SiRNA carrying an (E)-vinylphosphonate moiety at the 5? end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2.

ABSTRACT: Efficient gene silencing by RNA interference (RNAi) in vivo requires the recognition and binding of the 5?- phosphate of the guide strand of an siRNA by the Argonaute protein. However, for exogenous siRNAs it is limited by the rapid removal of the 5?- phosphate of the guide strand by metabolic enzymes. Here, we have determined the crystal structure of human Argonaute-2 in complex with the metabolically stable 5?-(E)-vinylphosphonate (5?-E-VP) guide RNA at 2.5-Å resolution. The structure demonstrates how the 5? binding site in the Mid domain of human Argonaute-2 is able to adjust the key residues in the 5?-nucleotide binding pocket to compensate for the change introduced by the modified nucleotide. This observation also explains improved binding affinity of the 5?-E-VP -modified siRNA to human Argonaute-2 in-vitro, as well as the enhanced silencing in the context of the trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNA in mice relative to the un-modified siRNA.

SUBMITTER: Elkayam E 

PROVIDER: S-EPMC5389677 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature