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.