Steric Protection of Phosphodiester Linkages by 5′-Geminal Substitution Enables Site-Selective Phosphorothioate Reduction in Gapmer-Type Antisense Oligonucleotides
4 affiliations
- 1Graduate School of Pharmaceutical Sciences, University of Osaka, 1-6 Yamadaoka, Suita, Osaka, Japan, 565-0871
- 2National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki, Kanagawa, Japan, 210-9501
- 3National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8, Saito-Asagi, Ibaraki, Osaka, Japan, 567-0085
- 4Institute for Open and Transdisciplinary Research Initiatives, University of Osaka, 1-1 Yamadaoka, Suita, Osaka, Japan, 565-0871
Abstract
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.
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BibTeX
@misc{yamaguchi2026steric,
title = {{Steric Protection of Phosphodiester Linkages by 5′-Geminal Substitution Enables Site-Selective Phosphorothioate Reduction in Gapmer-Type Antisense Oligonucleotides}},
author = {Takao Yamaguchi and Takaki Habuchi and Takaya Sugiura and Go Kato and Tokuyuki Yoshida and Kiyomi Sasaki and Kazuki Asada and Md Ariful Islam and Kenta Ishida and Kotomi Aso and Harumi Yamaguma and Yuuya Kasahara and Takao Inoue and Satoshi Obika},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15011106/v1},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15011106/v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Steric Protection of Phosphodiester Linkages by 5′-Geminal Substitution Enables Site-Selective Phosphorothioate Reduction in Gapmer-Type Antisense Oligonucleotides
AU - Takao Yamaguchi
AU - Takaki Habuchi
AU - Takaya Sugiura
AU - Go Kato
AU - Tokuyuki Yoshida
AU - Kiyomi Sasaki
AU - Kazuki Asada
AU - Md Ariful Islam
AU - Kenta Ishida
AU - Kotomi Aso
AU - Harumi Yamaguma
AU - Yuuya Kasahara
AU - Takao Inoue
AU - Satoshi Obika
PY - 2026
DA - 2026/10/27
PB - ChemRxiv
DO - 10.26434/chemrxiv.15011106/v1
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15011106/v1
AB - 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.
ER -