Reprogramming the genetic code with flexizymes

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.