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.