A 4'-Thioribouridine Modification Mimics 2'-O-Methoxyethyl in ASO-Like 20-Mers Targeting PTEN Knockdown

Gapmer antisense oligonucleotides (ASOs) represent a clinically validated therapeutic platform, with a 2'-O-methoxyethyl modification commonplace within the flanking regions. An alternative sugar modification, incorporation of 4'-thioribose, has demonstrated enhanced nuclease resistance in siRNAs and aptamers, but incorporation of this motif into gapmer architectures remains underexplored. Herein, we report the synthesis of a 4'-thiouridine phosphoramidite building block and its incorporation into gapmer ASOs targeting PTEN. Evaluation of sequences containing 4'-thiouridine in the flanking regions (compared with 2'-O-methoxyethyl) and varying phosphorothioate linkage composition demonstrates that the modified gapmers maintain equivalent knockdown efficacy under transfection and gymnotic delivery. These findings indicate 4'-thioribose as an alternative sugar modification for wider exploration within gapmer ASOs.