Research Project
8453115
DESCRIPTION (provided by applicant): RNAs and ribonucleoprotein complexes (RNPs) play central roles in biology and disease. Despite their importance to both basic and clinical biology, there is no general methodology to study the composition and function of RNPs isolated from their native context. To address this need, our Phase I goal is to develop a new and robust affinity separation technology for RNP complexes. In Phase II, we propose to develop the method into an easy-to-use kit that can be employed by researchers to detect and analyze specific RNPs in their laboratories without special expertise for a variety of applications. Our strategy is based on an endoribonuclease that binds a specific RNA sequence with very high affinity and cleaves this sequence at a single site. A mutant version of this protein can bind but not cleave target RNA. In our method, a target RNA will be expressed in vitro or in vivo with a 5' tag composed of the endoribonuclease target sequence. Cellular extracts will be applied to the mutant endoribonuclease immobilized on resin. The tagged RNA will be trapped by the mutant endoribonuclease. Under specific buffer conditions, cleavage activity of the mutant endoribonuclease can be reactivated, causing the tag to be cleaved off, liberating the target RNA and any protein partners bound to it. The isolated RNA and its associated RNA binding proteins can then be analyzed by a variety of molecular biological or biophysical techniques.
