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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.


  1. 1

    Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Reynolds, A. et al. Rational siRNA design for RNA interference. Nat. Biotechnol. 22, 326–330 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Khvorova, A., Reynolds, A. & Jayasena, S. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Naito, Y., Yamada, T., Ui-Tei, K., Morishita, S. & Saigo, K. siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference. Nucleic Acids Res. 32, W124–W129 (2004).

    CAS  Article  Google Scholar 

  5. 5

    Jagla, B. et al. Sequence characteristics of functional siRNAs. RNA 11, 864–872 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Huesken, D. et al. Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995–1001 (2005).

    CAS  Article  Google Scholar 

  7. 7

    Jackson, A.L. et al. Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635–637 (2003).

    CAS  Article  Google Scholar 

  8. 8

    Lin, X. et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res. 33, 4527–4535 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Elbashir, S.M., Harborth, J., Weber, K. & Tuschl, T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Amarzguioui, M., Holen, T., Babaie, E. & Prydz, H. Tolerance for mutations and chemical modifications in a siRNA. Nucleic Acids Res. 31, 589–595 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E. & Prydz, H. Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res. 30, 1757–1766 (2002).

    CAS  Article  Google Scholar 

  12. 12

    Altschul, S. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    CAS  Article  Google Scholar 

  13. 13

    Smith, T.F. & Waterman, M.S. Identification of common molecular subsequences. J. Mol. Biol. 147, 195–197 (1981).

    CAS  Article  Google Scholar 

  14. 14

    Lim, L. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Scaringe, S.A. Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis. Methods Enzymol. 317, 3–18 (2000).

    CAS  Article  Google Scholar 

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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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Corresponding author

Correspondence to Anastasia Khvorova.

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Competing interests

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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