4'- C-Acetamidomethyl-2'- O-methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs

  • J Org Chem. 2024 Mar 15;89(6):3747-3768. doi: 10.1021/acs.joc.3c02506.
Sumit Gangopadhyay  1 ,  Gourav Das  1 ,  Shalini Gupta  2 ,  Atanu Ghosh  2 ,  Siddharam Shivappa Bagale  3 ,  Pritam Kumar Roy  4 ,  Mahitosh Mandal  4 ,  S Harikrishna  5 ,  Surajit Sinha  2 ,  Kiran R Gore  1
Affiliations
  • 1. Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India.
  • 2. School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of Science, Kolkata, Jadavpur 700032, India.
  • 3. Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India.
  • 4. School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India.
  • 5. Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232, United States.
PMID: 38394362 DOI: 10.1021/acs.joc.3c02506
Abstract

In this study, we designed the 4'-C-acetamidomethyl-2'-O-methoxyethyl (4'-C-ACM-2'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'-C-ACM-2'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4'-C-ACM-2'-O-MOE thymidine modification at the penultimate position of the 3'-end of dT20 oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'-O-Me, 2'-O-MOE, and 2'-F. In gene silencing studies, we found that the 4'-C-ACM-2'-O-MOE uridine or thymidine modifications at the 3'-overhang in the passenger strand in combination with two 2'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4'-C-ACM-2'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4'-C-ACM-2'-O-MOE modification at the 3'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4'-C-ACM-2'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.

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