title: Single-stranded siRNAs activate RNAi in animals.
abstract: The therapeutic utility of siRNAs is limited by the requirement for complex formulations to deliver them to tissues. If potent single-stranded RNAs could be identified, they would provide a simpler path to pharmacological agents. Here, we describe single-stranded siRNAs (ss-siRNAs) that silence gene expression in animals absent lipid formulation. Effective ss-siRNAs were identified by iterative design by determining structure-activity relationships correlating chemically modified single strands and Argonaute 2 (AGO2) activities, potency in cells, nuclease stability, and pharmacokinetics. We find that the passenger strand is not necessary for potent gene silencing. The guide-strand activity requires AGO2, demonstrating action through the RNAi pathway. ss-siRNA action requires a 5' phosphate to achieve activity in vivo, and we developed a metabolically stable 5'-(E)-vinylphosphonate (5'-VP) with conformation and sterioelectronic properties similar to the natural phosphate. Identification of potent ss-siRNAs offers an additional option for RNAi therapeutics and an alternate perspective on RNAi mechanism.
authors.affiliation: Core Antisense Research, Isis Pharmaceuticals Inc., Carlsbad, CA 92010, USA.
authors.forename: Walt F
authors.initials: WF
authors.lastname: Lima
authors.forename: Thazha P
authors.initials: TP
authors.lastname: Prakash
authors.forename: Heather M
authors.initials: HM
authors.lastname: Murray
authors.forename: Garth A
authors.initials: GA
authors.lastname: Kinberger
authors.forename: Wenyu
authors.initials: W
authors.lastname: Li
authors.forename: Alfred E
authors.initials: AE
authors.lastname: Chappell
authors.forename: Cheryl S
authors.initials: CS
authors.lastname: Li
authors.forename: Susan F
authors.initials: SF
authors.lastname: Murray
authors.forename: Hans
authors.initials: H
authors.lastname: Gaus
authors.forename: Punit P
authors.initials: PP
authors.lastname: Seth
authors.forename: Eric E
authors.initials: EE
authors.lastname: Swayze
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D006367:HeLa Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D050356:Lipid Metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D063065:Organophosphonates / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D014753:Vinyl Compounds / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D063065:Organophosphonates; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D014753:Vinyl Compounds; C433467:vinylphosphonic acid