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Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries

From National Research Council Canada

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DOIResolve DOI: https://doi.org/10.1093/nar/gkn953
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Affiliation
  1. National Research Council of Canada. NRC Biotechnology Research Institute
FormatText, Article
Abstract
Use of highly potent small interfering RNAs (siRNAs) can substantially reduce dose-dependent cytotoxic and off-target effects. We developed a genetic forward approach by fusing the cytosine deaminase gene with targets for the robust identification of highly potent siRNAs from RNA interference (RNAi) libraries that were directly delivered into cells via bacterial invasion. We demonstrated that two simple drug selection cycles performed conveniently in a single container predominately enriched two siRNAs targeting the MVP gene (siMVP) and one siRNA targeting the egfp gene (siEGFP) in surviving cells and these proved to be the most effective siRNAs reported. Furthermore, the potent siRNAs isolated from the surviving cells possessed noncellular toxic characteristics. Interestingly, the length of highly potent siMVPs identified could be as short as 16-mer, and increasing the length of their native sequences dramatically reduced RNAi potency. These results suggest that the current approach can robustly discover the most potent and nontoxic siRNAs in the surviving cells, and thus has great potential in facilitating RNAi applications by minimizing the dose-dependent and sequence nonspecific side effects of siRNAs.
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PublisherOxford University Press
In
  • Nucleic Acids Research 37, no. 1, e8.
LanguageEnglish
Peer reviewedYes
NPARC number23004911
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Record identifier3fb6fff0-43ce-4d8b-81a9-745f21f47443
Record created2019-01-21
Record modified2020-05-30
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