In the canonical genetic code, the 61 sense codons are assigned to the 20 proteinogenic amino acids. Advancements in genetic code manipulation techniques have enabled the ribosomal incorporation of nonproteinogenic amino acids (npAAs). The critical molecule for translating messenger RNA (mRNA) into peptide sequences is aminoacyl-transfer RNA (tRNA), which recognizes the mRNA codon through its anticodon. Because aminoacyl-tRNA synthetases (ARSs) are highly specific for their respective amino acid–tRNA pairs, it is not feasible to use natural ARSs to prepare npAA-tRNAs. However, flexizymes are adaptable aminoacylation ribozymes that can be used to prepare diverse aminoacyl-tRNAs at will using amino acids activated with suitable leaving groups. Regarding recognition elements, flexizymes require only an aromatic ring in either the leaving group or side chain of the activated amino acid, and the conserved 3′-end CCA of the tRNA. Therefore, flexizymes allow virtually any amino acid to be charged onto any tRNA. The flexizyme system can handle not only l-α-amino acids with side chain modifications but also various backbone-modified npAAs. This Review describes the development of flexizyme variants and discusses their structure and mechanism and their applications in genetic code reprogramming for the synthesis of unique peptides and proteins.

在规范遗传密码中，61 个正义密码子被分配给 20 个蛋白原性氨基酸。遗传密码操作技术的进步使非蛋白原氨基酸 （npAA） 的核糖体掺入成为可能。将信使 RNA （mRNA） 翻译成肽序列的关键分子是氨酰转移 RNA （tRNA），它通过其反密码子识别 mRNA 密码子。由于氨酰基-tRNA 合成酶 （ARS） 对其各自的氨基酸-tRNA 对具有高度特异性，因此使用天然 ARS 制备 npAA-tRNA 是不可行的。然而，弹性酶是适应性强的氨酰化核酶，可用于使用用合适的离基激活的氨基酸随意制备不同的氨酰基-tRNA。关于识别元件，弹性酶只需要活化氨基酸的离去基团或侧链中的芳香环，以及 tRNA 的保守 3′-末端 CCA。因此，弹性酶几乎可以将任何氨基酸充电到任何 tRNA 上。flexizyme 系统不仅可以处理具有侧链修饰的 l-α-氨基酸，还可以处理各种骨架修饰的 npAA。本文介绍了 flexizyme 变体的发展，并讨论了它们的结构和机制及其在遗传密码重编程中用于合成独特肽和蛋白质的应用。