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

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.