Talk on Friday 01:15-01:30pm submitted by Stephanie Mack
Synthesis of mini-lariat RNA and use as a microRNA mimic for RNA interference
Stephanie Mack (Chemistry, Carnegie Mellon University), Jey Sabith Ebron (Biological Sciences, Cleveland State University), Girish C. Shukla (Biological Sciences, Cleveland State University), Subha R. Das (Chemistry, Carnegie Mellon University)
After the excision of intronic sequences as lariats in splicing, their fate is varied. Some introns are completely degraded into constituent nuclear bases and nucleotides recycled, while others enter into biogenesis and other pathways related to regulatory RNAs. MicroRNAs (miRNAs) derived from lariat introns, or mirtrons, represent a substantial amount of the total microRNA in a cell. These lariat introns are processed by lariat debranching enzyme, then enter the miRNA biogenesis pathway independent of drosha to generate miRNA duplexes that contain a guide and passenger strand. Of these, the guide strand is incorporated into the silencing complex for RNAi. Based on these lariat introns, here we synthesize single guide strands as mini-lariats for use as miRNA mimics. These mini-lariats are the size of a mature microRNA guide strand (19-21 mers) and are synthesized on the solid phase through the inclusion of a photo-cleavable protecting group at the 2' position of a desired residue. Following, photo-deprotection, the 2'-hydroxyl cyclizes with an activated 5'-phosphoramidite. Solid phase synthesis also allows for the incorporation of modified residues, which increase the stability of the RNA. These mini-lariats, with loops much smaller than natural lariat RNA, are opened up by debranching enzyme in vitro and effective in RNAi based knockdowns in mammalian cells.
Keywords: RNA interference, micro RNA