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

In this study, we designed the 4'--acetamidomethyl-2'--methoxyethyl (4'--ACM-2'--MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4'--ACM-2'--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'--ACM-2'--MOE thymidine modification at the penultimate position of the 3'-end of dT oligonucleotides in the presence of 3'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2'--Me, 2'--MOE, and 2'-F. In gene silencing studies, we found that the 4'--ACM-2'--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'--ACM-2'--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'--ACM-2'--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'--ACM-2'--MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.