Sahoo Avijit, Gupta Shalini, Das Gourav, Ghosh Atanu, Bagale Siddharam Shivappa, Sinha Surajit, Gore Kiran R
Department of Chemistry, Indian Institute of Technology Kharagpur, West Bengal 721302, India.
School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India.
ACS Med Chem Lett. 2024 Jul 16;15(8):1250-1259. doi: 10.1021/acsmedchemlett.4c00140. eCollection 2024 Aug 8.
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 mU-based destabilizing modifications. A nuclease stability study revealed that 2'-OMe-mU and 2'-OEt-mU modifications slightly improved the half-lives of siRNA strands in human serum. In the gene silencing assay, 2'-OMe-mU 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.
在此，我们已经证明，通过基于mU的去稳定修饰所引起的热力学不对称性，将引导链整合到RNA诱导沉默复合体（RISC）中，可以增强小干扰RNA（siRNA）的活性。一项核酸酶稳定性研究表明，2'-O-甲基-尿苷（2'-OMe-mU）和2'-O-乙基-尿苷（2'-OEt-mU）修饰略微提高了siRNA链在人血清中的半衰期。在基因沉默试验中，抗海肾荧光素酶（anti-renilla）和抗Bcl-2 siRNA双链体中过客链的3'-突出端和切割位点处的2'-OMe-mU修饰耐受性良好，并表现出改善的基因沉默活性。然而，当这些修饰掺入反义链种子区域的第3位时，基因沉默活性减弱。使用这些修饰在种子区域与hAGO2的MID结构域进行的分子建模研究解释说，2'-烷氧基与hAGO2蛋白的氨基酸残基发生空间相互作用。
