Asymmetric siRNA: New Strategy to Improve Specificity and Reduce Off-Target Gene Expression

Abstract

Small interfering RNAs (siRNAs) have been used extensively in reverse genetic research, and many have made their way into clinical trials. The most widely used siRNA structure consists of double-stranded RNA with 19 base pairs and 2-nucleotide overhangs at the 3'-end of both strands (19 + 2). Although widely used, this symmetric structure bears inherent disadvantages in both research and clinical applications. One of the most common caveats is the off-target effect leading to adverse effects in clinical application. In the current study, using C-C chemokine receptor (CCR5) as a target, we have shown that 19 + 2 siRNA could still cause considerable global off-target effects regardless of rational design based on its thermodynamic asymmetry. However, we demonstrated that structurally asymmetric siRNA targeting CCR5 could be adopted to improve the strand specificity and greatly reduce the off-target effects without significantly compromising its on-target effects. Data from microarray analysis suggest that an unidentified mechanism resulting in global gene down-regulation might be avoided through strand shortening. Taken together, our work suggested a promising and simple way to improve strand specificity and overcome the off-target gene-expression effects without introducing more complications while retaining the efficacy of siRNA.

Knockdown of glucose-regulated protein 78/binding immunoglobulin heavy chain protein expression by asymmetric small interfering RNA induces apoptosis in prostate cancer cells and attenuates migratory capability

“…They showed that asiRNA is superior to symmetrical siRNA for the purpose of gene silencing. A number of other studies have since confirmed this (18,(21)(22)(23). In addition to more efficient gene silencing, asiRNA typically causes fewer unpredictable off-target effects and produces a longer duration of knockdown compared with conventional symmetric siRNA (18,19,(21)(22)(23).…”

siRNA and RNAi optimization

“…Therefore, passenger shortening can be an intriguing approach to the elimination of passenger strand-mediated silencing. Asymmetric shorter duplex siRNA (asiRNA), asymmetric siRNA (aiRNA) and blunt end siRNA 61,[75][76][77] have exhibited silencing ability comparable to canonical siRNA structure and less siRNA-mediated off-target effects.…”

Strategic Applications of Gene Expression: From Drug Discovery/Development to Bedside

“…Many strategies have been adopted to improve RNAi specificity in order to reduce off-target gene expression and to reduce immune stimulation. For example, 2′-O-methyl ribosyl substitution at position 2 in the guide siRNA strand and structurally asymmetric siRNA design could be adopted to achieve improved siRNA specificity; in addition, Fucini et al showed that 2′-fluoro modification of adenosine significantly reduced cytokine induction by siRNA in human PBMC (45)(46)(47). Gene expression technologies have been the most widely used technology to monitor RNAi-induced off-target effects.…”