Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons

The genetic code in most organisms codes for 20 proteinogenic amino acids or translation stop. In order to encode more than 20 amino acids in the coding system, one of stop codons is usually reprogrammed to encode a non-proteinogenic amino acid. Although this approach works, usually only one amino acid is added to the amino acid repertoire. In this study, we incorporated non-proteinogenic amino acids into a protein by using a sense codon. As all the codons are allocated in the universal genetic code, we destroyed all the tRNA^Arg in a cell-free protein synthesis system by using a tRNA^Arg-specific tRNase, colicin D. Then by supplementing the system with tRNA_CCU, the translation system was partially restored. Through this creative destruction, reprogrammable codons were successfully created in the system to encode modified lysines along with the 20 proteinogenic amino acids.