Sar Santanu, Gupta Shalini, Das Gourav, Sharma Swrajit Nath, K Deepak, Ghosh Atanu, Bagale Siddharam Shivappa, Gangopadhyay Sumit, Sinha Surajit, Gore Kiran R
Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721302, India.
School of Applied and Interdisciplinary Sciences, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India.
Bioconjug Chem. 2025 Jun 18;36(6):1234-1246. doi: 10.1021/acs.bioconjchem.5c00079. Epub 2025 Jun 5.
Chemical modifications and targeted delivery through the conjugation of small molecules have transformed the potential of siRNA-based therapeutics. These advancements address key challenges, such as poor cellular uptake, low bioavailability, and limited metabolic stability, making siRNA delivery more efficient and clinically viable. Cholesterol-conjugated siRNA enables cellular uptake through lipoprotein pathways without transfection agents. In this study, we reported the synthesis of 4'--cholesterol-2'--methyl (4'--chol-2'-OMe) and 4'--methylpyridine-2'--methyl (4'--Mpy-2'-OMe) uridine conjugates via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and their incorporation at the 3'-overhangs of the siRNA duplex. A single incorporation of 4'--chol-2'-OMe or 4'--Mpy-2'-OMe uridine marginally increased the stability of the siRNA duplex. In the nuclease resistance assay, 4'--Mpy-2'-OMe modification at the penultimate position of the 3'-end of poly dT showed significant resistance against snake venom phosphodiesterase (SVPD), 3'-specific exonucleases. Gene silencing activity using anti- siRNA exhibited enhanced gene silencing activity when a single modification was incorporated at the 3'-overhang of the passenger strand. Similarly, 4'--Mpy-2'-OMe modification at the 3'-overhang of the passenger strand in anti--2 siRNA showed compatibility to RISC assembly and exhibited effective gene silencing against the endogenous gene. A molecular modeling study illustrated that the 4'--Mpy-2'-OMe uridine at the 3'-overhang of the guide strand shows minimal interaction with the PAZ domain of the hAgo2 protein. The dual incorporation of cholesterol modifications at the 3'-overhang of both strands resulted in 68% and 93% reductions in luciferase expression at 1000 nM concentration after 48 and 96 h, respectively, in a carrier-free system. This study demonstrated that C4'-cholesterol conjugation provides effective cellular uptake, high nuclease resistance, and prolonged silencing activity in carrier-free mode.
通过小分子偶联实现的化学修饰和靶向递送改变了基于小干扰RNA（siRNA）的治疗方法的潜力。这些进展解决了关键挑战，如细胞摄取差、生物利用度低和代谢稳定性有限等问题，使siRNA递送更高效且具有临床可行性。胆固醇偶联的siRNA能够通过脂蛋白途径实现细胞摄取，无需转染试剂。在本研究中，我们报道了通过铜（I）催化的叠氮化物-炔烃环加成反应（CuAAC）合成4'-胆固醇-2'-甲基（4'-chol-2'-OMe）和4'-甲基吡啶-2'-甲基（4'-Mpy-2'-OMe）尿苷缀合物，并将它们掺入siRNA双链体的3'端突出端。单个4'-chol-2'-OMe或4'-Mpy-2'-OMe尿苷的掺入略微提高了siRNA双链体的稳定性。在核酸酶抗性试验中，聚dT的3'端倒数第二位的4'-Mpy-2'-OMe修饰对蛇毒磷酸二酯酶（SVPD，一种3'特异性核酸外切酶）表现出显著抗性。当在过客链的3'端突出端进行单个修饰时，使用抗siRNA的基因沉默活性表现出增强的基因沉默活性。同样，抗-2 siRNA中过客链3'端突出端的4'-Mpy-2'-OMe修饰与RNA诱导沉默复合体（RISC）组装兼容，并对内源基因表现出有效的基因沉默作用。一项分子建模研究表明，引导链3'端突出端的4'-Mpy-2'-OMe尿苷与hAgo2蛋白的PAZ结构域相互作用最小。在无载体系统中，在两条链的3'端突出端双重掺入胆固醇修饰分别在48小时和96小时后，在1000 nM浓度下使荧光素酶表达降低了68%和93%。本研究表明，C4'-胆固醇偶联在无载体模式下提供了有效的细胞摄取、高核酸酶抗性和延长的沉默活性。
