Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3′ untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2–7 or 2–8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.
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We thank R. Knight for discussion and data analysis advice. We also thank the Dharmacon Production Team for synthesizing the siRNAs used in this work, and J. Kendall and A. O'Brien for providing assistance and direction in manuscript preparation.
The authors of this article are employed by Dharmacon or Agilent Technologies, which could potentially benefit from publication of this manuscript.
Variations of Smith-Waterman scoring parameters fail to improve the ability to distinguish off-targets from untargeted genes. (PDF 38 kb)
Web site tool for identification of potential off-targets based on 3UTR-hexamer seed matches. (PDF 49 kb)
Seed region-off-targeting association is not due to 3′ UTR length. (PDF 16 kb)
List of siRNA used in study. (PDF 37 kb)
Table of validated and predicted off-targets. (PDF 15 kb)
Custom S-W scoring parameters. (PDF 20 kb)
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Birmingham, A., Anderson, E., Reynolds, A. et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 3, 199–204 (2006). https://doi.org/10.1038/nmeth854
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