Reduced base-base interactions between the DNA seed and RNA target are the major determinants of a significant reduction in the off-target effect due to DNA-seed-containing siRNA

Previously, we showed that DNA replacement of the seed-containing region of class I siRNAs significantly reduced the off-target effect without substantial loss of target gene silencing activity (Ui-Tei et al, Nucleic Acids Res., 36, 2136–2151, 2008). Separately, we also showed that the level of the off-target effect due to non-modified class I siRNA is determined primarily by the calculated melting temperature (Tm) of the seed-target duplex, indicating that, at least in class I-siRNA-mediated gene silencing, Tm or thermodynamic stability of the complex between the siRNA seed and target mRNA is the most important factor determining the degree of off-target effects (Ui-Tei et al., Nucleic Acids Res., 36, 7100–7109, 2008). Here, using non-modified siRNAs other than class I siRNAs and DNA-seed-containing siRNAs, we examined the relationship between the thermodynamic stability of the seed-target duplex and the degree of the off-target effect. We found that the degree of the off-target effects is generally determined primarily by the Tm of the corresponding seed-target duplex, indicating that its thermodynamic stability is the most important general determinant of the degree of the off-target effect in gene silencing.