4'-C-Cholesterol/Pyridyl-2'-O-Methyl Uridine-Functionalized siRNA Enhances Stability and Carrier-Free Gene Silencing.

Abstract

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.