posted on 2024-02-23, 20:06authored bySumit Gangopadhyay, Gourav Das, Shalini Gupta, Atanu Ghosh, Siddharam Shivappa Bagale, Pritam Kumar Roy, Mahitosh Mandal, S. Harikrishna, Surajit Sinha, Kiran R. Gore
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.