siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
siRNA-mediated off-target gene silencing triggered by a 7 nt complementation.
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DOI：
10.1093/nar/gki762
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发表时间：
2005
影响因子：
14.9
通讯作者：
Shen, Y
中科院分区：
文献类型：
--
作者：
Lin, XY;Ruan, X;Anderson, MG;McDowell, JA;Kroeger, PE;Fesik, SW;Shen, Y
越来越多的证据表明，siRNA可以通过不同的机制产生脱靶效应。然而，在大规模siRNA文库筛选的背景下，脱靶基因调控对表型诱导以及相应地对数据解释的全面影响尚未报道。在这里，我们报告了在一个大规模的敲除实验中观察到的脱靶基因沉默效应，该实验旨在鉴定HIF-1通路的新型调节因子。来自我们筛选的所有三个“最高命中”已被证明是由脱靶基因沉默引起的。发现三个“siRNA命中”中的两个通过存在于hif-1α mRNA和siRNA中的7 nt基序AGGCAGT直接触发hif-1α mRNA的下调。进一步的分析表明，通过该7 nt基序产生脱靶基因沉默取决于靶mRNA的特征，包括互补区周围的序列背景、互补区在mRNA中的位置和互补区的拷贝数。有趣的是，针对hif-1α的脱靶siRNA也显示出触发mRNA降解的高概率，其他基因在3′-非翻译区具有多个AGGCAGT基序拷贝。从这项研究中吸取的经验教训将是一个宝贵的资产，以帮助设计siRNA与更严格的目标选择性和改善“命中后续”的战略，为未来的大规模敲除实验。
A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.
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DOI：
10.1073/pnas.1630797100
发表时间：
2003-08-19
影响因子：
11.1
作者：
Zeng, Y;Yi, R;Cullen, BR
通讯作者：
Cullen, BR
影响因子：
46.9
作者：
Jackson, AL;Bartz, SR;Linsley, PS
通讯作者：
Linsley, PS
影响因子：
64.8
作者：
Brummelkamp, TR;Nijman, SMB;Bernards, R
通讯作者：
Bernards, R
影响因子：
64.5
作者：
Lewis, BP;Shih, IH;Burge, CB
通讯作者：
Burge, CB
影响因子：
64.5
作者：
Schwarz, DS;Hutvágner, G;Zamore, PD
通讯作者：
Zamore, PD