Integrated DNA Technologies, Inc., Coralville, IA, USA.
Methods Mol Biol. 2020;2115:23-55. doi: 10.1007/978-1-0716-0290-4_2.
Chemically modified oligonucleotides (ONs) are routinely used in the laboratory to assess gene function, and clinical advances are rapidly progressing as continual efforts are being made to optimize ON efficacy. Over the years, RNA interference (RNAi) has become one of the main tools used to inhibit RNA expression across a wide variety of species. Efforts have been made to improve the exogenous delivery of the double-stranded RNA components to the endogenous intracellular RNAi machinery to direct efficacious degradation of a user-defined RNA target. More recently, synthetic RNA ONs are being used to mimic the bacterial-derived CRISPR/Cas system to direct specific editing of the mammalian genome. Both of these techniques rely on the use of various chemical modifications to the RNA phosphate backbone or sugar in specific positions throughout the ONs to improve the desired biological outcome. Relevant chemical modifications also include conjugated targeting ligands to assist ON delivery to specific cell types. Chemical modifications are most beneficial for therapeutically relevant ONs, as they serve to enhance target binding, increase drug longevity, facilitate cell-specific targeting, improve internalization into productive intracellular compartments, and mitigate both sequence-specific as well as immune-related off-target effects (OTEs). The knowledge gained from years of optimizing RNAi reagents and characterizing the biochemical and biophysical properties of each chemical modification will hopefully accelerate the CRISPR/Cas technology into the clinic, as well as further expand the use of RNAi to treat currently undruggable diseases. This review discusses the most commonly employed chemical modifications in RNAi reagents and CRISPR/Cas guide RNAs and provides an overview of select publications that have demonstrated success in improving ON efficacy and/or mitigating undesired OTEs.
化学修饰的寡核苷酸（ONs）在实验室中常用于评估基因功能，随着不断努力优化 ON 功效，临床进展迅速推进。多年来，RNA 干扰（RNAi）已成为抑制各种物种 RNA 表达的主要工具之一。人们一直在努力改进双链 RNA 成分的外源性递送到内源性细胞内 RNAi 机制，以有效地指导用户定义的 RNA 靶标降解。最近，合成 RNA ONs 被用于模拟细菌衍生的 CRISPR/Cas 系统，以指导哺乳动物基因组的特异性编辑。这两种技术都依赖于 RNA 磷酸骨架或 ON 中特定位置的糖的各种化学修饰，以改善所需的生物学结果。相关的化学修饰还包括共轭靶向配体，以协助 ON 递送到特定的细胞类型。化学修饰对治疗相关的 ONs 最有益，因为它们有助于增强靶标结合、增加药物寿命、促进细胞特异性靶向、改善进入生产性细胞内隔室的内化，并减轻序列特异性和免疫相关的脱靶效应（OTEs）。从多年来优化 RNAi 试剂和表征每种化学修饰的生化和物理特性中获得的知识，有望加速 CRISPR/Cas 技术进入临床，并进一步扩展 RNAi 用于治疗目前无法治疗的疾病的用途。这篇综述讨论了 RNAi 试剂和 CRISPR/Cas 指南 RNA 中最常用的化学修饰，并概述了一些已证明成功提高 ON 功效和/或减轻不良 OTEs 的出版物。
