Steric Protection of Phosphodiester Linkages by 5′-Geminal Substitution Enables Site-Selective Phosphorothioate Reduction in Gapmer-Type Antisense Oligonucleotides

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Phosphorothioate (PS) linkages are widely incorporated into antisense oligonucleotides (ASOs) to enhance nuclease resistance, but their nonspecific interactions with proteins can contribute to toxicity. Selectively replacing PS linkages with phosphodiester (PO) linkages without compromising ASO activity remains challenging because of the susceptibility of PO linkages to nuclease degradation. Here, we introduce a strategy for site-selective PS reduction based on steric protection of adjacent PO linkages through 5′-geminal substitution. We designed and synthesized 5′-cyclopropylene (5′-cp)- and 5′-dimethyl (5′-diMe)-modified DNAs to investigate this concept. These modifications preserved or caused only a slight reduction in duplex-forming ability toward complementary RNA. Oligonucleotides bearing these modifications at the 3′ terminus exhibited greater resistance to 3′-exonuclease digestion than the corresponding PS-modified oligonucleotides. When incorporated at selected positions within the gap region of model hepatotoxic gapmer ASOs, 5′-cp/PO and 5′diMe/PO motifs enabled replacement of PS linkages while retaining substantial antisense activity and attenuating elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in vivo. These findings establish 5′-geminal substitution as a promising molecular design strategy for protecting selected PO linkages and reducing PS content in ASOs.