RNAi off-targeting: Light at the end of the tunnel

The broad post-transcriptional gene regulation induced by RNA interference (RNAi) is mediated by microRNAs (miRNAs). These small, highly structured, non-coding RNAs undergo extensive processing in both the nucleus and cytoplasm before partnering with the RNA Induced Silencing Complex (RISC) to target the 3′ UTR of messenger RNAs for degradation and/or translation attenuation. Synthetic 19 base pair (bp) RNA duplexes (small interfering RNAs, siRNAs) can enter RISC directly and induce RNAi-mediated cleavage of targeted transcripts without additional processing. Though this ability has enormous potential, expansion of RNAi technology requires overcoming obstacles associated with siRNA functionality and specificity. Initial problems associated with inconsistent gene knockdown were found to be inherent in the sequence and thermodynamic properties of the siRNA and were addressed by development of algorithms that enabled the selection of highly functional sequences. In contrast, three separate attributes appear to contribute to overall siRNA specificity, thus making precise knockdown a surprisingly onerous problem. Two of the features that contribute to a lack of siRNA specificity were found to be non-specific in nature. As demonstrated by Federov et al (Fedorov et al, 2005), cationic lipids typically used to deliver siRNA can induce broad changes in gene expression profiles. Separately, multiple labs have observed that particular sequence motifs as well as dsRNA lengths can induce broad, non-specific changes in gene expression that have origins in the induction of the interferon response pathway (Hornung et al, 2005; Judge et al, 2005). While these unanticipated effects can be eliminated by adopting stringent siRNA design filters and optimizing lipid concentrations and/or compositions, the most recent challenge, siRNA-mediated off-target effects, has required a more thorough understanding of the mechanism underlying RNAi.