Effect of target secondary structure on RNAi efficiency
Effect of target secondary structure on RNAi efficiency
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作者：
Shao, Yu;Chan, Chi Yu;Ding, Ye
由小干扰RNA （sirna）或短发夹RNA （shRNAs）介导的RNA干扰（RNAi）已成为基因敲低研究的有力工具。然而，基因敲除的水平差异很大。在这里，我们研究了靶标破坏能量（一种新的靶标可及性测量方法）的影响，以及其他可能影响RNAi效率的参数。根据Sfold程序预测的目标二级结构，目标破坏能表示局部改变目标结构以允许siRNA引导链结合目标的自由能成本。在针对103个内源性人类基因的100个siRNA和101个shrna的分析中，我们发现破坏能量是RNAi活性的重要决定因素，siRNA双工不对称的不对称性对于促进rna诱导沉默复合物（RISC）的组装是重要的。我们估计，目标可及性和双工不对称可以显著提高目标击倒水平，分别提高近40%和26%。在RNAi途径中，RISC组装先于siRNA引导链结合靶标。因此，我们的研究结果表明，双不对称对RISC组装具有显著的上游影响，而目标可及性对目标识别具有强烈的下游影响。分析结果为改进sirna和shrna的设计提供了标准。
RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. However, the levels of knockdown vary greatly. Here, we examine the effect of target disruption energy, a novel measure of target accessibility, along with other parameters that may affect RNAi efficiency. Based on target secondary structures predicted by the Sfold program, the target disruption energy represents the free energy cost for local alteration of the target structure to allow target binding by the siRNA guide strand. In analyses of 100 siRNAs and 101 shRNAs targeted to 103 endogenous human genes, we find that the disruption energy is an important determinant of RNAi activity and the asymmetry of siRNA duplex asymmetry is important for facilitating the assembly of the RNA-induced silencing complex (RISC). We estimate that target accessibility and duplex asymmetry can improve the target knockdown level significantly by nearly 40% and 26%, respectively. In the RNAi pathway, RISC assembly precedes target binding by the siRNA guide strand. Thus, our findings suggest that duplex asymmetry has significant upstream effect on RISC assembly and target accessibility has strong downstream effect on target recognition. The results of the analyses suggest criteria for improving the design of siRNAs and shRNAs.