Institute of Molecular Life Sciences, University of Zurich, CH-8057 Zurich, Switzerland.
Swiss Institute of Bioinformatics, University of Zurich, CH-8057 Zurich, Switzerland.
Nucleic Acids Res. 2018 Oct 12;46(18):9309-9320. doi: 10.1093/nar/gky798.
Perturbation of gene expression by means of synthetic small interfering RNAs (siRNAs) is a powerful way to uncover gene function. However, siRNA technology suffers from sequence-specific off-target effects and from limitations in knock-down efficiency. In this study, we assess a further problem: unintended effects of siRNA transfections on cellular fitness/proliferation. We show that the nucleotide compositions of siRNAs at specific positions have reproducible growth-restricting effects on mammalian cells in culture. This is likely distinct from hybridization-dependent off-target effects, since each nucleotide residue is seen to be acting independently and additively. The effect is robust and reproducible across different siRNA libraries and also across various cell lines, including human and mouse cells. Analyzing the growth inhibition patterns in correlation to the nucleotide sequence of the siRNAs allowed us to build a predictor that can estimate growth-restricting effects for any arbitrary siRNA sequence. Competition experiments with co-transfected siRNAs further suggest that the growth-restricting effects might be linked to an oversaturation of the cellular miRNA machinery, thus disrupting endogenous miRNA functions at large. We caution that competition between siRNA molecules could complicate the interpretation of double-knockdown or epistasis experiments, and potential interactions with endogenous miRNAs can be a factor when assaying cell growth or viability phenotypes.
通过合成的小干扰 RNA（siRNA）来干扰基因表达是一种揭示基因功能的有效方法。然而，siRNA 技术受到序列特异性的脱靶效应和敲低效率的限制。在本研究中，我们评估了另一个问题：siRNA 转染对细胞适应性/增殖的非预期影响。我们表明，siRNA 在特定位置的核苷酸组成对培养中的哺乳动物细胞具有可重复的生长限制作用。这可能与依赖杂交的脱靶效应不同，因为每个核苷酸残基似乎独立且累加地发挥作用。这种效应在不同的 siRNA 文库和不同的细胞系（包括人和鼠细胞）中都是稳健且可重复的。通过分析与 siRNA 核苷酸序列的相关性，我们构建了一个预测器，可以估计任何任意 siRNA 序列的生长限制作用。与共转染的 siRNA 的竞争实验进一步表明，生长限制作用可能与细胞 miRNA 机制的过饱和有关，从而破坏了大量内源性 miRNA 的功能。我们提醒注意，siRNA 分子之间的竞争可能会使双敲除或上位性实验的解释复杂化，并且在检测细胞生长或活力表型时，与内源性 miRNA 的潜在相互作用可能是一个因素。
