Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5'-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3'-termini of both strands, the addition of a UNA at the 5'-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.     

siRNA-mediated off-target gene silencing triggered by a 7 nt complementation

A growing body of evidence suggests that siRNA could generate off-target effects through different mechanisms. However, the full impact of off-target gene regulation on phenotypic induction and accordingly on data interpretation in the context of large-scale siRNA library screen has not been reported. Here we report on off-target gene silencing effects observed in a large-scale knockdown experiment designed to identify novel regulators of the HIF-1 pathway. All of the three ‘top hits’ from our screen have been demonstrated to result from off-target gene silencing. Two of the three ‘siRNA hits’ were found to directly trigger down-regulation of hif-1α mRNA through a 7 nt motif, AGGCAGT, that is present in both the hif-1α mRNA and the siRNAs. Further analysis revealed that the generation of off-target gene silencing via this 7 nt motif depends on the characteristics of the target mRNA, including the sequence context surrounding the complementary region, the position of the complementary region in the mRNA and the copy number of the complementary region. Interestingly, the off-target siRNA against hif-1α was also shown to trigger mRNA degradation with high probability of other genes that possess multiple copies of the AGGCAGT motif in the 3′-untranslated region. Lessons learned from this study will be a valuable asset to aid in designing siRNAs with more stringent target selectivity and improving ‘hits-follow-up’ strategies for future large-scale knockdown experiments.