Nucleic Acids Research Advance Access originally published online on November 28, 2008
Nucleic Acids Research 2009 37(2):346-353; doi:10.1093/nar/gkn958
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Nucleic Acids Research, 2009, Vol. 37, No. 2 346-353
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RNA major groove modifications improve siRNA stability and biological activity
Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA
*To whom correspondence should be addressed. Tel: +1 650 724 4741; Fax: +1 650 725 0259; Email: kool{at}stanford.edu
Received October 20, 2008. Revised November 10, 2008. Accepted November 11, 2008.
RNA 5-methyl and 5-propynyl pyrimidine analogs were substituted into short interfering RNAs (siRNAs) to probe major groove steric effects in the active RNA-induced silencing complex (RISC). Synthetic RNA guide strands containing varied combinations of propynyl and methyl substitution revealed that all C-5 substitutions increased the thermal stability of siRNA duplexes containing them. Cellular gene suppression experiments using luciferase targets in HeLa cells showed that the bulky 5-propynyl modification was detrimental to RNA interference activity, despite its stabilization of the helix. Detrimental effects of this substitution were greatest at the 5'-half of the guide strand, suggesting close steric approach of proteins in the RISC complex with that end of the siRNA/mRNA duplex. However, substitutions with the smaller 5-methyl group resulted in gene silencing activities comparable to or better than that of wild-type siRNA. The major groove modifications also increased the serum stability of siRNAs.