Engineering of tRNAPro1E2 anticodon stem enhances multiple/consecutive ribosomal incorporation of <i>N</i> -methyl- <scp>l</scp> -α-amino acids and <scp>d</scp> -α-amino acids

Transfer RNA (tRNA) structures influence the incorporation efficiency of amino acids, particularly when nonproteinogenic amino acids (npAAs), such as N-methyl-l-α-amino acids (MeAAs) and d-α-amino acids (d-AAs), are charged. Such npAAs are generally far poorer substrates than the 20 canonical α-amino acids for translation. However, we have shown that their incorporation efficiencies could be improved by using a chimeric tRNA, termed tRNAPro1E2, bearing optimal T-stem and D-arm. Here we report that the engineering anticodon stem of tRNAPro1E2 further enhances multiple/consecutive elongation of MeAAs and d-AAs. By screening 149 types of anticodon stem mutants, we found eleven tRNAPro1E2 variants capable of enhancing the incorporation of N-methyl-l-leucine (MeLeu) or d-phenylalanine (d-Phe) at six codons by up to 4.5- and 4.4-fold, respectively. Interestingly, 7 out of the 11 variants showed different mobilities compared to the parental tRNAsPro1E2 in native polyacrylamide gel electrophoresis analysis, indicating that their unique conformations contributed to promoting peptidyl transfer reaction. These tRNAs exhibited increased incorporation efficiencies of not only MeLeu and d-Phe but also diverse MeAAs and d-AAs. Ribosomal synthesis of a model macrocyclic peptide containing three consecutive MeAAs and three d-AAs was also demonstrated with high quality, indicating their usefulness for the peptide library construction containing multiple exotic amino acids.