siRNA Seed Region Is Divided into Two Functionally Different Domains in RNA Interference in Response to 2′-OMe Modifications

In RNA interference (RNAi), small interfering RNA (siRNA) functions to suppress the expression of its target mRNA with perfect sequence complementarity. In a mechanism different from above, siRNA also suppresses unintended mRNAs with partial sequence complementarities, mainly to the siRNA seed region (nucleotides 2-8). This mechanism is largely utilized by microRNAs (miRNAs) and results in siRNA-mediated off-target effects. Thus, the siRNA seed region is considered to be involved in both RNAi and off-target effects. In this study, we revealed that the impact of 2'-O-methyl (2'-OMe) modification is different according to the nucleotide positions. The 2'-OMe modifications of nucleotides 2-5 inhibited off-target effects without affecting on-target RNAi activities. In contrast, 2'-OMe modifications of nucleotides 6-8 increased both RNAi and off-target activities. The computational simulation revealed that the structural change induced by 2'-OMe modifications interrupts base pairing between siRNA and target/off-target mRNAs at nucleotides 2-5 but enhances at nucleotides 6-8. Thus, our results suggest that siRNA seed region consists of two functionally different domains in response to 2'-OMe modifications: nucleotides 2-5 are essential for avoiding off-target effects, and nucleotides 6-8 are involved in the enhancement of both RNAi and off-target activities.