The siRNA Off-Target Effect Is Determined by Base-Pairing Stabilities of Two Different Regions with Opposite Effects

In RNA interference (RNAi), small interfering RNA (siRNA) suppresses the expression of its target mRNA with a perfect complementary sequence. In addition, siRNA also suppresses the expression of unintended mRNAs with partially complementary sequences mainly within the siRNA seed region (nucleotides 2–8). This mechanism is highly similar to microRNA (miRNA)-mediated RNA silencing, and known as the siRNA-mediated off-target effect. Previously, we revealed that the off-target effect is induced through stable base-pairing between the siRNA seed region and off-target mRNAs, but not induced through unstable base-pairing. However, in our recent study, we found that the siRNA seed region consists of two functionally different domains: nucleotides 2–5, essential for off-target effects, and nucleotides 6–8, involved in both RNAi and off-target effects. In this study, we investigated the most responsible region for the off-target effect by conducting a comprehensive analysis of the thermodynamic properties of all possible siRNA subregions that involved a machine learning technique using a random sampling procedure. As a result, the thermodynamic stability of nucleotides 2–5 showed the highest positive correlation with the off-target effect, and nucleotides 8–14 showed the most negative correlation. Thus, it is revealed that the siRNA off-target effect is determined by the base-pairing stabilities of two different subregions with opposite effects.