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Strand-specific 5'-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity

¹ Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York, 10021, USA
² Munich Center for Integrated Protein Science (CIPSM), Max Planck Institute of Biochemistry, D-82152 Martinsried, Germany
³ Bioinformatics, Biozentrum der Universität Basel and Swiss Institute of Bioinformatics, CH-4056 Basel, Switzerland

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide catalytic sequence-specific cleavage of fully or nearly fully complementary target mRNAs or control translation and/or stability of many mRNAs that share 6–8 nucleotides (nt) of complementarity to the siRNA and miRNA 5' end. siRNA- and miRNA-containing ribonucleoprotein silencing complexes are assembled from double-stranded 21- to 23-nt RNase III processing intermediates that carry 5' phosphates and 2-nt overhangs with free 3' hydroxyl groups. Despite the structural symmetry of a duplex siRNA, the nucleotide sequence asymmetry can generate a bias for preferred loading of one of the two duplex-forming strands into the RNA-induced silencing complex (RISC). Here we show that the 5'-phosphorylation status of the siRNA strands also acts as an important determinant for strand selection. 5'-O-methylated siRNA duplexes refractory to 5' phosphorylation were examined for their biases in siRNA strand selection. Asymmetric, single methylation of siRNA duplexes reduced the occupancy of the silencing complex by the methylated strand with concomitant elimination of its off-targeting signature and enhanced off-targeting signature of the phosphorylated strand. Methylation of both siRNA strands reduced but did not completely abolish RNA silencing, without affecting strand selection relative to that of the unmodified siRNA. We conclude that asymmetric 5' modification of siRNA duplexes can be useful for controlling targeting specificity.

Keywords: RNA interference; RNAi; off-target effects; gene silencing; siRNA; RISC

⁴ These authors contributed equally to this work.

⁵ Present address: RNA Therapeutics, Merck & Co., Inc., WP26-410, West Point, PA 19486, USA.

Reprint requests to: Thomas Tuschl, Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10021, USA; e-mail: ttuschl{at}rockefeller.edu; fax: (212) 327-7652; or Gunter Meister, Munich Center for Integrated Protein Science (CIPSM), Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany; e-mail: meister{at}biochem.mpg.de; fax: 49-89-8578-3430.

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.789808.

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