Tsiamantas Christos, Otero-Ramirez Manuel E, Suga Hiroaki
Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, Japan.
Methods Mol Biol. 2019;2001:299-315. doi: 10.1007/978-1-4939-9504-2_14.
Flexizymes, highly flexible tRNA aminoacylation ribozymes, have enabled charging of virtually any amino acid (including non-proteogenic ones) onto tRNA molecules. Coupling to a custom-made in vitro translation system, namely the flexible in vitro translation (FIT) system, has unveiled the remarkable tolerance of the ribosome toward molecules, remote from what nature has selected to carry out its elaborate functions. Among the very diverse molecules and chemistries that have been ribosomally incorporated, a plethora of entities capable of mediating intramolecular cyclization have revolutionized the design and discovery of macrocyclic peptides. These macrocyclization reactions (which can be spontaneous, chemical, or enzymatic) have all served as tools for the discovery of peptides with natural-like structures and properties. Coupling of the FIT system and mRNA display techniques, known as the random non-standard peptide integrated discovery (RaPID) system, has in turn allowed for the simultaneous screening of trillions of macrocyclic peptides against challenging biological targets. The macrocyclization methodologies are chosen depending on the structural and functional characteristics of the desired molecule. Thus, they can emanate from the peptide's N-terminus or its side chains, attributing flexibility or rigidity, or even result in the installation of fluorescent probes.
柔性酶，即高度灵活的tRNA氨酰化核酶，能够将几乎任何氨基酸（包括非蛋白质ogenic氨基酸）加载到tRNA分子上。与定制的体外翻译系统，即柔性体外翻译（FIT）系统相结合，揭示了核糖体对分子的显著耐受性，这些分子与自然界选择用于执行其精细功能的分子截然不同。在众多已通过核糖体掺入的分子和化学物质中，大量能够介导分子内环化的实体彻底改变了大环肽的设计和发现。这些环化反应（可以是自发的、化学的或酶促的）都已成为发现具有天然结构和性质的肽的工具。FIT系统与mRNA展示技术的结合，即随机非标准肽整合发现（RaPID）系统，进而允许针对具有挑战性的生物靶标同时筛选数万亿种大环肽。大环化方法根据所需分子的结构和功能特征进行选择。因此，它们可以源自肽的N端或其侧链，赋予其灵活性或刚性，甚至导致荧光探针的安装。
