1. Single-Stranded siRNAs Activate RNAi in Animals
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Garth A. Kinberger, Alfred E. Chappell, Punit P. Seth, Cheryl S. Li, Wenyu Li, Stanley T. Crooke, Eric E. Swayze, Walt F. Lima, Thazha P. Prakash, Hans Gaus, Heather M. Murray, and Susan F. Murray
- Subjects
Male ,Small interfering RNA ,Vinyl Compounds ,Molecular Sequence Data ,Organophosphonates ,Biology ,General Biochemistry, Genetics and Molecular Biology ,RNAi Therapeutics ,Mice ,RNA interference ,Gene expression ,Gene silencing ,Animals ,Humans ,RNA-Induced Silencing Complex ,RNA, Small Interfering ,Cells, Cultured ,Nuclease ,Mice, Inbred BALB C ,Base Sequence ,Biochemistry, Genetics and Molecular Biology(all) ,RNA ,Argonaute ,Lipid Metabolism ,Cell biology ,Biochemistry ,Liver ,Argonaute Proteins ,biology.protein ,Hepatocytes ,RNA Interference ,HeLa Cells - Abstract
SummaryThe 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.
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