Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

The impact of target site accessibility on the design of effective siRNAs

Nature Biotechnology volume 26, pages 578–583 (2008)Cite this article

Abstract

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Application of RNAplfold to separate functional from nonfunctional siRNAs.
Figure 2: Correlation plots for different design criteria for 2,433 siRNAs from data set 1.
Figure 3: Performance of RNAxs on a set of 360 siRNAs targeting the four genes firefly luciferase, PPIB, ALPPL2 and DBI.

Similar content being viewed by others

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Tomari, Y. & Zamore, P.D. Perspective: machines for RNAi. Genes Dev. 19, 517–529 (2005).

    Article  CAS  Google Scholar 

  2. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).

    Article  CAS  Google Scholar 

  3. Ameres, S.L., Martinez, J. & Schroeder, R. Molecular basis for target RNA recognition and cleavage by human RISC. Cell 130, 101–112 (2007).

    Article  CAS  Google Scholar 

  4. Bernhart, S.H., Hofacker, I.L. & Stadler, P.F. Local RNA base pairing probabilities in large sequences. Bioinformatics 22, 614–615 (2006).

    Article  CAS  Google Scholar 

  5. Ding, Y. & Lawrence, C.E. Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond. Nucleic Acids Res. 29, 1034–1046 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Takyar, S., Hickerson, R.P. & Noller, H.F. mRNA helicase activity of the ribosome. Cell 120, 49–58 (2005).

    Article  CAS  Google Scholar 

  8. Doshi, K.J., Cannone, J.J., Cobaugh, C.W. & Gutell, R.R. Evaluation of the suitability of free-energy minimization using nearest-neighbor energy parameters for RNA secondary structure prediction. BMC Bioinformatics 5, 105 (2004).

    Article  Google Scholar 

  9. Haley, B. & Zamore, P.D. Kinetic analysis of the RNAi enzyme complex. Nat. Struct. Mol. Biol. 11, 599–606 (2004).

    Article  CAS  Google Scholar 

  10. Brennecke, J., Stark, A., Russell, R.B. & Cohen, S.M. Principles of microRNA-target recognition. PLoS Biol. 3, e85 (2005).

    Article  Google Scholar 

  11. Doench, J.G. & Sharp, P.A. Specificity of microRNA target selection in translational repression. Genes Dev. 18, 504–511 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. Lai, E.C. Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation. Nat. Genet. 30, 363–364 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Patzel, V. et al. Design of siRNAs producing unstructured guide-RNAs results in improved RNA interference efficiency. Nat. Biotechnol. 23, 1440–1444 (2005).

    Article  CAS  Google Scholar 

  18. Ding, Y. & Lawrence, C.E. A statistical sampling algorithm for RNA secondary structure prediction. Nucleic Acids Res. 31, 7280–7301 (2003).

    Article  CAS  Google Scholar 

  19. Yu, Y. & Sato, J.D. MAP kinases, phosphatidylinositol 3-kinase, and p70 S6 kinase mediate the mitogenic response of human endothelial cells to vascular endothelial growth factor. J. Cell. Physiol. 178, 235–246 (1999).

    Article  CAS  Google Scholar 

  20. Ladunga, I. More complete gene silencing by fewer siRNAs: transparent optimized design and biophysical signature. Nucleic Acids Res. 35, 433–440 (2007).

    Article  CAS  Google Scholar 

  21. Lu, Z. & Mathews, D.H. Efficient siRNA selection using hybridization dynamic. Nucleic Acids Res. 36, 640–647 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank A. Khvorova for critical discussions during the project as well as Dharmacon Inc. for providing siRNA knockdown data. H.T. is supported by Siemens and the Wiener Wissenschafts-, Technologie-, und Forschungsfonds. S.L.A. is funded by the Austrian Science Fund FWF through WK001. J.M. is a Junior Group Leader at IMBA, the Institute of Molecular Biotechnology supported by the Austrian Academy of Sciences. Funding by the Austrian Government's GEN-AU is acknowledged by R.S., J.M. and I.L.H. We thank the members of the Hofacker, Schroeder and Martinez labs for encouragement, helpful discussions and comments on the manuscript.

Author information

Author notes

  1. Stefan L Ameres

    Present address: Present address: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605-2324, USA.,

  2. Hakim Tafer, Stefan L Ameres and Gregor Obernosterer: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Theoretical Biochemistry (TBI), University of Vienna, Währingerstraße 17, Vienna, 1090, Austria

    Hakim Tafer & Ivo L Hofacker

  2. Max F. Perutz Laboratories (MFPL), University of Vienna, Dr. Bohr-Gasse 9/5, Vienna, 1030, Austria

    Stefan L Ameres & Renée Schroeder

  3. Institute of Molecular Biotechnology (IMBA), Austrian Academy of Sciences, Dr. Bohr-Gasse 3, Vienna, 1030, Austria

    Gregor Obernosterer, Christoph A Gebeshuber & Javier Martinez

Authors
  1. Hakim Tafer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan L Ameres
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gregor Obernosterer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christoph A Gebeshuber
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Renée Schroeder
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Javier Martinez
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ivo L Hofacker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.T., S.L.A. and G.O. initiated research, designed and performed the experiments and analyzed data. I.L.H. designed algorithms, supervised implementation and analysis. C.A.G. performed western blot analysis. R.S. supervised experimental work and analysis. J.M. supervised experimental work and analysis and procured access to the Dharmacon data. All authors contributed to the writing of the manuscript.

Corresponding authors

Correspondence to Javier Martinez or Ivo L Hofacker.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Tables 1–4 (PDF 1299 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tafer, H., Ameres, S., Obernosterer, G. et al. The impact of target site accessibility on the design of effective siRNAs. Nat Biotechnol 26, 578–583 (2008). https://doi.org/10.1038/nbt1404

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1404

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing