Herein, we report the synthesis of 2′-O-alkyl/2′-fluoro-N³-methyluridine (2′-O-alkyl/2′-F-m³U) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2′-O-alkyl/2′-F-m³U modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT₂₀ with 2′-O-alkyl-m³U modification exhibited excellent nuclease resistance when incubated with 3′-exonucleases (SVPD) or 5′-exonucleases (PDE-II) as compared to m³U, 2′-F, 2′-OMe modified oligonucleotides. MD simulation studies with RNA tetradecamer duplexes illustrated that the m³U and 2′-O-methyl-m³U modifications reduce the duplex stabilities by disrupting the Watson-Crick hydrogen bonding and base-stacking interactions. Further molecular modelling investigations demonstrated that the 2′-O-propyl-m³U modification exhibits steric interactions with amino acid residues in the active site of 3′- and 5′-exonuclease, leading to enhanced stability. These combined data indicate that the 2′-modified-m³U nucleotides can be used as a promising tool to enhance the stability, silencing efficiency, and drug-like properties of antisense/siRNA-based therapeutics.

在此，我们报道了2'- O-烷基/2'-氟-N ^{3 -}甲基尿苷（2'- O-烷基/2'-Fm ³ U）亚磷酰胺的合成及其在DNA和RNA寡核苷酸中的掺入。双链体结合亲和力和碱基区分研究表明，所有2′- O-烷基/2′-Fm ³ U修饰均显着降低了热稳定性和碱基配对区分能力。^{与 m 3} U, 2 相比，具有 2'- O -烷基-m 3 U修饰的 dT ₂₀在与 3'-核酸外切酶 (SVPD) 或 5'-核酸外切酶 (PDE-II) 一起孵育时表现出优异的核酸^酶抗性'-F，2'-OMe 修饰的寡核苷酸。RNA 十四聚体双链体的 MD 模拟研究表明，m ³ U 和 2′- O -甲基-m ³ U 修饰通过破坏 Watson-Crick 氢键和碱基堆积相互作用来降低双链体稳定性。进一步的分子模型研究表明，2′- O-丙基-m ³ U 修饰与 3′- 和 5′- 核酸外切酶活性位点的氨基酸残基表现出空间相互作用，从而增强了稳定性。这些综合数据表明，2'-修饰-m ³ U 核苷酸可用作增强反义/siRNA 疗法的稳定性、沉默效率和药物样特性的有前途的工具。