Abstract：
In this study, we designed the 4\'-C-acetamidomethyl-2\'-O-methoxyethyl (4\'-C-ACM-2\'-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4\'-C-ACM-2\'-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4\'-C-ACM-2\'-O-MOE thymidine modification at the penultimate position of the 3\'-end of dT20 oligonucleotides in the presence of 3\'-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2\'-O-Me, 2\'-O-MOE, and 2\'-F. In gene silencing studies, we found that the 4\'-C-ACM-2\'-O-MOE uridine or thymidine modifications at the 3\'-overhang in the passenger strand in combination with two 2\'-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3\'-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4\'-C-ACM-2\'-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4\'-C-ACM-2\'-O-MOE modification at the 3\'-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4\'-C-ACM-2\'-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs.
摘要：
在这项研究中,我们设计了4'-C-乙酰氨基甲基-2'-O-甲氧基乙基（4'-C-ACM-2'-O-MOE）尿苷和胸苷修饰，旨在将它们测试为小干扰RNA。热解链研究表明，在DNA双链体中掺入单个4'-C-ACM-2'-O-MOE修饰会降低热稳定性。相比之下,当在DNA:RNA杂交体和siRNA中引入修饰时,观察到热稳定性增加。DNA双链体中的热不稳定归因于不利的熵，这在一定程度上主要由焓因子补偿。在存在3'特异性外切核酸酶的情况下，在dT20寡核苷酸的3'末端的倒数第二个位置进行单个4'-C-ACM-2'-O-MOE胸苷修饰，蛇毒磷酸二酯酶（SVPD），与包括2'-O-Me的单体修饰相比，证明了显着的稳定性，2\'-O-MOE,和2'-F。在基因沉默研究中,我们发现4'-C-ACM-2'-O-MOE尿苷或胸苷修饰与两个2'-F修饰结合在一起显示出优异的RNAi活性。结果表明，双重修饰在过客链的3'端具有良好的耐受性，这反映了更好的siRNA稳定性和沉默活性。有趣的是,4'-C-ACM-2'-O-MOE修饰的siRNA即使在转染后96小时也显示出相当大的基因沉默;它表明我们的修饰可以诱导由于改善的代谢稳定性而延长的基因沉默。分子建模研究表明，在siRNA引导链的3'端引入4'-C-ACM-2'-O-MOE修饰有助于将该链锚定在hAgo2蛋白的PAZ结构域内。总体结果表明，4'-C-ACM-2'-O-MOE尿苷和胸苷修饰是有希望的修饰，以提高稳定性，效力，效力和siRNA的hAgo2结合。
