Patent ID: 9447454
Filing Date: 2016-09-20
CPC Classification: C12N,C12Q

Claim Text:
1. A method for isolating one or more test RNA molecules having one or more binding sites interacting with an RNA binding protein (RBP) of interest from a biological sample, said method comprising the steps of: (a) irradiating a test biological sample to create an irreversible covalent crosslinking between the test RNA molecules and RBPs of interest in said test biological sample, thereby generating a sample of covalently-linked RBP-test RNA complexes which is substantially absent from a control biological sample which was not subjected to irradiation, wherein the one or more test RNA molecules are not modified with a chemical group to facilitate crosslinking with the RBP prior to irradiating the test biological sample; (b) cleaving the test RNA molecules of said covalently-linked RBP-test RNA complexes by contacting said sample of covalently-linked RBP-test RNA complexes with a nuclease, thereby generating a first population of covalently-linked RBP-test RNA fragment complexes; (c) generating a second population of covalently-linked RBP-test RNA fragment complexes by selecting covalently-linked RBP-test RNA fragment complexes in said first population by utilizing a molecule that specifically interacts with a component of said covalently-linked RBP-test RNA fragment complexes in said first population; (d) purifying said second population of covalently-linked RBP-test RNA fragment complexes obtained in step (c) under stringent conditions comprising the consecutive steps of: (e) digesting said RBP with a protease to liberate said fragments of test RNA from said purified second population of covalently-linked RBP-test RNA fragment complexes; and (f) amplifying said fragments of test RNA obtained in step (e), thereby isolating the one or more test RNA molecules having the one or more binding sites interacting with the RNA binding protein of interest.