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

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-angstrom 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.