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3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets

A Corrigendum to this article was published on 01 June 2007

An Addendum to this article was published on 01 June 2006


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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Figure 1: Microarray analysis identifies off-targeted genes.
Figure 2: Maximum sequence alignment does not accurately predict off-targeted gene regulation by RNAi.
Figure 3: Systematic single base pair–mismatch analysis of siRNA functionality.
Figure 4: Exact complementarity between the siRNA seed region and the 3′ UTR (but not 5′ UTR or ORF) distinguishes off-targeted from untargeted genes.


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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.

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Correspondence to Anastasia Khvorova.

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The authors of this article are employed by Dharmacon or Agilent Technologies, which could potentially benefit from publication of this manuscript.

Supplementary information

Supplementary Fig. 1

Variations of Smith-Waterman scoring parameters fail to improve the ability to distinguish off-targets from untargeted genes. (PDF 38 kb)

Supplementary Fig. 2

Web site tool for identification of potential off-targets based on 3UTR-hexamer seed matches. (PDF 49 kb)

Supplementary Fig. 3

Seed region-off-targeting association is not due to 3′ UTR length. (PDF 16 kb)

Supplementary Table 1

List of siRNA used in study. (PDF 37 kb)

Supplementary Table 2

Table of validated and predicted off-targets. (PDF 15 kb)

Supplementary Table 3

Custom S-W scoring parameters. (PDF 20 kb)

Supplementary Note (DOC 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).

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