Poster abstracts

Poster number 48 submitted by Debasish Grahacharya

Solid-phase synthesis of backbone branched RNA sequences: access to branch modifications

Debasish Grahacharya (Department of Chemistry, Carnegie Mellon University), Sourav Kumar Dey (Department of Chemistry, Carnegie Mellon University), Eduardo Paredes (Department of Chemistry, Carnegie Mellon University), Subha R. Das (Department of Chemistry, Carnegie Mellon University)

Abstract:
Backbone branching occurs in RNA sequences during splicing. In splicing, the protein coding regions (exons) are joined and the introns are removed as lariats to generate the correct mRNA. The lariat structure includes a 2’,5’-branch. Thus, access to backbone branched RNA (bbRNA) is important to investigate splicing, debranching and other regulatory processes. There have been significant efforts to obtain bbRNAs yet, the ability to obtain natural and the modified sequences have been elusive. We report a simple strategy for the solid-phase synthesis (SPS) of bbRNAs using a photolabile protecting group. Synthesis of bbRNAs starts with incorporation of 2'-photoprotected amidite in the stem as the branching point. Photodeprotection followed by incorporation of reverse amidites provide the bbRNAs. However, to include modifications in the 2'- branch reverse amidites with the desired modification are required. As these are not available, we used a branching amidite with 3'-photoprotecting group which enable us to avoid modified reverse amidites. We observed 2'- to 3'- branch migration (and vice versa) during the photodeprotection step. That could be resolved by the selective removal of cyanoethyl group to generate phospodiester linkage (before the photodeprotection step and branch synthesis). Debranching of these modified bbRNAs by Dbr is presented. This synthetic approach will provide an insight into the role of bbRNAs in debranching, splicing and other regulatory processes.

Keywords: splicing, debranching enzyme, backbonebranched RNA, lariat