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While microRNAs (miRNAs) are recognized as playing a critical role in regulating eukaryotic gene expression, both the mechanism by which these small, noncoding RNAs function and the genes they target remain elusive. Previous studies have shown that short, single-stranded 2′-O-methyl-modified oligonucleotides that are complementary to mature microRNA sequences can interact with the miRNA–RISC nucleoprotein complex and weakly inhibit miRNA function. Here we report the identification of secondary structural elements that enhance the potency of these molecules. Incorporation of highly structured, double-stranded flanking regions around the reverse complement core significantly increases inhibitor function and allows for multi-miRNA inhibition at subnanomolar concentrations. The improved functionality of these double-stranded miRNA inhibitors may provide insights into the miRNA mechanism by suggesting the possible importance of such structures in or near endogenous miRNA target sites.

Long (27–29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell type-specific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.