Compositions for detection and analysis of polynucleotides using light harvesting multichromophores

• US 8,101,416 B2
• Filed: 05/08/2007
• Issued: 01/24/2012
• 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): Wallenhorst, Maureen
• Application Number:
  US11/746,055
• Publication Number:
  US 20080280369A1
• Time in Patent Office: 1,722 Days
• Field of Search: 436/63, 436/94, 436/164, 436/166, 436/172, 422/61, 422/82.05, 422/82.08, 422/82.09, 422/430, 435/6, 435/6.1, 435/6.19, 536/23.1, 536/26.6
• US Class Current: 436/94
• 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. A signaling chromophore absorbs energy from the excited multichromophore and emits light in the presence of the target polynucleotide. The methods can be used in multiplex form. Kits having reagents for performing such methods are also provided.

Claims

1. An assay method comprising:

i. providing a sample that is suspected of comprising a target polynucleotide;

ii. providing a conjugated polymer that upon excitation is capable of transferring energy to a signaling chromophore when in proximity thereto;

iii. providing a sensor peptide nucleic acid (PNA) and a signaling chromophore, wherein the sensor PNA is single-stranded and is complementary to the target polynucleotide;

iv. contacting the sample, the sensor PNA and signaling chromophore and the conjugated polymer in a solution under conditions in which the sensor PNA can hybridize to the target polynucleotide, if present; and

v. determining if the sensor PNA and target polynucleotide have hybridized by detecting if light is produced from the signaling chromophore upon excitation of the conjugated polymer, indicating that the conjugated polymer and signaling chromophore are in proximity and associated with the target polynucleotide.

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

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

4. 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.

5. The method of claim 1, wherein the signaling chromophore is a fluorophore selected from a semiconductor nanocrystal, a fluorescent dye, a green fluorescent protein and a lanthanide chelate.

6. The method of claim 5, wherein the signaling chromophore is a fluorescent dye.

7. 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.

8. 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.

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

10. The method of claim 9, wherein the substrate is conjugated to a plurality of different sensor PNAs.

11. The method of claim 1, wherein a greater amount of light is produced from the signaling chromophore upon excitation of the conjugated polymer than from direct excitation.

12. The method of claim 11, wherein greater than eight times the amount of light is produced from the signaling chromophore upon excitation of the conjugated polymer than from direct excitation.

20. A signaling complex formed by the method of claim 1, comprising a sensor peptide nucleic acid, a complementary target polynucleotide, a conjugated polymer, and a signaling chromophore that receives energy from an excited state of the conjugated polymer.

21. The signaling complex of claim 20, wherein light emission produced from the signaling chromophore in the signaling complex upon receiving energy from an excited state of the conjugated polymer is greater than eight times larger than that obtained from direct excitation of the signaling chromophore.

22. The signaling complex of claim 20, wherein the signaling complex is present on a substrate.

23. The signaling complex of claim 22, wherein the substrate is selected from the group consisting of a chip, a microarray and a bead.

13. A polynucleotide sensing solution comprising:

a sensor peptide nucleic acid (PNA), a signaling chromophore, a conjugated polymer, and a solvent, wherein the sensor PNA is complementary to a target polynucleotide, and the conjugated polymer can interact with the phosphate backbone of the target polynucleotide and is capable of transferring energy to the signaling chromophore upon excitation when in proximity thereto upon hybridization of the sensor PNA to the target polynucleotide, 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 than from direct excitation of the signaling chromophore.

14. The solution of claim 13, wherein the emission from the signaling chromophore is greater than eight times larger than that obtained from direct excitation of the signaling chromophore.

15. A kit for assaying a sample for a target polynucleotide comprising:

a sensor peptide nucleic acid (PNA), a signaling chromophore, and a conjugated polymer, wherein the sensor PNA is single-stranded and is complementary to a target polynucleotide, and the conjugated polymer can interact with the phosphate backbone of the target polynucleotide and is capable of transferring energy to the signaling chromophore upon excitation when in proximity thereto upon hybridization of the sensor PNA to the target polynucleotide, 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 than from direct excitation of the signaling chromophore.

16. An energy transfer assay method for detecting a target polynucleotide in a sample comprising contacting a sample with a light harvesting luminescent conjugated polymer and a sensor peptide nucleic acid (PNA) in a predominantly aqueous solution in the presence of a signaling chromophore, wherein emission of a wavelength of light characteristic of the signaling chromophore upon excitation of the luminescent conjugated polymer indicates the presence in the sample of the target polynucleotide, and wherein the emission from the signaling chromophore is greater than that obtained from direct excitation of the signaling chromophore.

17. An energy transfer complex comprising a sensor peptide nucleic acid, a target polynucleotide, a conjugated polymer, and a signaling chromophore capable of receiving energy from an excited state of the conjugated polymer.

18. The complex of claim 17, wherein light emission from the signaling chromophore upon receiving energy from the conjugated polymer is greater than eight times larger than that obtained from direct excitation of the signaling chromophore.

19. A method of detecting complex formation via fluorescence energy transfer comprising (a) adding a conjugated polymer to a solution comprising a target polynucleotide, a peptide nucleic acid (PNA) and a signaling chromophore, thereby forming a complex comprising the conjugated polymer and the signaling chromophore;

and (b) detecting light emission from the signaling chromophore by exciting the conjugated polymer, wherein the light emission from the complex is greater than the amount of light emission obtained upon direct excitation of the signaling chromophore.