Poster abstracts

Poster number 47 submitted by Sourav Kumar Dey

Solid phase synthesis of lariat RNAs for RNA interference

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

Abstract:
To generate the correct sequence for coding proteins, the pre-mRNA has to remove its non-coding intronic sequences and join the coding exons through the process of splicing. During the process of splicing, the introns are removed as a special lariat structure where the 5'-end of the RNA is tied back to the 2'-OH of an internal adenosine nucleotide. Recent reports indicate that some of these lariat introns after getting linearized by the lariat debranching enzyme (Dbr) can form active pre-microRNA. Therefore we sought to synthesize siRNAs (21-22 mer) where only the guide or the anti-sense strand of the siRNA is in a lariat form. As the 5'-end of these lariats are not free, they are more stable towards nuclease degradation. We used a 2'-photoprotected adenosine phosphoramidite at the branch point of the growing RNA strand to make the lariat. After the synthesis of the linear strand, we generated a 5'-phosphoramidte in the solid phase using literature adapted method. Then selective unmasking of the 2'-OH of the branch point adenosine using UV light followed by coupling of the 5'-phosphoramidite and the deprotected 2'-OH resulted in the lariat RNAs. These synthetic lariats are substrates for the lariat debranching enzyme (Dbr) in vitro. We tested the activity of these lariat RNAs in Drosophila S2 cells using a dual luciferase reporter system. Our positive results suggest that synthetic mini lariat RNAs may represent a significant new class of Drosha and Dicer independent RNA silencing agent.

References:
1. Ruby, J.G., Jan, C.H., and Bartel, D.P. Intronic microRNA precursors that bypass Drosha processing. Nature 448: 83-86 (2007).

Keywords: Lariat Intron, Debranching enzyme(Dbr), siRNA