Structural and Functional Insights Into ( S )‐5′‐ C ‐Guanidinopropyl Uridine‐Modified Oligonucleotides With 2′‐ O

Guanidinium moieties are important structural motifs for improving the physicochemical and biological properties of oligonucleotides. Here, two novel uridine analogs, ( S )‐5′‐ C ‐guanidinopropyl‐2′‐ O ‐methyluridine (5′‐Gp‐2′‐MoU, 2 ) and ( S )‐5′‐ C ‐guanidinopropyl‐2′‐fluorouridine (5′‐Gp‐2′‐FU, 3 ), were synthesized and incorporated into RNA oligonucleotides. Thermal denaturation studies revealed that single incorporation of 2 slightly reduced duplex stability, whereas 3 produced modest stabilization; however, multiple incorporations markedly decreased melting temperatures, indicating cumulative destabilization. Thermodynamic analyses showed that this effect was primarily enthalpy‐driven and partially compensated by favorable entropy contributions. Circular dichroism spectroscopy confirmed preservation of the A‐form helical structure, although slight reductions in negative band intensity suggested localized conformational perturbations. siRNAs containing terminal or overhang modifications retained typical A‐form signatures without detectable spectral changes. Mismatch discrimination studies demonstrated enhanced sequence selectivity, particularly against U:U and U:G mismatches. Serum stability assays further revealed substantially improved nuclease resistance, with 5′‐Gp‐2′‐MoU ( 2 ) exhibiting the highest stability, comparable to the previously reported 5′‐Ap‐2′‐MoU ( 1 ). Moreover, KNTC2 ‐targeting siRNAs containing these modifications maintained potent gene‐silencing activity in HCT116 cells under lipofection conditions, demonstrating compatibility with RNA‐induced silencing complex (RISC) function. These findings identify 5′‐guanidinopropyl‐modified uridine analogs as promising nucleoside modifications for improving the stability and therapeutic potential of RNA‐based therapeutics.