Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores

• US 7,214,489 B2
• Filed: 06/20/2003
• Issued: 05/08/2007
• Est. Priority Date: 06/20/2002
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Bazan, Guillermo C., Gaylord, Brent S.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/600,286
• Publication Number:
  US 20040219556A1
• Time in Patent Office: 1,418 Days
• Field of Search: 435/6, 536/23.1, 536/26.6
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods, compositions and articles of manufacture for assaying a sample for a target polynucleotide are provided. A sample suspected of containing the target polynucleotide is contacted with a polycationic multichromophore and a sensor PNA complementary to the target polynucleotide. The sensor PNA comprises a signaling chromophore to absorb energy from the excited multichromophore and emit light in the presence of the target polynucleotide. The methods can be used in multiplex form. Kits comprising reagents for performing such methods are also provided.

Claims

1. An assay method comprising:

providing a sample that is suspected of containing a target polynucleotide;

providing a polycationic multichromophore that electrostatically interacts with the target polynucleotide and upon excitation is capable of transferring energy to a signaling chromophore;

providing a sensor peptide nucleic acid (PNA) that is single-stranded and is complementary to the target polynucleotide, said sensor PNA conjugated to the signaling chromophore;

contacting the sample with the sensor PNA and the multichromophore in a solution under conditions in which the sensor PNA can hybridize to the target polynucleotide, if present;

applying a light source that can excite the multichromophore; and

detecting whether light is emitted from the signaling chromophore.

2. The method of claim 1, wherein the multichromophore comprises a structure selected from a saturated polymer, a conjugated polymer, a dendrimer, and a semiconductor nanocrystal.

3. The method of claim 2, wherein the multichromophore comprises a saturated polymer.

4. The method of claim 2, wherein the multichromophore comprises a dendrimer.

5. The method of claim 2, wherein the multichromophore comprises a semiconductor nanocrystal.

6. The method of claim 2, wherein the multichromophore comprises a conjugated polymer.

7. The method of claim 6, wherein the conjugated polymer has the structure

8. The method of claim 6, wherein the conjugated polymer has the structure

9. The method of claim 1, wherein the sample is contacted with the sensor PNA and the multichromophore in the presence of a sufficient amount of an organic solvent to decrease hydrophobic interactions between the sensor PNA and the multichromophore.

10. The method of claim 1, wherein the sample is contacted with a plurality of different sensor PNAs having corresponding different sequences, said different sensor PNAs comprising a corresponding different signaling chromophore, wherein each of said different sensor PNAs can selectively hybridize to a corresponding different target polynucleotide.

11. The method of claim 1, wherein the signaling chromophore is a fluorophore.

12. The method of claim 11, wherein the fluorophore is selected from a semiconductor nanocrystal, a fluorescent dye, and a lanthanide chelate.

13. The method of claim 12, wherein the fluorophore is a semiconductor nanocrystal.

14. The method of claim 12, wherein the fluorophore is a fluorescent dye.

15. The method of claim 14, wherein the fluorescent dye is fluorescein.

16. The method of claim 12, wherein the fluorophore is a lanthanide chelate.

17. The method of claim 1, wherein the target polynucleotide is DNA.

18. The method of claim 1, wherein the target polynucleotide is RNA.

19. The method of claim 1, wherein the sample comprises single-stranded target polynucleotide.

20. The method of claim 1, wherein the sample comprises double-stranded target polynucleotide.

21. The method of claim 1, wherein the target polynucleotide is produced via an amplification reaction.

22. The method of claim 1, wherein light emitted from the signaling chromophore above a threshold level indicates that the target polynucleotide is present in the sample.

23. The method of claim 1, wherein the amount of light emitted from the signaling chromophore is quantitated and used to determine the amount of the target polynucleotide in the sample.

24. The method of claim 11, wherein the fluorophore.is a green fluorescent protein.

25. The method of claim 1, wherein the target polynucleotide is not amplified.

26. The method of claim 1, wherein the method is performed on a substrate.

27. The method of claim 26, wherein the substrate is conjugated to a plurality of different sensor PNAs.

28. The method of claim 1, wherein the amount of light emitted from the signaling chromophore upon excitation of the multichromophore is greater than the amount of light obtained upon direct excitation of the signaling chromophore.

29. The method of claim 28, wherein the amount of light emitted from the signaling chromophore upon excitation of the multichromophore is at least eight-fold greater than the amount of light obtained upon direct excitation of the signaling chromophore.