Poster abstracts

Poster number 28 submitted by Debasish Grahacharya

Solid-phase synthesis of natural and unnatural branched RNA sequences

Debasish Grahacharya (Department of Chemistry), Eduardo Paredes (Department of Chemistry), Molly Evans (Department of Chemistry), Subha R. Das (Department of Chemistry)

Abstract:
The process of splicing joins the protein coding regions of transcripts (exons) and removes the non-coding region (intron) as a lariat RNA that includes a highly conserved 2'-5'-branched RNA sequence with a branch point adenosine. The lariat branched structure is debranched by debranching enzyme after which the intronic RNA can enter critical regulatory processes such as miRNA biogenesis, retrotransposition and snoRNAs biogenesis pathways. The significance of branched RNAs in these critical regulatory processes have led to significant efforts to obtain branched RNA, yet access either to the natural conserved sequences or the ability to readily modify sequences have been elusive. We describe a straightforward strategy for the solid-phase synthesis of branched RNAs using a photolabile protecting group for reagentless and selective unmasking of an internal 2'-hydroxyl for branch synthesis. We describe the synthesis of the four photoprotected phosphoramidite compounds required for the synthesis of the conserved adenosine branched structure and three branch point analogues, guanosine, cytosine and uracil. Our method allows for rapid access to branched RNAs of any sequence and other synthetic modifications. A preliminary investigation of debranching enzyme with these branched RNAs is presented. We envision that our approach to branched RNA synthesis will aid in the molecular level understanding of the central role branched RNAs play in debranching, splicing and related critical regulatory processes.

Keywords: lariat, branched RNA, splicing