Nucleic acid hairpin probes and uses thereof

• US 6,696,255 B2
• Filed: 11/29/2001
• Issued: 02/24/2004
• Est. Priority Date: 07/14/2000
• Status: Expired due to Fees

• Current Assignee: Applied Gene Technologies Incorporated
• Original Assignee: Applied Gene Technologies Incorporated
• Inventors: Dattagupta, Nanibhushan
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/995,686
• Publication Number:
  US 20020110826A1
• Time in Patent Office: 817 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.15
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/30 Detection characterised by liberation or release of label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates generally to nucleic acid hybridization analysis. More specifically, oligonucleotide probes containing hairpin structures, or arrays of such oligonucleotide probes immobilized on a solid support, that are suitable for hybridization analysis are provided. Methods for nucleic acid hybridization analysis using the probes or array of immobilized probes are also provided.

Claims

1. A method for detecting a target nucleotide sequence in a sample comprising the steps of:

a) providing an oligonucleotide probe comprising a nucleotide sequence that forms a hairpin structure having a stem region comprising a double stranded segment under suitable conditions, wherein the double stranded segment is formed between two complementary nucleotide sequences, and wherein at least a portion of at least one of the nucleotide sequences located within the double stranded segment is complementary to the target nucleotide sequence to be detected;

b) contacting the probe provided in step a) with a sample containing or suspected of containing the target nucleotide sequence under conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself, whereby the contacting breaks the intramolecular hybridization of the probe itself by dissociating the double stranded segment of the probe and forms a first intermolecular hybrid between the target and a region consisting of at least a portion of one strand of said stem that is complementary to the target nucleotide sequence; and

c) assessing the first intermolecular hybrid formed in step b), wherein the assessing is effected by addition of a secondary probe that specifically hybridizes with at least a portion of the first intermolecular hybrid, thereby forming a secondary intermolecular hybrid formed between the secondary probe and the first intermolecular hybrid wherein the detection of the secondary intermolecular hybrid indicates the presence and/or amount of the target nucleotide sequence in the sample.

2. The method of claim 1, wherein the nucleotide sequence complementary to the target nucleotide sequence to be detected is completely located within the double stranded segment.

3. The method of claim 1, wherein the conditions that favor intermolecular hybridization between the probe and the target nucleotide sequence over intramolecular hybridization of the probe itself is achieved by controlling compositions of the probe and the target nucleotide sequence so that the Tm of the intermolecular hybrid is higher than the Tm of the intramolecular hybrid.

4. The method of claim 3, wherein the Tm of the intermolecular hybrid is at least 2°

C. higher than the Tm of the intramolecular hybrid.

5. The method of claim 3, wherein the intermolecular hybrid is a RNA:

RNA hybrid whereas the intramolecular hybrid is a RNA;

DNA.

6. The method of claim 3, wherein the intermolecular hybrid is a RNA:

DNA hybrid whereas the intramolecular hybrid is a DNA;

DNA hybrid.

7. The method of claim 1, further comprising a step of crosslinking the stem region of the probe that has not been hybridized with the target after the formation of the first intermolecular hybrid but before the addition of the secondary probe.

8. The method of claim 7, wherein the crosslinking step is effected via addition of a crosslinking agent subsequent to hybridization of the probe with the target nucleotide sequence.

9. The method of claim 1, wherein the hairpin probe further comprises an element or a modification that facilitates intramolecular crosslinking of the probe upon a suitable treatment.

10. The meted of claim 9, wherein the element is a crosslinking agent that is activated by chemical or photoactive txeatment.

11. The method of claim 1, wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is not involved in the hybridization of the target nucleotide sequence and the probe.

12. The method of claim 1, wherein the secondary probe specifically hybridizes with a portion of the target nucleotide sequence that is involved in the hybridization of the target nucleotide sequence and the probe and the nucleotide sequence in the probe that is complementary to the same portion of the target nucleotide sequence is removed concurrently with the addition of the secondary probe.

13. The method of claim 11, wherein the hairpin structure in the probe is formed between a DNA strand that contains methylphosphonates and a RNA strand that is complementary to the target nucleotide sequence and wherein the RNA strand, after forming a hybrid with the target nucleotide sequence but before the addition of the secondary probe, is removed by a RNase H treatment.

14. The method of claim 1, wherein the secondary probe specifically hybridizes with a portion of the probe that is involved in the formation of the intramolecular hybrid but is not involved in the formation of the first intermolecular hybrid with the target nucleotide sequence and wherein the portion of the probe dissociates as a single strand.

15. The method of claim 1, wherein the secondary probe is a specific probe.

16. The method of claim 1, wherein the secondary probe is a degenerate probe.

17. The method of claim 1, wherein the probe is immobilized on a solid support.

18. The method of claim 1, wherein the sample is a biosample.

19. The method of claim 1, wherein a plurality of samples is assayed simultaneously.

20. The method of claim 1, wherein the secondary probe has a detectable label.

21. The method of claim 20, wherein the label is selected from the group consisting of a chemical, an enzymatic, a radioactive, a fluorescent, a luminescent and a FRET label.

22. The method of claim 20, wherein the detectably labeled secondary probe is crosslinked to the first intermolecular hybrid before the secondary intermolecular hybrid is formed between the secondary probe and the first intermolecular hybrid.

23. The method of claim 1, wherein the probe further comprises an element that is sensitive to nuclease digestion or a modification that is resistant to nuclease digestion.

24. The method of claim 23, wherein the nuclease digestion is effected via a restriction enzyme or a RNase H.