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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.