Small interfering RNAs (siRNAs) enable efficient gene silencing through RNA interference (RNAi) mechanisms. The RNAi machinery relies on an RNA-guided nuclease, Argonaute-2 (Ago2), which preferentially selects a single strand from an siRNA duplex. Complementarity between the selected strand and an RNA target strand leads to silencing through cleavage. The U.S. Food and Drug Administration's recent approval of two siRNA drugs has reignited optimism for RNAi therapeutics. Despite this recent success in the field, off-target effects are still a major concern; however, chemical modifications have shown promise in mitigating some off-target gene silencing. To evaluate the impact of novel chemical modifications on strand selection, we developed a quantitative polymerase chain reaction-based assay that is compatible with several pre-existing siRNA libraries and was used to characterize chemically modified siRNAs. siRNAs bearing azobenzene and propargyl modifications at the central region of the passenger strand significantly improved strand selection. On the other hand, folic acid-modified siRNAs improved strand selection best when placed at the 3′ terminus. This study highlights the development and utility of a convenient method to evaluate the impact that novel chemical modifications have on strand-specific gene silencing of siRNAs.

中文翻译：

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化学修饰对哺乳动物细胞中 siRNA 链选择的影响。

小干扰 RNA (siRNA) 通过 RNA 干扰 (RNAi) 机制实现有效的基因沉默。RNAi 机制依赖于 RNA 引导的核酸酶 Argonaute-2 (Ago2)，它优先从 siRNA 双链体中选择单链。所选链和 RNA 目标链之间的互补性导致通过切割沉默。美国食品和药物管理局最近批准了两种 siRNA 药物，重新点燃了对 RNAi 疗法的乐观情绪。尽管最近在该领域取得了成功，但脱靶效应仍然是一个主要问题。然而，化学修饰已显示出减轻某些脱靶基因沉默的希望。为了评估新型化学修饰对链选择的影响，我们开发了一种基于定量聚合酶链反应的检测方法，该检测方法与多个预先存在的 siRNA 文库兼容，并用于表征化学修饰的 siRNA。在过客链的中心区域带有偶氮苯和炔丙基修饰的 siRNA 显着改善了链的选择。另一方面，当放置在 3' 末端时，叶酸修饰的 siRNA 可以最好地改善链选择。本研究强调了一种评估新型化学修饰对 siRNA 的链特异性基因沉默的影响的便捷方法的开发和实用性。当放置在 3' 末端时，叶酸修饰的 siRNA 可以最好地改善链选择。本研究强调了一种评估新型化学修饰对 siRNA 的链特异性基因沉默的影响的便捷方法的开发和实用性。当放置在 3' 末端时，叶酸修饰的 siRNA 可以最好地改善链选择。本研究强调了一种评估新型化学修饰对 siRNA 的链特异性基因沉默的影响的便捷方法的开发和实用性。