim 29, wherein both the first arm and the second arm have lengths in the range of from 6-8 bases.

34. The hybridization probe of claim 24, wherein said pair of interactive labels is a pair of FRET interactive labels.

35. The hybridization probe of claim 24, wherein said pair of interactive labels is a pair of non-FRET interactive labels.

36. The hybridization probe of claim 35, wherein one member of said pair of non-FRET interactive labels comprises fluorescein.

37. The hybridization probe of claim 32, wherein said pair of interactive labels is a pair of FRET interactive labels.

38. The hybridization probe of claim 32, wherein said pair of interactive labels is a pair of non-FRET interactive labels.

39. A method of determining whether a test sample contains a target polynucleotide, comprising the steps of:

(a) providing a hybridization probe in accordance with claim 1;

(b) contacting said hybridization probe with any of said target polynucleotide that may be present in the test sample under hybridization-promoting conditions; and

(c) detecting the formation of hybrid duplexes comprising said hybridization probe and said target polynucleotide as an indication of the presence of said target polynucleotide sequence in said test sample.

40. A kit for detecting a target polynucleotide sequence using a hybridization assay, comprising:

(a) a hybridization probe in accordance with claim 1;

and

(b) a positive-control target polynucleotide having a sequence complementary to said target-complementary sequence of bases of said loop.

41. The kit of claim 40, further comprising

(c) a hybridization solution.