Oligonucleotides comprising a molecular switch

• US 20050042638A1
• Filed: 04/30/2004
• Published: 02/24/2005
• Est. Priority Date: 05/01/2003
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Brown, Bob D., Arnold, Lyle J. Jr.
• Granted Patent: US 8,679,789 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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

Abstract

This invention relates to oligonucleotides comprising a molecular switch which may exist in an “open” or “closed” position. The molecular switch portion of the probe is particularly sensitive to the identity of sequences complementary to the molecular switch. Oligonucleotides containing a molecular switch are applicable to all kinds of hybridization processes. Due to the sensitivity of the switch domain of the oligonucleotide, probes containing a molecular switch are particularly useful in the identification of single point mismatches. More specifically, a portion, but not all, of the oligonucleotide becomes unbound from a mismatched target. The invention further relates to methods of using said oligonucleotides for research reagents, and clinical diagnostics. An exemplary oligonucleotide comprises a first hybridizable domain, a second bridging block domain, and a third binding domain.

Claims

1. An oligonucleotide comprising:

(a) a nucleic acid region complementary to a first sequence of nucleic acid residues of a target nucleic acid, and

(b) a switch domain comprising at least one bridging domain and at least one binding domain, wherein said switch domain is able to discriminate between (i) a second sequence of nucleic acid residues of said target nucleic acid that is complementary to said binding domain and (ii) a second sequence of nucleic acid residues of said target nucleic acid that contains at least one nucleic acid residue that is not complementary to said binding domain;

under conditions wherein said region (a) forms a stable duplex with said first sequence of nucleic acid residues of said target nucleic acid.

2. An oligonucleotide according to claim 1, further comprising at least one detectable label.

3. An oligonucleotide according to claim 2, wherein the amount of signal detected from said detectable label is determinative of the hybridization status of said switch domain.

4. An oligonucleotide according to claim 2, wherein said at least one detectable label is a fluorescent label.

5. An oligonucleotide according to claim 4, further comprising a quencher, wherein said quencher and said fluorescent label interact to modulate the amount of signal detected from said fluorescent label.

6. An oligonucleotide according to claim 2, further comprising a quencher and a second fluorescent label, wherein the amount of signal detected from said second fluorescent label is determinative of the hybridization status of said region (a).

7. An oligonucleotide according to claim 3, wherein the amount of signal detected from said at least one detectable label is decreased when the switch domain is not hybridized to said target nucleic acid, relative to the amount of signal detected from said at least one detectable label when the switch domain is hybridized to said target nucleic acid.

8. An oligonucleotide according to claim 2, wherein said at least one detectable label is a first fluorescent label and a second fluorescent label, wherein said first and said second fluorescent labels interact to modulate the amount of signal detected from said first and/or said second fluorescent label dependent on the hybridization status of said switch domain.

9. An oligonucleotide according to claim 2, wherein said at least one detectable label is a chemiluminescent label.

10. An oligonucleotide according to claim 5, wherein said chemiluminescent label is an acridinium ester.

11. An oligonucleotide according to claim 2, wherein said at least one detectable label is a single electron conductor.

13. A tandem oligonucleotide assembly comprising at least two oligonucleotides according to claim 1.

14. A tandem oligonucleotide assembly according to claim 13, further comprising a first detectable label associated with a first oligonucleotide and a second detectable label associated with a second oligonucleotide, wherein the amount of signal detected from said first and/or said second detectable label is altered when said tandem oligonucleotide assembly is hybridized to a target nucleic acid, relative to the amount of signal detected from said first and/or said second detectable label detected when either oligonucleotide is hybridized to said target nucleic acid individually.

15. A tandem oligonucleotide assembly according to claim 14, wherein said first and said second detectable labels are both fluorescent labels, further wherein said first fluorescent label transfers energy non-radiatively to said second fluorescent label when said first and said second oligonucleotides are both hybridized to said target nucleic acid.

16. A tandem oligonucleotide assembly according to claim 13, wherein the binding domains of said first oligonucleotide and said second oligonucleotide hybridize at least in part with overlapping regions of the target nucleic acid.

17. A tandem oligonucleotide assembly according to claim 13, wherein said first oligonucleotide is attached to a solid support.

18. An oligonucleotide according to claim 1, further comprising a 5′

modification, wherein said modified oligonucleotide is resistant to digestion by enzymes possessing 5′

nuclease activity.

19. An oligonucleotide according to claim 1, wherein said region (a) is 15-150 nucleotides in length.

20. An oligonucleotide according to claim 1, wherein said oligonucleotide suitable for use as a primer in an enzymatic reaction, and wherein the switch domain is positioned on the 3′

terminus such that the oligonucleotide does not support 3′

extension when the binding domain of said switch domain is not complementary to the target nucleic acid, but does support extension when the binding domain of said switch domain is complementary to the target nucleic acid.

21. An oligonucleotide according to claim 1, wherein said oligonucleotide is attached to an electron conducting solid surface, and wherein the amount of hybridization to said target nucleic acid controls the amount of current flow.

22. A method for determining in a sample the amount of target sequence that is complementary to the binding domain of the oligonucleotide of claim 1, said method comprising:

contacting the oligonucleotide of claim 1 with a sample containing said target nucleic acid under conditions suitable for hybridization; and

determining the hybridization status of said switch domain as a measure of the amount of target nucleic acid sequence in said sample that is complementary to the binding domain of said switch domain.

23. A method for determining in a sample the amount of target sequence that is complementary to the binding domain of the first oligonucleotide of the tandem oligonucleotide assembly of claim 15 and the amount of target sequence that is complementary to the binding domain of the second oligonucleotide of the tandem oligonucleotide assembly of claim 15, said method comprising:

contacting the tandem oligonucleotide assembly of claim 15 with a sample containing said target nucleic acid under conditions suitable for hybridization; and

measuring energy transfer from said first fluorescent label to said second fluorescent label to determine the hybridization status of each switch domain.

12. An oligonucleotide comprising:

(a) a nucleic acid region complementary to a first sequence of nucleic acid residues of a double-stranded target nucleic acid, and

(b) a switch domain comprising at least one bridging domain and at least one binding domain, wherein said switch domain is able to discriminate between (i) a second sequence of nucleic acid residues of said double-stranded target nucleic acid that is complementary to said binding domain and (ii) a second sequence of nucleic acid residues of said double-stranded target nucleic acid that contains at least one nucleic acid residue that is not complementary to said binding domain;

under conditions wherein said region (a) forms a stable triple-stranded nucleic acid with said first sequence of nucleic acid residues of said double-stranded target nucleic acid.