Activatable probes and methods for in vivo gene detection

• US 20050287548A1
• Filed: 01/21/2005
• Published: 12/29/2005
• Est. Priority Date: 06/25/2001
• Status: Active Grant

• Current Assignee: Georgia Tech Research Corporation (University System of Georgia)
• Original Assignee: Georgia Tech Research Corporation (University System of Georgia)
• Inventors: Nitin, Nitin, Bao, Gang
• Granted Patent: US 7,297,494 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• 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 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

Probes for detecting a target polynucleotide are provided. One aspect provides a molecular beacon probe set wherein the donor molecular beacon comprises a quantum dot and an acceptor molecular beacon comprises at least one reporter. The probes optionally comprise a protein transduction domain, targeting signal, or a combination thereof. Methods for detecting target polynucleotides using the disclosed probes are also provided.

Claims

1. A probe set comprising:

(a) a donor polymer comprising

(i) a first polynucleotide binding domain complementary to a first region of a target polynucleotide flanked by first and second stem regions which hybridize in the absence of the target polynucleotide to form a stem-loop or random-coil structure, and

(ii) a quantum dot; and

(b) an acceptor polymer comprising

(i) a second polynucleotide binding domain complementary to a second region of the target polynucleotide flanked by first and second stem regions which hybridize in the absence of the target polynucleotide to form a stem-loop or random-coil structure, and

(ii) at least one reporter;

wherein energy transfer occurs between the donor and the at least one reporter when the donor polymer and the acceptor polymer hybridize to the target polynucleotide and the quantum dot is exposed to an exciting amount of energy.

2. The probe set of claim 1, wherein the reporter does not emit a detectable signal when exposed to an exciting amount of energy sufficient to cause the donor to emit energy.

3. The probe set of claim 1, wherein the acceptor probe comprises at least two reporters.

4. The probe set of claim 1, wherein the polymers specifically hybridize to the target polynucleotide under stringent conditions.

5. The probe set of claim 1, wherein the polymers specifically hybridize to the target polynucleotide in vivo.

6. The probe set of claim 1, wherein the polymers further comprise a protein transduction domain, a targeting signal, a fragment thereof or a combination thereof.

7. The probe set of claim 1, wherein at least one polymer comprises a plurality of natural or non-natural nucleotides.

8. The probe set of claim 1, wherein at least one polymer comprises at least one peptide nucleic acid.

9. The probe set of claim 1, wherein at least one polymer comprises a plurality of quantum dots.

10. The probe set of claim 1, wherein at least one linkage between subunits of at least one of the polymers is modified to resist enzymatic cleavage.

11. The probe set of claim 1, wherein at least one of the polymers further comprises at least one quencher.

12. The probe set of claim 1, wherein the acceptor polymer comprises a quencher that quenches the reporter in the absence of the target polynucleotide.

13. The probe set of claim 1, wherein the quantum dot comprises ZnS, CdS, ZnSe, CdSe, CdTe, CdSe_xTe_1-x, InAs, InP, PbTe, PbSe, PbS, HgS, HgSe, HgTe, CdHgTe, GaAs, or combinations thereof.

14. The probe set of claim 1, wherein the quantum dot is encapsulated in a micelle or polymer coating, or chemically linked coatings such as silica-based coatings or DHLA (Lipoic Acid) and Mercaptoacetic Acid coatings.

15. The probe set of claim 14, wherein micelle comprises a lipid, phospholipid, or a combination thereof.

16. The probe set of claim 14, wherein the polymer coating comprises polyethylene glycol or derivatives thereof.

17. The probe set of claim 1, wherein the acceptor comprises Cy-3, ROX, Texas Red, Red Phycoerythrin (RPE), Blue Phycoerythrin (BPE), or Allophycocyanin (APC), or a fragment thereof.

18. The probe set of claim 1, further comprises at least a second set of probes specific for a second target polynucleotide.

44. A method for identifying modulators of gene expression comprising:

(a) contacting a cell with a test compound;

(b) contacting the cell of step (a) with the probe set of claim 1, wherein each probe is individually, operably linked to a protein transduction domain, a targeting signal, or a combination thereof;

(c) irradiating the cell with an exciting amount of radiation;

(d) detecting electromagnetic emissions from the cell; and

(e) selecting the test compound that induces a change in the electromagnetic emissions of the cell contacted with the test compound compared to a control sample.

19-34. -34. (canceled)

35. A method of detecting a target polynucleotide comprising:

(a) delivering at least one probe pair to cell lysates, tissue extracts or the interior of a cell, wherein the probe pair comprises

(i) a donor probe comprising a quantum dot and forms a stem-loop (or random coil) structure in the absence of a target polynucleotide and

(ii) an acceptor probe comprising at least one reporter and forms a stem-loop (or random coil) structure in the absence of the target polynucleotide; and

(b) exposing the quantum dot to an exciting amount of energy, wherein energy transfer occurs between the quantum dot and the reporter to produce a detectable signal when the donor probe and the acceptor probe hybridize to the target polynucleotide.

36-43. -43. (canceled)

45-48. -48. (canceled)