5'-end uridine-derived base modification for siRNAs potency reinforcement.

Small interfering RNAs (siRNAs), bound by Argonaute, efficiently cleave target mRNAs through full complementary base pairing, achieving potent gene silencing. While chemical modifications are widely employed to improve siRNA stability and binding affinity, modifying the 5'-end nucleotide of the guide strand remains a primary strategy for enhancing potency. In this study, we performed a structural analysis of the 5'-end binding pocket of the RISC complex to design a novel series of uridine-derived modifications and screen a library including 001U-018U and vs30U as candidates. We further tested the 001 ~ 003 μm-modified siRNA binding activity via in vitro RNA-induced silencing complex (RISC) loading in HepG2 cells and in vivo RISC loading in mice. These function valiation results indicated a significant improvement in gene silencing activity for the modified siRNAs compared to unmodified control. Additionally, preliminarily safety evaluations demonstrated the modified nucleoside carry no detectable cytotoxicity or genomic insertion risks. Our findings expand the chemical space of siRNA modification and provide new insights into the design of high-potency siRNA drugs.