Poster abstracts
Poster number 34 submitted by Sourav Kumar Dey
Single Molecule Fluorescence Studies on Conformation of Backbone Branched RNA
Sourav K. Dey (Carnegie Mellon University), Linda A. Peteanu (Carnegie Mellon University), Subha R. Das (Carnegie Mellon University)
Abstract:
Lariat RNAs generated during splicing include a branch-point adenosine residue which has a unique 2'-5'-phosphodiester linkage. This 2'-5' linkage in the lariat RNA backbone can be specifically cleaved by the lariat debranching enzyme (Dbr1p). Following debranching, some lariat introns participate in important biological processes like snoRNA or microRNA biogenesis. We have developed a solid phase synthesis method to generate 2'-5'-phosphodiester linked RNAs which are mimics of the lariat RNAs. Our method of backbone branched RNA (bbRNA) synthesis allows us to incorporate modifications into the 5'- and 3'- ends and within the branch. A bbRNA, dual-labeled with fluorescent dyes (Alexa488 and Alexa594) will allow us to examine fluctuations in its conformation using single molecule FRET (smFRET). We have also synthesized an unnatural 'click-branched' RNA where the native 2'-5'-phosphodiester bond is replaced by a triazole linkage that is non-cleavable by Dbr1p. This non-native bbRNA with the same dual labels can be used to identify conformational changes due to binding of Dbr1p. Together, these bbRNAs with native and non-cleavable linkages will be used to study the conformations and dynamics pertaining to the Dbr1p debranching reaction at the single molecule level.
Keywords: Backbone branched RNA, Dbr1p, Single molecule FRET