Thermodynamic Stabilities in the siRNA Seed and Non- seed Regions Regulate Off-target Effect in Opposite Direction

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA) suppresses the expression of its target mRNA with a perfect complementary sequence. However, siRNA also suppresses the expression of unintended mRNAs with partial sequence complementarities mainly within the siRNA seed region (nucleotides 2–8 from the 5’ end of the guide strand). This mechanism is highly similar to microRNA (miRNA)-mediated RNA silencing, and known as the siRNA-mediated off-target effect. However, in RNAi, the off-target effect is an unwanted side effect that suppresses the expression of the genes other than the target, so it should be avoided. In this study, we investigated the detailed mechanism of off-target effect in order to avoid it efficiently. Previously, we revealed that the off-target effect is induced through thermodynamically stable base-pairing between the siRNA seed region and off-target mRNAs, but not induced through unstable base-pairing. However, we recently discovered that the siRNA seed region is divided into two functional domains: nucleotides 2–5, which are required for off-target effects, and nucleotides 6–8, which are implicated in both RNAi and off-target effects. We studied the most responsible location for the off-target effect in this study by employing a machine learning technique and a random sampling procedure to conduct a comprehensive examination of the thermodynamic properties of all conceivable siRNA subregions.As a result, nucleotides 2–5 in the seed region had the strongest positive association with the off-target effect, while nucleotides 8–14 in the non-seed region had the strongest negative correlation. As a result, the base-pairing stabilities of two separate subregions with opposite effects define the siRNA off-target effect.