title: 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.
authors.affiliation: Keck Structural Biology Lab, Cold Spring Harbor, NY 11724, USA.|Howard Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA.|Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
authors.forename: Elad
authors.initials: E
authors.lastname: Elkayam
authors.affiliation: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
authors.forename: Rubina
authors.initials: R
authors.lastname: Parmar
authors.affiliation: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Brown
authors.affiliation: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Willoughby
authors.affiliation: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Theile
authors.affiliation: Alnylam Pharmaceuticals, 300 Third Street, Cambridge, MA 02142, USA.
authors.forename: Muthiah
authors.initials: M
authors.lastname: Manoharan
authors.affiliation: Keck Structural Biology Lab, Cold Spring Harbor, NY 11724, USA.|Howard Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA.|Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
authors.forename: Leemor
authors.initials: L
authors.lastname: Joshua-Tor
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D063065:Organophosphonates / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism*; D017457:Receptors, Albumin / Q000235:genetics / Q000378:metabolism; D014753:Vinyl Compounds / Q000737:chemistry*; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D060565:Argonaute Proteins; D063065:Organophosphonates; D034741:RNA, Small Interfering; D017457:Receptors, Albumin; D014753:Vinyl Compounds; D000094704:RNA, Guide, CRISPR-Cas Systems; C466422:AGO2 protein, human; C091986:transthyretin receptor; C433467:vinylphosphonic acid