Expression of model peptides containing nPAA. (a) Model mRNA (mR2–mR5) and the corresponding peptide sequences (rP2–rP5) used for nPAA incorporation. Codons used for nPAA incorporation are indicated in bold. Macrocyclizations of peptides between ^ClAcd-Phe and d-Cys are indicated by arrows. The amino acid sequence of flag is Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys. (b) Structures of macrocyclic peptides rP4 and (c) rP5. (d) Expression levels of model peptides rP2-d-Ala₂, (e) rP2-β-hMet₂, (f) rP3-β-hMet_n, and (g) rP3-(1S,2S)-2-ACPC_n. Black bars indicate EF-P(+) translation, and white bars indicate EF-P(−) translation. Numbers above the bars indicate the ratio of EF-P(+) experiment to EF-P(−). Error bars, s.d. (n = 3).