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

Elkayam, E., Parmar, R., Brown, C. R., Willoughby, J. L., Theile, C. S., Manoharan, M., Joshua-Tor, L. (2017) siRNA carrying an (E)-vinylphosphonate moiety at the 5' end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2. Nucleic Acids Res, 45 (6). pp. 3528-3536. ISSN 1362-4962 (Electronic)0305-1048 (Linking)

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URL: https://www.ncbi.nlm.nih.gov/pubmed/27903888
DOI: 10.1093/nar/gkw1171

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-A 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.

Item Type: Paper
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > argonaute proteins
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > siRNA
structural biology
Investigative techniques and equipment > x ray crystallography
CSHL Authors:
Communities: CSHL labs > Joshua-Tor lab
Depositing User: Matt Covey
Date Deposited: 08 Dec 2016 17:46
Last Modified: 08 Nov 2017 15:04
PMCID: PMC5389677
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Dataset ID:
URI: http://repository.cshl.edu/id/eprint/33930

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