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