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

[Image: see text] 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.