1′-Sugar-Tethered Cholesterol-Modified siRNAs with Variable Linker Lengths for Carrier-Free Delivery and Enhanced RNAi Efficiency

Abstract

Small interfering RNAs (siRNAs) hold significant therapeutic potential but require chemical modifications to achieve efficient delivery and robust RNA interference (RNAi) in target cells. Here, we report a series of newly developed cholesterol-conjugated siRNAs engineered to enable carrier-free cellular uptake and potent gene silencing. Novel 1′-sugar-linked cholesterol derivatives featuring alkyl linkers of 0, 2, 4, or 6 carbons were designed and synthesized. These derivatives (Y1–Y4; Chol-C0, Chol-C2, Chol-C4, and Chol-C6) were conjugated to the 3′ end of the passenger strand to evaluate how linker length affects siRNA duplex stability and RNAi activity. Thermal melting analysis revealed that all cholesterol modifications increased duplex stability, with the Y1 (Chol-C0) conjugate exhibiting the highest T (m), consistent with rigid, linker-free cholesterol positioning at the terminus. X-ray crystallographic studies of Y1-modified oligonucleotides further demonstrated defined cholesterol orientations and extensive sterol–sterol and sterol–nucleobase interactions, providing a structural basis for enhanced duplex stabilization. Functional evaluation in HCT116 colon cancer cells under lipofection-free conditions revealed that cholesterol conjugation markedly improved cellular uptake and gene silencing in a linker-length-dependent manner. The four-carbon linker (Y3: Chol-C4) achieved the most efficient KNTC2 knockdown (∼62% at 200 nM, ∼80% at 500 nM, and ∼93% at 1250 nM), followed closely by the two-carbon linker (Y2: Chol-C2), while shorter (Y1: Chol-C0) or longer (Y4: Chol-C6) linkers exhibited reduced activity. Quantitative IC(50) analysis further supported these findings, with Chol-C4 showing the lowest IC(50) value (141.5 nM), followed by Chol-C2 (188.4 nM), Chol-C6 (265.4 nM), and Chol-C0 (600.5 nM). This study demonstrates that siRNAs bearing 1′-sugar-tethered cholesterol with optimized linker lengths achieve efficient carrier-free delivery and potent RNAi activity, offering significant promise for advancing siRNA-based therapeutics.