Structural and Functional Insights Into (S)-5'-C-Guanidinopropyl Uridine-Modified Oligonucleotides With 2'-O-Methyl...

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.