Methods for detection and analysis of polynucleotide-binding protein interactions

摘要

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 PBP that can bind to the target polynucleotide. The sensor PBP 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.

主权项

1. A method of assaying a sample for the presence of a target polynucleotide that binds to a polynucleotide binding protein selected from the group consisting of Tat, matrix protein M1, and hnRNP U, the method comprising:
(a) contacting the sample with:
(i) a sensor polynucleotide binding protein (sensor PBP) that comprises a polynucleotide binding protein selected from the group consisting of Tat, matrix protein M1, and hnRNP U linked to a signaling chromophore, wherein the signaling chromophore comprises a fluorophore; and(ii) a conjugated polymer that interacts with the target polynucleotide and upon excitation transfers energy to the signaling chromophore when the sensor PBP, target polynucleotide and conjugated polymer are present in a complex, wherein the conjugated polymer has the structure wherein m=1 or 2; (b) applying a light source to the sample that excites the conjugated polymer of the complex if present; (c) detecting light emitted from the signaling chromophore if emitted; and (d) evaluating whether the light emitted from the signaling chromophore is above a threshold level that indicates that the target polynucleotide is present in the sample.