Abstract
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.
Highlights
Despite immense progress in gene editing technologies such as CRISPR/Cas9 [1,2], transient small interfering RNAs (siRNAs) transfection remains a powerful technique for perturbing gene function in the lab
A full understanding of the unintended side-effects of siRNA transfections is important in the context of therapeutic applications, or in quantitative genetics experiments
Our cotransfection experiments suggest that competition between co-transfected siRNAs may reduce the efficacy of any individual siRNA in a given experiment, the results of double-knockdown epistasis perturbations need to be interpreted with caution
Summary
Despite immense progress in gene editing technologies such as CRISPR/Cas9 [1,2], transient siRNA transfection remains a powerful technique for perturbing gene function in the lab. The siRNAs (short interfering RNAs) bind target mRNAs via sequence complementarity, and interfere post-transcriptionally with gene expression by inducing mRNA degradation and/or by blocking translation [3,4,5]. To achieve delivery of siRNAs into the cell, a number of approaches have been established, including expression from within the genome as short hair-pin precursor molecules (shRNA), or transfection of synthetic mixtures of double-stranded RNA molecules derived from longer precursors (esiRNA). The most widely used delivery approach relies on commercially available libraries consisting of defined, double-stranded siRNA oligonucleotides (oligos). These oligos are designed to uniquely bind to a sin-
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