Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries.
Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries.
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DOI：
10.1093/nar/gkn953
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发表时间：
2009-01
影响因子：
14.9
通讯作者：
Shen, Shi-Hsiang
中科院分区：
文献类型：
--
作者：
Li, Zhen;Fortin, Yves;Shen, Shi-Hsiang
使用高效的小干扰RNA(SiRNAs)可以显著减少剂量依赖的细胞毒性和靶外效应。我们开发了一种遗传正向方法，将胞嘧啶脱氨酶基因与靶标融合，从通过细菌入侵直接进入细胞的RNA干扰(RNAi)文库中可靠地识别高效siRNAs。我们证明了在一个容器中方便地进行两个简单的药物选择周期，在存活细胞中主要富含两个针对MVP基因的siRNA(SiMVP)和一个针对EGFP基因的siRNA(SiEGFP)，这被证明是已报道的最有效的siRNA。此外，从存活细胞中分离的有效siRNAs具有非细胞毒性特征。有趣的是，所鉴定的高效力siMVP的长度可能短至16聚体，而增加其固有序列的长度显著降低了RNAi效力。这些结果表明，目前的方法可以在存活细胞中强有力地发现最有效和无毒的siRNAs，因此通过最小化siRNAs的剂量依赖和序列非特异性副作用，在促进RNAi应用方面具有巨大的潜力。
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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影响因子：
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作者：
Elbashir, SM;Martinez, J;Tuschl, T
通讯作者：
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作者：
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通讯作者：
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DOI：
10.1073/pnas.152327299
发表时间：
2002-07-23
影响因子：
11.1
作者：
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通讯作者：
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