Off-Target Effects: Disturbing the Silence of RNA interference (RNAi)

Figure 1. Small RNA molecules, miRNA and siRNA, regulate mRNA translation through RNA interference (RNAi). Depending on the level of complementarity between the small RNA molecule and mRNA, the mRNA will be silenced (complete) or translationally repressed (partial). siRNA off-targeting effects occur through partial complementarity of the siRNA with unintended mRNA targets. Since the discovery that siRNAs can enter the RNAi pathway and mediate gene knockdown, researchers have invested considerable effort in optimizing the technology for functional genomics studies. As is the case with most new technologies, the transition of RNAi into a research tool has had its obstacles. Initial problems associated with inconsistent siRNA functionality were addressed by detailed studies that identified functional attributes associated with duplex performance.1,2 More recently, widespread, non-specific effects that complicate the interpretation of data generated from siRNA-mediated knockdown studies have been observed. Off-target effects occur when an siRNA is processed by the RNA-Induced Silencing Complex (RISC) and down-regulates unintended targets. As these changes in gene expression can lead to measurable phenotypes (i.e. false positives) it is of great importance to understand the mechanism behind off-targeting so that strategies can be developed to minimize their effects. Dharmacon has discovered that bioinformatics, novel chemical modifications, and siRNA pooling significantly decrease off-target effects. These three strategies are incorporated into the new ON-TARGETplusTM SMARTpool® siRNA reagents. Non-specific effects resulting from the introduction of siRNA appear to have three separate origins 3: