METHOD OF IDENTIFYING HAIRPIN DNA PROBES BY PARTIAL FOLD ANALYSIS

• US 20070166731A1
• Filed: 10/27/2006
• Published: 07/19/2007
• Est. Priority Date: 01/02/2004
• Status: Active Grant

• Current Assignee: University of Rochester
• Original Assignee: University of Rochester
• Inventors: STROHSAHL, Christopher, MILLER, Benjamin
• Granted Patent: US 7,442,510 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide

Abstract

Methods of identifying molecular beacons in which a secondary structure prediction algorithm is employed to identify oligonucleotide sequences within a target gene having the requisite hairpin structure. Isolated oligonucleotides, molecular beacons prepared from those oligonucleotides, and their use are also disclosed.

Claims

1. An isolated molecular beacon comprising:

a hairpin nucleic acid molecule that hybridizes over its full length to a target nucleic acid sequence;

a label tethered to one terminus of the hairpin nucleic acid molecule; and

a quenching agent tethered to the other terminus of the hairpin nucleic acid molecule.

2. The isolated molecular beacon according to claim 1, wherein the label is tethered to the 5′

terminus and the quenching agent is tethered to the 3′

terminus.

3. The isolated molecular beacon according to claim 1, wherein the label is tethered to the 3′

terminus and the quenching agent is tethered to the 5′

terminus.

4. The isolated molecular beacon according to claim 1, wherein the quenching agent is a solid surface.

5. The isolated molecular beacon according to claim 1, wherein the quenching agent is a micro- or nano-particle.

6. The isolated molecular beacon according to claim 1, wherein the label is a fluorescent dye, semiconductor quantum dot, lanthanide atom-containing complex, or fluorescent protein.

7. The isolated molecular beacon according to claim 1, wherein the quenching agent is a metal or 4-([4-(Dimethylamino)phenyl]azo)benzoic acid.

8. The isolated molecular beacon according to claim 1, wherein the metal is gold, silver, platinum, copper, cobalt, iron, or iron-platinum.

9. The isolated molecular beacon according to claim 1, wherein the hairpin nucleic acid molecule is characterized by a predicted E value of at most about −

3 kcal/mol.

10. The isolated molecular beacon according to claim 9, wherein the predicted E value is between about −

4 kcal/mol and about −

12 kcal/mol.

11. The isolated molecular beacon according to claim 1, wherein the hairpin nucleic acid molecule is between about 12 and about 60 nucleotides in length.

12. The isolated molecular beacon according to claim 1, wherein hybridization between the hairpin nucleic acid molecule and a target nucleic acid sequence is predicted to have a lowest free energy value that is at least about a two-fold increase over the lowest predicted energy value of the hairpin nucleic acid molecule alone.

13. The isolated molecular beacon according to claim 1, wherein the hairpin nucleic acid molecule includes first and second portions that hybridize to form a stem when the hairpin nucleic acid molecule is in a hairpin configuration, and the first and second portions contain no exogenous nucleotides.

14. The isolated molecular beacon according to claim 1, wherein the hairpin nucleic acid molecule consists of a nucleotide sequence that is 100% complementary to a target nucleic acid sequence.

15. A method of detecting a target nucleic acid molecule in a sample comprising:

providing a sample that may contain a target nucleic acid molecule having a target nucleic acid sequence;

introducing the isolated molecular beacon of claim 1 into the sample; and

determining whether the label can be detected within the sample, wherein detection of the label indicates the presence of the target nucleic acid molecule in the sample.

16. The method according to claim 15, wherein said determining comprises:

exposing the sample comprising the isolated molecular beacon to a source of light suitable to induce fluorescent emissions by the label; and

measuring fluorescent emissions by the label.

17. An isolated nucleic acid molecule comprising first and second regions that are capable of self-hybridizing, under appropriate conditions, into a hairpin configuration, wherein the nucleic acid molecule has a nucleotide sequence that hybridizes over its full length to a target nucleic acid sequence.

18. The isolated nucleic acid molecule according to claim 17, wherein the nucleic acid molecule consists of a nucleotide sequence that is 100% complementary to a target nucleic acid sequence.

19. The isolated nucleic acid molecule according to claim 17, wherein the nucleic acid molecule is DNA complementary to the target nucleic acid sequence and contains no exogenous nucleotides.

20. The isolated nucleic acid molecule according to claim 17, wherein the nucleic acid molecule contains between about 12 and about 60 nucleotides.