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

• US 8,227,187 B2
• Filed: 12/07/2009
• Issued: 07/24/2012
• Est. Priority Date: 08/26/2002
• Status: Expired due to Term

• 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., Wang, Shu
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/632,734
• Publication Number:
  US 20100136702A1
• Time in Patent Office: 960 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 435/6
• 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 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2563/107 fluorescence
• 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 polynucleotide complementary to the target polynucleotide. The sensor polynucleotide comprises a signaling chromophore to receive energy from the excited multichromophore and increase emission 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 for detecting polynucleotide binding comprising:

a. providing a sample that is suspected of containing a first polynucleotide;

b. providing a second polynucleotide that is complementary to the first polynucleotide;

c. providing a signaling chromophore;

d. providing a conjugated polymer that upon excitation is capable of transferring energy to the signaling chromophore;

e. contacting the sample with the second polynucleotide, the signaling chromophore and the conjugated polymer under conditions in which the first and second polynucleotides can hybridize to form a signaling complex further comprising the conjugated polymer and the signaling chromophore; and

f. determining if the first and second polynucleotides have hybridized by detecting if light is produced from the signaling chromophore upon excitation of the conjugated polymer.

2. The method of claim 1, wherein the amount of emitted light produced from the signaling chromophore upon excitation of the signaling complex is greater than that obtained from direct excitation of the signaling chromophore.

3. The method of claim 1, wherein the conjugated polymer has the structure

4. The method of claim 1, wherein the conjugated polymer is an aggregate of conjugated molecules.

5. The method of claim 1, wherein the sample is contacted with the second polynucleotide and the conjugated polymer in the presence of a sufficient amount of an organic solvent to decrease hydrophobic interactions with the conjugated polymer.

6. The method of claim 1, wherein the method is performed in a multiplex format.

7. The method of claim 6, wherein the sample is contacted with a plurality of different second polynucleotides having corresponding different sequences, wherein each of said different second polynucleotides can selectively hybridize to a corresponding different complementary first polynucleotide resulting in different signaling complexes, wherein each of said different signaling complexes comprises a corresponding different signaling chromophore.

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

9. The method of claim 7, wherein the fluorophore is selected from a semiconductor nanocrystal, a fluorescent dye, a green fluorescent protein, and a lanthanide chelate, and a polynucleotide-specific dye.

10. The method of claim 8, wherein the fluorophore is a fluorescent dye.

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

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

13. The method of claim 12, wherein the substrate is selected from the group consisting of a bead, a chip, a microsphere, a slide, a multiwell plate, an optical fiber, an optionally porous gel matrix, a photodiode, and an optoelectronic device.

14. The method of claim 13, wherein the substrate is a bead.

15. The method of claim 13, wherein the substrate is a slide and detecting polynucleotide binding comprises performing fluorescence in situ hybridization.

16. The method of claim 12, wherein the substrate is nanoaddressable or microaddressable.

17. The method of claim 12, wherein the substrate is conjugated to a plurality of different second polynucleotides having corresponding different sequences, wherein each of said different sensor polynucleotides can selectively hybridize to a corresponding different first polynucleotide to form a signaling complex with the conjugated polymer.

22. A signaling complex formed by the method of claim 1.

18. A polynucleotide sensing solution comprising:

a signaling chromophore; and

a conjugated polymer that is capable of transferring energy to the signaling chromophore upon excitation when in proximity thereto, wherein a greater amount of energy is emitted from the signaling chromophore upon excitation of the conjugated polymer in the presence of the polynucleotide being sensed.

19. A method of performing fluorescence energy transfer comprising:

a. adding a conjugated polymer, a sensor polynucleotide and a signaling chromophore to a biological sample; and

b. detecting emission from the signaling chromophore by exciting the conjugated polymer, wherein the emission from the signaling chromophore is greater than that obtained from direct excitation.

20. The method of claim 19, wherein the emission is at least 4 times larger than that obtained from direct excitation of the signaling chromophore.

21. A composition comprising:

a. a conjugated polymer, and

b. a signaling chromophore,

wherein the conjugated polymer when excited transfers energy to the signaling chromophore, upon which the signaling chromophore emits a signal at least four-fold greater than when directly excited.