Method of purifying RNA binding protein-RNA complexes

• US 9,447,454 B2
• Filed: 12/12/2013
• Issued: 09/20/2016
• Est. Priority Date: 10/23/2003
• Status: Active Grant

• Current Assignee: Rockefeller University
• Original Assignee: Rockefeller University
• Inventors: Darnell, Robert, Jensen, Kirk, Ule, Jernej
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US14/104,581
• Publication Number:
  US 20140378316A1
• Time in Patent Office: 1,013 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection

Abstract

The present invention provides methods for purifying RNA molecules interacting with an RNA binding protein (RBP), and the use of such methods to analyze a gene expression profile of a cell. The invention also provides sequences of RNA molecules that mediate binding to an RBP, proteins encoded by the sequences, a method of identifying the sequences, and the use of the sequences in a screen to identify bioactive molecules. The invention also provides RNA motifs found among the sequences and compounds that bind the RNA motifs. In addition, the invention provides methods of treating diseases associated with a function of an RNA binding protein.

Claims

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;

(i) washing the complexes with buffer;

(ii) boiling the complexes in a denaturing ionic detergent;

(iii) separating the complexes by SDS-PAGE; and

(iv) transferring said complexes to a substrate that preferentially binds test RNA covalently crosslinked to protein over test RNA not covalently crosslinked to protein;

(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.

2. The method of claim 1, wherein said nuclease is RNAse T1.

3. The method of claim 1, further comprising labeling said test RNA molecule.

4. The method of claim 1, wherein said substrate is a nitrocellulose membrane.

5. The method of claim 1, wherein the irradiating of the biological sample comprises the input of sufficient energy to produce at least about a 4-fold enrichment in the frequency of occurrence of the one or more RBP binding sites in the second population of covalently-linked RBP-test RNA fragment complexes as compared with the expected random occurrence of the one or more RBP binding sites.

6. The method of claim 1, wherein the irradiating of the biological sample comprises the input of sufficient energy to produce at least about a 10-fold enrichment in the frequency of occurrence of the one or more RBP binding sites in the second population of covalently-linked RBP-test RNA fragment complexes as compared with the expected random occurrence of the one or more RBP binding sites.

7. The method of claim 1, wherein the irradiating of the biological sample comprises the input of sufficient energy to produce at least about a 4-fold to an at least about a 10-fold enrichment in the frequency of occurrence of the one or more RBP binding sites in the second population of covalently-linked RBP-test RNA fragment complexes as compared with the expected random occurrence of the one or more RBP binding sites.

8. The method of claim 1, wherein the irradiating of the biological sample comprises the input of about 400 milliJoules per square centimeter of the biological sample.

9. The method of claim 1, wherein the fragments of the one or more test RNA molecules obtained in step (e) are amplified by ligating nucleotide linkers to said fragments of one or more test RNA molecules and subjecting the fragments of the one or more test RNA molecules to reverse transcriptase-polymerase chain reaction (RT-PCR).

10. The method of claim 9, wherein said nucleotide linkers are directionally oriented.

11. The method of claim 1, wherein the molecule that specifically interacts with the component of said RBP-test RNA fragment complexes is an antibody.

12. The method of claim 1, wherein the one or more binding sites on the one or more test RNA molecules interacting with an RNA binding protein of interest comprise an RNA motif which exhibits a particular structure.

13. The method of claim 12, wherein the RNA motif comprises a recurring RNA element, structure or sequence.

14. The method of claim 1, wherein the sequence of the one or more test RNA molecules interacting with an RNA binding protein of interest is determined.

15. The method of claim 1, wherein the one or more test RNA molecules interacting with an RNA binding protein of interest is subjected to RNA footprinting.

16. The method of claim 1, wherein the method is an unbiased screen.

17. The method of claim 16, wherein the one or more test RNA molecules interacting with an RNA binding protein of interest was not predetermined.

18. The method of claim 17, wherein the sequence of the one or more test RNA molecules interacting with an RNA binding protein of interest is determined.

19. The method of claim 18, wherein the fragments of the one or more test RNA molecules obtained in step (e) are amplified by ligating nucleotide linkers to said fragments of RNA and subjecting the test RNA fragments to reverse transcriptase-polymerase chain reaction (RT-PCR).

20. The method of claim 19, wherein said nucleotide linkers are directionally oriented.

21. The method of claim 1, wherein the test biological sample is selected from the group consisting of whole tissue biopsy, tissue sample biopsy and whole organ.

22. The method of claim 1, wherein the test RNA fragment is derived from a transfer RNA, a small nuclear RNA, a ribosomal RNA, a messenger RNA, an antisense RNA, a small inhibitory RNA, a micro RNA or a ribozyme.