Rational siRNA design for RNA interference


Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi)1,2,3,4. RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi5,6. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3′-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.

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Figure 1: The test panel of 180 siRNAs, targeting firefly luciferase (left) and human cyclophilin B (right).
Figure 2: Development of a multicomponent algorithm.
Figure 3: Validation of the multi-component rational design algorithm.

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We would like to acknowledge Jason Spellberg and Julia Kendall for assistance with manuscript preparation and Carl Novina and Alexey Wolfson for helpful discussions. This work was supported in part by the National Science Foundation under grant no. 0320480.

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Correspondence to Anastasia Khvorova.

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Authors are employed by Dharmacon, which is involved in marketing RNAi-based technologies.

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Reynolds, A., Leake, D., Boese, Q. et al. Rational siRNA design for RNA interference. Nat Biotechnol 22, 326–330 (2004). https://doi.org/10.1038/nbt936

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