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.