3′‐C‐Extended TNA: De Novo Synthesis, Enhanced Exonuclease Resistance, and Functional siRNA 3′‐Overhang Modifications Without Compromising Gene Silencing

Abstract The 3′‐overhang of siRNAs is crucial for RISC assembly and stability, yet it is susceptible to nuclease degradation, significantly limiting its therapeutic applications. To address this challenge, we developed 3′‐C‐extended threose nucleic acid (TNA) analogs (ApioNA and BpioNA) using an asymmetric aldol synthesis strategy. Our results demonstrate that the BpioNA modification provides exceptional nuclease resistance, exhibiting a 60‐fold increase in half‐life compared to dT, while maintaining full gene silencing activity. Structural modeling revealed that these novel analogs preserve critical interactions with the Ago2 PAZ domain through adaptive binding geometries. This work not only addresses the stability‐activity trade‐off in siRNA design but also provides fundamental insights into the structure‐activity relationships (SARs) at the 3′‐terminus, paving the way for the development of durable RNAi therapeutics.