Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries.
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 targets the MVP gene (siMVP) and one siRNA targets 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.
Li
Zhen
Z
Department of Medicine, McGill University, Montréal, Québec, Canada.
Fortin
Yves
Y
Shen
Shi-Hsiang
SH
D002460:Cell Line; D043525:Cytosine Deaminase / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D050939:Gene Fusion; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000633:toxicity; D020394:Vault Ribonucleoprotein Particles / Q000235:genetics; D000099187:Major Vault Protein
D043525:Cytosine Deaminase; D049452:Green Fluorescent Proteins; D034741:RNA, Small Interfering; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein; C485184:enhanced green fluorescent protein