Consecutive Elongation of D-Amino Acids in Translation

Recent progress in the field of genetic code reprogramming using a reconstituted cell-free translation system has made it possible to incorporate a wide array of non-proteinogenic amino acids, including N-methyl-amino acids and D-amino acids. Despite the fact that up to ten N-methyl-amino acid residues can be continuously elongated, the successive incorporation of even two D-amino acids into a nascent peptide chain remains a formidable challenge, thus far being nearly impossible. Here we report achievement of continuous D-amino acid elongation by the use of engineered tRNAs and optimized concentrations of translation factors, enabling us to incorporate up to ten consecutive D-Ser residues into a nascent peptide chain. We have also expressed macrocyclic peptides consisting of four or five consecutive D-amino acids consisting of D-Phe, D-Ser, D-Ala, or D-Cys closed by either a disulfide bond or a thioether bond.