In this study, we explore the effect of a library of 2'-, 4'-, and 2',4'-modified uridine nucleosides and their impact on silencing firefly luciferase and on down-regulated in renal cell carcinoma (DRR) gene targets. The modifications studied were 2'-F-ribose, 2'-F-arabinose, 2'-OMe-ribose, 2'-F,4'-OMe-ribose, 2'-F,4'-OMe-arabinose, and 2'-OMe,4'-F-ribose. We found that 2',4'-modifications are well tolerated within A-form RNA duplexes, leading to virtually no change in melting temperature as assessed by UV thermal melting. The impact of the dual (2',4') modification was assessed by comparing gene silencing ability to 2'- or 4'- (singly) modified siRNA counterparts. siRNAs with (2',4')-modified overhangs generally outperformed the native siRNA as well as siRNAs with a 2'- or 4'-modified overhang, suggesting that 2',4'-modified nucleotides interact favorably with Argonaute protein's PAZ domain. Among the most active siRNAs were those with 2'-F,4'-OMe-ribose or 2'-F,4'-OMe-arabinose at the overhangs. When modifications were placed at both overhangs and internal positions, a duplex with the 2'-F (internal) and 2'-F,4'-OMe (overhang) combination was found to be the most potent, followed by the duplex with 2'-OMe (internal) and 2',4'-diOMe (overhang) modifications. Given the nuclease resistance exhibited by 2',4'-modified siRNAs, particularly when the modification is placed at or near the overhangs, these findings may allow the creation of superior siRNAs for therapy.

中文翻译：

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糖2'，4'-修饰对siRNA双链体基因沉默活性的影响。

在这项研究中，我们探讨了2'-，4'-和2'，4'-修饰的尿苷核苷文库的作用及其对沉默萤火虫荧光素酶的影响以及对肾细胞癌（DRR）基因下调的影响目标。研究的修饰是2'-F-核糖，2'-F-阿拉伯糖，2'-OMe-核糖，2'-F，4'-OMe-核糖，2'-F，4'-OMe-阿拉伯糖和2'-OMe，4'-F-核糖。我们发现2'，4'-修饰在A型RNA双链体中具有很好的耐受性，导致通过UV热熔解评估的解链温度几乎没有变化。通过将基因沉默能力与2'-或4'-（单）修饰的siRNA对应物进行比较，评估了双重（2'，4'）修饰的影响。具有（2'，4' ）-修饰的突出端通常胜过天然siRNA以及具有2'-或4'-修饰的突出端的siRNA，这表明2'，4'-修饰的核苷酸与Argonaute蛋白的PAZ结构域相互作用良好。siRNA中活性最高的是突出端带有2'-F，4'-OMe-核糖或2'-F，4'-OMe-阿拉伯糖的siRNA。当在悬突和内部位置均进行修饰时，发现具有2'-F（内部）和2'-F，4'-OMe（突出）组合的双链体是最有效的，其次是带有2'-F（4'-OMe）的双链体。 '-OMe（内部）和2'，4'-diOMe（突出）修饰。鉴于2'，4'修饰的siRNA表现出的核酸酶抗性，尤其是当修饰位于突出端或突出端附近时，