Nucleic Acids Research Advance Access published online on February 11, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn042
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Functional dissection of siRNA sequence by systematic DNA substitution: modified siRNA with a DNA seed arm is a powerful tool for mammalian gene silencing with significantly reduced off-target effect
1Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 and 2Molecular Cellular Oncology and Microbiology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan
*To whom correspondence should be addressed. Tel: +81 3 5841 3043; Fax: +81 3 5841 3044; Email: ktei{at}biochem.s.u-tokyo.ac.jp
Received December 11, 2007. Revised January 21, 2008. Accepted January 22, 2008.
Short interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene knockdown in mammalian cells. To clarify the position-dependent functions of ribonucleotides in siRNA, siRNAs with various DNA substitutions were constructed. The following could be simultaneously replaced with DNA without substantial loss of gene-silencing activity: the seed arm, which occupies positions 2–8 from the 5'end of the guide strand; its complementary sequence; the 5'end of the guide strand and the 3'overhang of the passenger strand. However, most part of the 3' two-thirds of the guide strand could not be replaced with DNA, possibly due to binding of RNA-recognition proteins such as TRBP2 and Ago2. The passenger strand with DNA in the 3'end proximal region was incapable of inducing off-target effect. Owing to lesser stability of DNA–RNA hybrid than RNA duplex, modified siRNAs with DNA substitution in the seed region were, in most cases, incapable to exert unintended gene silencing due to seed sequence homology. Thus, it may be possible to design DNA–RNA chimeras which effectively silence mammalian target genes without silencing unintended genes.
Present address: Shuhei Zenno, Department of Biotechnology, Faculty of Engineering, Maebashi Institute of Technology, 460-1 Kamisadori-cho, Maebashi-shi, Gunma 371-0816, Japan
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