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'-C-cholesterol-2'-O-methyl (4'-C-chol-2'-OMe) and 4'-C-methylpyridine-2'-O-methyl (4'-C-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'-C-chol-2'-OMe or 4'-C-Mpy-2'-OMe uridine marginally increased the stability of the siRNA duplex. In the nuclease resistance assay, 4'-C-Mpy-2'-OMe modification at the penultimate position of the 3'-end of poly dT₂₀ showed significant resistance against snake venom phosphodiesterase (SVPD), 3'-specific exonucleases. Gene silencing activity using anti-Renilla siRNA exhibited enhanced gene silencing activity when a single modification was incorporated at the 3'-overhang of the passenger strand. Similarly, 4'-C-Mpy-2'-OMe modification at the 3'-overhang of the passenger strand in anti-Bcl-2 siRNA showed compatibility to RISC assembly and exhibited effective gene silencing against the endogenous Bcl-2 gene. A molecular modeling study illustrated that the 4'-C-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 Renilla 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.

化学修饰和通过小分子偶联实现的靶向递送已经变革了基于siRNA的治疗潜力。这些进展解决了诸如细胞摄取不良、生物利用度低以及代谢稳定性差等关键挑战，使siRNA递送更加高效并具有临床可行性。含有胆固醇的siRNA能够通过脂蛋白途径在没有转染剂的情况下实现细胞内吞。在这项研究中，我们报道了4'-C-胆固醇-2'-O-甲基（4'-C-chol-2'-OMe）和4'-C-甲基吡啶-2'-O-甲基（4'-C-Mpy-2'-OMe）尿苷缀合物的合成方法，并通过铜(I)催化的叠氮-炔烃环加成反应（CuAAC）将其在siRNA双链体的3'突出部分进行引入。单一地引入4'-C-chol-2'-OMe或4'-C-Mpy-2'-OMe尿苷对siRNA双链体的稳定性仅略有提高。在核酸酶抗性实验中，当4'-C-Mpy-2'-OMe修饰位于poly dT₂₀ 3'端倒数第二个位置时，其对蛇毒磷酸二酯酶（SVPD）和3'-特异性外切核酸酶显示出显著的抵抗能力。使用anti-Renilla siRNA进行基因沉默活性测试时，在乘客链的3'突出部分引入单一修饰后表现出增强的基因沉默效果。类似地，当在anti-Bcl-2 siRNA的乘客链的3'突出部分进行4'-C-Mpy-2'-OMe修饰后显示出与RISC组装的兼容性，并对内源性的Bcl-2基因表现出了有效的基因沉默作用。分子模拟研究表明，在导向链的3'突出部分含有4'-C-Mpy-2'-OMe尿苷与hAgo2蛋白的PAZ结构域之间存在最小的相互作用。在两股双链体的3'突出部分进行胆固醇修饰的双重引入后，无载体系统中Renilla荧光素酶表达分别在给药48小时和96小时后的1000 nM浓度时降低了68%和93%。本研究证明了C4'-胆固醇偶联能够提供有效的细胞摄取、高核酸酶抗性以及非载体制剂下的长时间沉默效果。