Elkayam, E., Parmar, R., Brown, C. R., Willoughby, J. L., Theile, C. S., Manoharan, M., Joshua-Tor, L. (April 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) (Public Dataset)
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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 CSHL Cancer Center Program > Gene Regulation and Inheritance Program |
| Depositing User: | Matt Covey |
| Date: | 7 April 2017 |
| Date Deposited: | 08 Dec 2016 17:46 |
| Last Modified: | 26 Oct 2020 21:21 |
| PMCID: | PMC5389677 |
| Related URLs: | |
| Dataset ID: | |
| URI: | https://repository.cshl.edu/id/eprint/33930 |
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