Method of assaying for RNA: protein interactions

• US 6,261,772 B1
• Filed: 01/29/1999
• Issued: 07/17/2001
• Est. Priority Date: 01/30/1998
• Status: Expired due to Fees

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Cullen, Bryan R., Blair, Wade S.
• Primary Examiner(s): YUCEL, IREM
• Application Number:
  US09/239,523
• Time in Patent Office: 900 Days
• Field of Search: 435/6, 435/7.2, 435/7.21, 536/23.1, 536/24.1
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The present invention relates, in general to an assay method, and, in particular, to an in vivo method of assaying the interaction of RNA with proteins. The invention relates to a kit suitable for use in connection with such method.

Claims

1. A method for screening a test compound for its ability to modulate a RNA:

protein interaction comprising;

i) preparing a reporter construct comprising, in order, a Tat-responsive promoter operably linked to a sequence encoding a heterologous RNA binding site, and an indicator gene,

ii) preparing an effector construct comprising a promoter operably linked to a nucleic acid sequence encoding a fusion protein comprising a Tat protein activation domain and a protein cognate of said heterologous RNA binding sites,

iii) introducing said reporter construct and said effector construct into a vertebrate host cell and culturing said host cell in the presence and absence of said test compound, and

iv) measuring the level of expression of said indicator gene in the presence and absence of said test compound, a difference in the level of expression of said indicator gene in the presence of said test compound, as compared to the level of expression of said indicator gene in the absence of said test compound, being indicative of a test compound that modulates interaction of said heterologous RNA binding site with said protein cognate.

2. The method according to claim 1 wherein said TAT responsive promoter is a lentivirus LTR.

3. The method according to claim 1 wherein the reporter construct comprises a minimum Tat-responsive LTR promoter.

4. The method according to claim 1 wherein the reporter construct further comprises an internal ribosome entry site between the heterologous RNA binding site and the indicator gene and in operable linkage therewith.

5. The method according to claim 1 wherein the reporter construct comprises the RNA stem-loop binding site for polio and rhino-viral 3C proteins, or the RNA binding site from a picornavirus.

6. The method according to claim 1 wherein the effector construct comprises a cytomegalovirus intermediate early promoter, a SV40 early promoter or a retroviral LTR promoter.

7. A vertebrate host cell comprising:

i) a reporter construct comprising, in order, a Tat-responsive promoter functional in said host cell operably linked to a sequence encoding a heterologous RNA binding site, and an indicator gene, and

ii) an effector construct comprising a promoter functional in said host cell operably linked to a nucleic acid sequence encoding a fusion protein comprising a Tat protein activation domain and a protein cognate of said heterologous RNA binding site,

wherein said host cell expresses the protein product of said indicator gene.

8. The host cell according to claim 7 wherein said TAT responsive promoter is a lentivirus LTR.

9. The host cell according to claim 7 wherein wherein the reporter construct comprises a minimum Tat-responsive LTR promoter.

10. The host cell according to claim 7 wherein the reporter construct further comprises an internal ribosome entry site between the heterologous RNA binding site and the indicator gene and in operable linkage therewith.

11. The host cell according to claim 7 wherein said host cell is a mammalian cell.

12. The host cell according to claim 11 wherein said host cell is a human cell.

13. The host cell according to claim 11 wherein said host cell is a human fibroblast cell, a human T-cell, a rodent cell or a monkey cell.

14. The method according to claim 7 wherein the reporter construct comprises the RNA stem-loop binding site for polio and rhino-viral 3C proteins, or the RNA binding site from a picornavirus.

15. The method according to claim 7 wherein the effector construct comprises a cytomegalovirus intermediate early promoter, a SV40 early promoter or a retroviral LTR promoter.

16. A kit comprising a reporter construct disposed with a container means and an effector construct disposed within a container means wherein

the reporter construct comprising, in order, a Tat-responsive promoter operably linked to a site suitable for insertion of a sequence encoding a heterologous RNA binding site, and an indicator gene, and

the effector construct comprising a promoter operably linked to a nucleic acid sequence encoding a Tat protein activation domain, said nucleic acid sequence comprising a site 3′

or 5′

to said Tat protein activation domain encoding sequence, suitable for insertion of a sequence encoding a protein cognate of said heterologous RNA binding site.

17. The kit according to claim 16 wherein said TAT responsive promoter is a lentivirus LTR.

18. The kit according to claim 16 wherein the reporter construct comprises a minimum Tat-responsive LTR promoter.

19. The kit according to claim 16 wherein the reporter construct further comprises an internal ribosome entry site between the heterologous RNA binding site and the indicator gene and in operable linkage therewith.

20. The kit according to claim 16 wherein the reporter construct comprises the RNA stem-loop binding site for polio and rhino-viral 3C proteins, or the RNA binding site from a picornavirus.

21. The kit according to claim 16 wherein the effector construct comprises a cytomegalovirus intermediate early promoter, a SV40 early promoter or a retroviral LTR promoter.