Methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated RNA decay

• US 20060134681A1
• Filed: 12/22/2005
• Published: 06/22/2006
• Est. Priority Date: 12/14/1999
• Status: Abandoned Application

• Current Assignee: Holger Beckmann, Kevin Czaplinski, Marc Learned, Stuart Peltz
• Original Assignee: Holger Beckmann, Kevin Czaplinski, Marc Learned, Stuart Peltz
• Inventors: Beckmann, Holger, Peltz, Stuart, Czaplinski, Kevin, Learned, Marc
• Application Number:
  US11/318,940
• Publication Number:
  US 20060134681A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 2333/70503 Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
• CPC Class Codes: G01N 2333/90 Enzymes; Proenzymes
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Abstract

The present application provides methods of assaying for compounds that inhibit premature translation termination and nonsense mediated RNA decay in cells.

Claims

1. A method of in vitro screening for compounds that modulate premature translation termination and nonsense-mediated mRNA decay, the method comprising the steps of:

(i) incubating a translation assay, the assay comprising an in vitro translation cellular extract;

a nucleic acid encoding a polypeptide, wherein the coding sequence for the polypeptide comprises a premature stop codon; and

a candidate modulator compound; and

(ii) detecting the polypeptide translated from the nucleic acid.

2. The method of claim 1, wherein the nucleic acid encodes an enzyme.

3. The method of claim 1, wherein the nucleic acid encodes an immunoglobin.

4. The method of claim 1, wherein the nucleic acid encodes luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or β

-galactosidase.

6. The method of claim 1, wherein the cellular extract is from yeast, plants, mammals, or amphibians.

7. The method of claim 1, wherein the cellular extract is a eukaryotic reticulocyte lysate.

9. The method of claim 1, wherein the cellular extract is a mammalian tissue culture cell extract.

11. The method of claim 1, wherein the polypeptide is detected by ELISA, light emission, colorimetric measurements, enzymatic activity, or radioactivity.

12. The method of claim 1, wherein the assay is performed in a well of a microtiter dish.

5. (canceled)

8. (canceled)

10. (canceled)

13-15. -15. (canceled)

16. The method of in vivo screening for compounds that modulate premature translation termination and nonsense-mediated mRNA decay, the method comprising the steps of:

(i) expressing in a cell a nucleic acid encoding a polypeptide, wherein the coding sequence for the polypeptide comprises a premature stop codon;

(ii) contacting the cell with a candidate modulator compound; and

(iii) detecting either the polypeptide translated from the nucleic acid or RNA transcribed from the nucleic acid.

17. The method of claim 16, wherein the nucleic acid comprises a promoter operably linked to a heterologous nucleic acid encoding the polypeptide.

18. The method of claim 17, wherein the heterologous nucleic acid encoding the polypeptide comprises an intron and at least two exons comprising coding sequence.

20. The method of claim 18, wherein the heterologous nucleic acid encodes a chimeric polypeptide.

22. The method of claim 17, wherein the nucleic acid encodes an enzyme.

23. The method of claim 17, wherein the nucleic acid encodes luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or β

-galactosidase.

24. The method of claim 17, wherein the nucleic acid encodes an immunoglobin.

25. The method of claim 16, wherein the nucleic acid is an endogenous gene.

26. The method of claim 25, wherein the endogenous gene is α

-globin, β

-globin, factor VIII, factor IX, vWF, p53, dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1, hexosaminidase A, neurofibromin 1, or neurofibromin 2.

27. The method of claim 25, wherein the endoegenous gene encodes an immunoglobin.

28. The method of claim 16, wherein the polypeptide is detected by ELISA, light emission, colorimetric measurements, enzymatic activity, drug resistance, FACS, or radioactivity.

29. The method of claim 16, wherein the assay is performed in a well of a microtiter dish.

35. The method of claim 16, wherein the cell is a human cell or a mouse cell.

36. The method of claim 16, wherein the cell is stably transfected with the nucleic acid.

19. (canceled)

21. (canceled)

30-34. -34. (canceled)

37. A kit for screening for compounds that modulate translation termination and nonsense-mediated mRNA decay, the kit comprising a nucleic acid encoding a polypeptide, wherein the polypeptide coding sequence comprises a premature stop codon;

instructions for practicing a method of screening for compounds that inhibit translation termination at premature stop codons and nonsense mediated RNA decay; and

a control compound that inhibits nonsense-mediated RNA decay.

38-43. -43. (canceled)