Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mrna decay
First Claim
1. A method for identifying a compound that binds to a target RNA, said method comprising (a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA:
- compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and
(b) detecting the formation of a detectably labeled target RNA;
compound complex.
6 Assignments
0 Petitions
Accused Products
Abstract
The present invention relates to a method for screening and identifying compounds that modulate premature translation termination and/or nonsense-mediated messenger ribonucleic acid (“mRNA”) by interacting with a preselected target ribonucleic acid (“RNA”). In particular, the present invention relates to identifying compounds that bind to regions of the 28S ribosomal RNA (“rRNA”) and analogs thereof. Direct, noncompetitive binding assays are advantageously used to screen libraries of compounds for those that selectively bind to a preselected target RNA. Binding of target RNA molecules to a particular compound is detected using any physical method that measures the altered physical property of the target RNA bound to a compound. The structure of the compound attached to the labeled RNA is also determined. The methods used will depend, in part, on the nature of the library screened. The methods of the present invention provide a simple, sensitive assay for high-throughput screening of libraries of compounds to identify pharmaceutical leads.
-
Citations
15 Claims
-
1. A method for identifying a compound that binds to a target RNA, said method comprising
(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA: - compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and
(b) detecting the formation of a detectably labeled target RNA;
compound complex. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
- compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and
-
2. A method for identifying a compound to test for its ability to modulate premature translation termination or nonsense-mediated mRNA decay, said method comprising:
-
(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;
compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and
(b) detecting a detectably labeled target RNA;
compound complex formed in step(a), so that if a target RNA;
compound complex is detected then the compound identified is tested for its ability to modulate premature translation or nonsense-mediated mRNA delay.
-
-
3. A method for identifying a compound that binds to a target RNA, said method comprising detecting the formation of a detectably labeled target RNA:
- compound complex formed from contacting a detectably labeled RNA with a member of a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;
compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon.
- compound complex formed from contacting a detectably labeled RNA with a member of a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;
-
4. A method of identifying a compound that modulates premature translation termination or nonsense-mediated mRNA decay, said method comprising:
-
(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;
compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and
(b) detecting a detectably labeled target RNA;
compound complex formed in step(a), so that if a target RNA;
compound complex is detected, then(c) contacting the compound with a cell-free translation mixture and a nucleic acid sequence comprising a regulatory element operably linked to a reporter gene, wherein the reporter gene contains a premature stop codon; and
(d) detecting the expression of the reporter gene, wherein a compound that modulates premature translation termination or nonsense-mediated mRNA decay is identified if the expression of the reporter gene in the presence of the compound is altered relative to the expression of the reporter gene in the absence of the compound or the presence of a negative control.
-
-
5. A method of identifying a compound that modulates premature translation termination or nonsense-mediated mRNA decay, said method comprising:
-
(a) contacting a detectably labeled target RNA molecule with a library of compounds under conditions that permit direct binding of the labeled target RNA to a member of the library of compounds and the formation of a detectably labeled target RNA;
compound complex, wherein the target RNA is a region of 28S rRNA or contains a premature stop codon; and
(b) detecting a detectably labeled target RNA;
compound complex formed in step(a), so that if a target RNA;
compound complex is detected, then(c) contacting the compound with a cell containing a nucleic acid sequence comprising a regulatory element operably linked to a reporter gene, wherein the reporter gene contains a premature stop codon; and
(d) detecting the expression of the reporter gene, wherein a compound that modulates premature translation termination or nonsense-mediated mRNA decay is identified if the expression of the reporter gene in the presence of the compound is altered relative to the expression of the reporter gene in the absence of the compound or the presence of a negative control.
-
Specification