DNA-modified siRNA-dependent gene silencing with reduced off-target effect is induced through a pathway parallel to that for siRNA-mediated RNA interference

RNA interference (RNAi) is mediated by 21 nucleotides of short interfering RNA (siRNA) through sequence-specific cleavage of the cognate transcript. The position-dependent function of ribonucleotide residues in siRNA was analyzed by systematic DNA substitution. The results indicated that eight nucleotides from the 5´ end of the guide strand and its complementary sequence are replaceable with DNA counterparts without any substantial loss of gene silencing activity. However, the remaining duplex including the 3´ end of the guide strand could not be replaced with DNA, probably because of binding of RNA-binding proteins such as Argonaute and TRBP2. In addition, due to the reduced stability of a DNA-RNA hybrid other than the RNA duplex, previous studies have reported that the guide strand of the DNA-modified siRNA was, in most cases, incapable of exerting unintended off-target gene silencing (Ui-Tei et al., Nucleic Acids Res. 36, 2136-2151, 2008). Argonaute and TRBP2 were found to be necessary for inducing both nonmodified and DNA-modified siRNA-mediated gene silencing. Although the major target cleavage sites by nonmodified and DNA-modified siRNAs were identical to each other, transcript cleavage at minor sites was prevented by the presence of the DNA arm. Unlike the 5´ end of the nonmodified-siRNA guide strand, the 5´ end of the guide strand of DNA-modified siRNA appeared dispensable for seed positioning on the Argonaute surface.