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A protocol for designing siRNAs with high functionality and specificity

Abstract

Effective gene silencing by the RNA interference (RNAi) pathway requires a comprehensive understanding of the elements that influence small interfering RNA (siRNA) functionality and specificity. These include (i) sequence space restrictions that define the boundaries of siRNA targeting, (ii) structural and sequence features required for efficient siRNA performance, (iii) mechanisms that underlie nonspecific gene modulation and (iv) additional features specific to the intended use (i.e., inclusion of native sugar or base chemical modifications for increased stability or specificity, vector design, etc.). Attention to each of these factors enhances siRNA performance and heightens overall confidence in the output of RNAi-mediated functional genomic studies. Here, we provide a detailed protocol explaining the methodologies used for manual and web-based design of siRNAs.

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Figure 1: RNAi mechanism: design elements for each step in the process.
Figure 2: Work flow for siRNA design and selection.

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Acknowledgements

We acknowledge the manufacturing team for Dharmacon RNA products at Thermo Fisher Scientific for siRNA synthesis and Brian Ferguson for help with implementation of the web interface for the Thermo Fisher Scientific siDESIGN Center.

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Correspondence to Anastasia Khvorova.

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Competing interests

All authors are employed by Thermo Fisher scientific, manufacturer of RNAi products.

Supplementary information

Supplementary Table 1

This Excel file provides a tool for generating all sliding-window 19 nucleotide sequences for a given oligonucleotide target. (XLS 10 kb)

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Birmingham, A., Anderson, E., Sullivan, K. et al. A protocol for designing siRNAs with high functionality and specificity. Nat Protoc 2, 2068–2078 (2007). https://doi.org/10.1038/nprot.2007.278

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