2'- O -Alkyl- N 3 -Methyluridine Functionalized Passenger Strand Improves RNAi Activity by Modulating the Thermal Stability.

Herein, we have demonstrated that the siRNA activity could be enhanced by incorporating the guide strand in the RISC complex through thermodynamic asymmetry caused by m 3 U-based destabilizing modifications. A nuclease stability study revealed that 2'-OMe-m 3 U and 2'-OEt-m 3 U modifications slightly improved the half-lives of siRNA strands in human serum. In the in vitro gene silencing assay, 2'-OMe-m 3 U modification at the 3'-overhang and cleavage site of the passenger strand in anti-renilla and anti-Bcl-2 siRNA duplexes were well-tolerated and exhibited improved gene silencing activity. However, gene silencing activity was attenuated when these modifications were incorporated at position 3 in the seed region of the antisense strand. The molecular modeling studies using these modifications at the seed region with the MID domain of hAGO2 explained that the 2'-alkoxy group makes steric interactions with the amino acid residues of the hAGO2 protein.