Published online 21 March 2005
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A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites
Center for Genomics and Bioinformatics, Karolinska Institutet 171 77 Stockholm, Sweden 1Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet Stockholm 171 77, Sweden
*To whom correspondence should be addressed. Tel: +46 8 5248 6381; Fax: +46 8 327934; Email: quan.du{at}cgb.ki.se
Received December 20, 2004. Revised February 14, 2005. Accepted March 2, 2005.
The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificity on all possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.
Correspondence may also be addressed to Zicai Liang. Tel: +46 8 5248 6371; Fax: +46 8 327934; Email: zicai.liang{at}cgb.ki.se
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