Method for identifying accessible binding sites on RNA

US6562570B1 · kind B1 · utility

Assignee

• City of Hope · US

Inventors

• John J. Rossi · Goleta, US
• Michaela Scherr · Hemmingen, DE
• Arthur D. Riggs · La Verne, US

Key dates

• Filing date: Mar 28, 2000
• Grant date: May 13, 2003
• Expiry date: Mar 28, 2020

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/6846
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method for identifying sites on a target RNA which are accessible to pairing by antisense DNA, ribozymes or DNAzymes. Native or in vitro-synthesized target RNA is incubated with defined ODNs and RNase H, or with a random or semi-random ODN library and RNase H, or with defined ribozymes or DNAzymes, or with a semi-random ribozyme or DNAzyme library, in a cell extract containing endogenous RNA-binding proteins, or in a medium which mimics a cell extract due to presence of one or more RNA-binding proteins. Any antisense ODN, ribozyme or DNAzyme which is complementary to an accessisble site in the target RNA hybridizes to that site and the RNA is cleaved at that site. Reverse transcription can be used to generate a first strand DNA from the RNA cleavage product, and terminal deoxynucleotidyl transferase-dependent polymerase chain reaction (TDPCR) can be used to identify sites on target RNA to which antisense ODNs, ribozymes or DNAzymes have bound and cleavage has occurred.