Methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated RNA decay
View Patent ↗The present application provides methods of assaying for compounds that inhibit premature translation termination and nonsense mediated RNA decay in cells.
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 nucleic acid comprises an intron and at least two exons and 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 cellular extract is from plants, mammals or amphibians.
3. The method of claim 1 , wherein the cellular extract is a eukaryotic reticulocyte lysate or a rabbit reticulocyte lysate.
4. The method of claim 1 , wherein the cellular extract is a mammalian tissue culture cell extract.
5. The method of claim 1 , wherein the cellular extract is a HeLa cell S100 extract.
6. The method of claim 1 , wherein the polypeptide is detected by ELISA, light emission, colorimetric measurements or enzymatic activity.
7. The method of claim 1 , wherein the incubating and detecting steps are performed in a well of a microtiter dish.
8. The method of claim 7 , wherein the microtiter dish has 96 or 384 wells.
9. The method of claim 7 , wherein a plurality of candidate compounds are screened in parallel with each other and steps (i)–(ii) are repeated sequentially in a microtiter plate format, and wherein said plurality is between at least about 100 and at least about 6,000 different compounds.
10. The method of claim 7 , wherein the incubating and detecting steps are performed in a high throughput integrated system comprising an automatic pipetting station, a robotic armature, and a robotic controller.
11. The method of claim 1 , wherein the compound inhibits premature translation termination thereby increasing the amount of full-length polypeptide translated from the nucleic acid.
12. The method of claim 11 , wherein the nucleic acid encodes an enzyme or an immunoglobulin.
13. The method of claim 11 , wherein the nucleic acid encodes a luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or β-Galactosidase.
14. The method of claim 11 , wherein the nucleic acid encodes α-globin, β-globin, factor VIII, factor IX, vWF, p53, dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1, hexosaminidase A, neurofibromin 1, or neurofibromin 2.
15. The method of claim 1 , wherein the nucleic acid encodes a chimeric polypeptide.
16. The method of claim 1 , wherein the polypeptide contains a detectable moiety.
17. The method of claim 16 , wherein the detectable moiety is a radiolabel.
18. The method of claim 1 , wherein the polypeptide is detected using radioactivity.