Assessment of RNA interference effectiveness mediated by siRNA sequences and structures in Drosophila S2 cells

Extensive research has clarified the mechanisms of RNA interference (RNAi) in insects. Although double-stranded RNA (dsRNA) effectively induces RNAi in insects, comprehensive studies on how small interfering RNA (siRNA) structural characteristics influence messenger RNA (mRNA) cleavage efficiency are limited. This study systematically examined the impact of diverse sequence and structural modifications on the efficiency of siRNA-mediated knockdown of target genes in Drosophila melanogaster . Our findings indicate that siRNA efficacy drastically decreased at a length of 17 nucleotides (nt), but was restored by extending to 19 base pairs (bp) with random sequences. Additionally, siRNAs with 2-nt overhangs demonstrated greater efficacy compared to blunt-ended structures. We found that the knockdown efficiency varies depending on the secondary structure characteristics of the mRNA region to which siRNA binds. Furthermore, next-generation sequencing was employed to map predicted siRNA distributions to actual dsRNA processing patterns, allowing detailed profiling of cleavage depth and sequence preferences. Collectively, these results enhance our understanding of the relationship between siRNA sequence design parameters and RNAi efficiency in Drosophila , providing a significant advance in siRNA-based RNAi research in insects. This study also offers valuable insights into dsRNA off-target effects.