Institut Pasteur, Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR3523, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France; Sorbonne Université, Collège doctoral, F-75005 Paris, France.
Institut Pasteur, Department of Structural Biology and Chemistry, Laboratory for Bioorganic Chemistry of Nucleic Acids, CNRS UMR3523, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France.
Methods. 2019 May 15;161:64-82. doi: 10.1016/j.ymeth.2019.03.018. Epub 2019 Mar 21.
RNA is often considered as being the vector for the transmission of genetic information from DNA to the protein synthesis machinery. However, besides translation RNA participates in a broad variety of fundamental biological roles such as gene expression and regulation, protein synthesis, and even catalysis of chemical reactions. This variety of function combined with intricate three-dimensional structures and the discovery of over 100 chemical modifications in natural RNAs require chemical methods for the modification of RNAs in order to investigate their mechanism, location, and exact biological roles. In addition, numerous RNA-based tools such as ribozymes, aptamers, or therapeutic oligonucleotides require the presence of additional chemical functionalities to strengthen the nucleosidic backbone against degradation or enhance the desired catalytic or binding properties. Herein, the two main methods for the chemical modification of RNA are presented: solid-phase synthesis using phosphoramidite precursors and the enzymatic polymerization of nucleoside triphosphates. The different synthetic and biochemical steps required for each method are carefully described and recent examples of practical applications based on these two methods are discussed.
RNA 通常被认为是遗传信息从 DNA 传递到蛋白质合成机制的载体。然而，除了翻译，RNA 还参与了广泛的基本生物学功能，如基因表达和调控、蛋白质合成，甚至化学反应的催化。这种多样化的功能与复杂的三维结构以及在天然 RNA 中发现的 100 多种化学修饰相结合，需要对 RNA 进行化学修饰的方法，以研究它们的机制、位置和确切的生物学作用。此外，许多基于 RNA 的工具，如核酶、适体或治疗性寡核苷酸，需要额外的化学功能来增强核碱基对降解的抵抗力或增强所需的催化或结合特性。本文介绍了两种主要的 RNA 化学修饰方法：使用亚磷酰胺前体的固相合成和核苷三磷酸的酶聚合。仔细描述了每种方法所需的不同合成和生化步骤，并讨论了基于这两种方法的最新实际应用实例。
