We designed and synthesized 3′-C-extended TNA analogs (ApioNA and BpioNA) through asymmetric aldol synthesis. These analogs, modified at the 3′-terminal of oligonucleotides, display significant exonuclease resistance, particularly BpioNA, which has a 60-fold increase in half-life compared to dT. Furthermore, both analogs maintain full gene-silencing efficacy when integrated into the 3′-overhang positions of siRNA, highlighting their potential in nucleic acid therapeutics.

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.