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On the art of identifying effective and specific siRNAs

Abstract

Small interfering RNAs (siRNAs) have been widely exploited for sequence-specific gene knockdown, predominantly to investigate gene function in cultured vertebrate cells, and also hold promise as therapeutic agents. Because not all siRNAs that are cognate to a given target mRNA are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective siRNAs. Furthermore, because target-complementary siRNAs can also target other mRNAs containing sequence segments that are partially complementary to the siRNA, most computational tools include ways to reduce potential off-target effects in the siRNA selection process. Though these methods facilitate selection of functional siRNAs, they do not yet alleviate the need for experimental validation. This perspective provides a practical guide based on current wisdom for selecting siRNAs.

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Figure 1: A scheme for siRNA-mediated gene silencing.
Figure 2: siRNA and target mRNA structures.

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Acknowledgements

We apologize to authors whose works are not cited owing to space limitations. We thank C. Echeverri at Cenix Bioscience for valuable discussion. We also thank M. Landthaler, P. Landgraf, J. Brennecke and C. Rogler for critical reading of the manuscript. Y.P. is supported by the Ruth L. Kirschstein Fellowship from the US National Institutes of Health–National Institute of General Medical Sciences.

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Correspondence to Thomas Tuschl.

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T.T. is a cofounder of Alnylam Therapeuticals and serves on its Scientific Advisory Board.

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Pei, Y., Tuschl, T. On the art of identifying effective and specific siRNAs. Nat Methods 3, 670–676 (2006). https://doi.org/10.1038/nmeth911

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