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.