Faculty of Science, University of Ontario Institute of Technology, Oshawa, Canada.
Nucleic Acid Ther. 2020 Aug;30(4):229-236. doi: 10.1089/nat.2020.0848. Epub 2020 Mar 12.
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.
小干扰 RNA（siRNA）通过 RNA 干扰（RNAi）机制实现高效基因沉默。RNAi 机制依赖于一种 RNA 指导的核酸内切酶，Argonaute-2（Ago2），它优先从 siRNA 双链体中选择一条单链。所选链与 RNA 靶链之间的互补性导致通过切割进行沉默。美国食品和药物管理局最近批准了两种 siRNA 药物，这重新点燃了人们对 RNAi 治疗的乐观情绪。尽管该领域最近取得了成功，但脱靶效应仍然是一个主要关注点；然而，化学修饰已显示出减轻一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测定法，该方法与几种现有的 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 显著改善了链选择。另一方面，当叶酸修饰的 siRNA 放在 3'末端时，对链选择的改善最佳。这项研究强调了开发和利用一种方便的方法来评估新型化学修饰对 siRNA 特定链基因沉默的影响。
