4'-O-methoxyethyl-2'-deoxy-uridine and cytidine ribonucleotides improve duplex stability, nuclease resistance, and elicit efficient knockdown of Bcl-2 expression

  • Bioorg Med Chem Lett. 2025 Dec 1:128:130329. doi: 10.1016/j.bmcl.2025.130329.
Sumit Gangopadhyay  1 ,  Gourav Das  1 ,  Swrajit Nath Sharma  2 ,  Atanu Ghosh  2 ,  Siddharam Shivappa Bagale  3 ,  Surajit Sinha  2 ,  Kiran R Gore  4
Affiliations
  • 1. Department of Chemistry, Indian Institute of Technology, Kharagpur, West Bengal 721302, India.
  • 2. School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India.
  • 3. Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India.
  • 4. Department of Chemistry, Indian Institute of Technology, Kharagpur, West Bengal 721302, India. Electronic address: [email protected].
PMID: 40633810 DOI: 10.1016/j.bmcl.2025.130329
Abstract

In this study, we report the synthesis of 4'-O-methoxyethyl deoxyuridine and 4'-O-methoxyethyl deoxycytidine phosphoramidites and their incorporation into oligonucleotides. The 3'-exonuclease experiment indicated a significant increase in stability with the incorporation of 4'-O-MOE dU alone (T1/2 = 178 min) and in combination with phosphorothioate modification (T1/2 = 13 h) in dT10-mer. Western blot studies demonstrated that 4'-O-MOE-dU and 4'-O-MOE-dC modifications were well-tolerated when placed at both overhang regions and multiple positions within the passenger strand of anti-Bcl2 siRNAs. Moreover, overhang-modified siRNA duplexes showed no significant change in the IFNα level compared to native siRNA. Molecular modelling studies revealed that the 4'-O-MOE group makes multiple steric interactions with 3'-exonuclease residues, contributing to improved nuclease resistance. Additionally, this modification accommodates well within the PAZ domain, potentially influencing RNAi activity. Overall, the modified deoxynucleotides could enhance the stability and efficacy of nucleic acid drugs.

Keywords
3′-Exonuclease; 4′-O-methoxyethyl modification; RNA interference; hAGO2; siRNA.
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