Optimal codon choice can improve the efficiency and fidelity of N-methyl amino acid incorporation into peptides by in-vitro translation.

The generation of peptides containing N-methyl amino acids (N-Me AAs) by in-vitro translation is of interest as a potential route to the directed evolution of pharmaceutically useful modified peptides. However, N-Me AAs are suboptimal substrates for the translational apparatus, and as a result, premature termination of translation can occur or incorrect amino acids can be misincorporated in place of the desiredNMe AA, resulting in reduced incorporation efficiency and fidelity of the N-Me AA. We sought to determine the main cause of such misincorporation and truncation events, and then to optimize the incorporation of N-Me AAs. We examined the efficiency and fidelity with which three different N-Me AAs (N-Me Leu, N-Me Thr, and N-Me Val) are incorporated at a single position in a short peptide (MH6MXmEP, Xm=N-Me AA, M=Met, E=Glu, P=Pro, H=His) using each of the codons of the corresponding natural amino acid (Figure 1). We showed previously that these three N-Me AAs can be incorporated into peptides by in-vitro translation using the fully reconstituted PURE system for translation (PURE= protein synthesis using recombinant elements, an in vitro system containing only purifies components). We directed the incorporation of these N-Me AAs into peptides by supplying in-vitro translation reaction mixtures with total tRNA that had been enzymatically precharged with Leu, Thr, or Val and then chemically N-methylated as previously described (see Experimental Section). The presence of the other 19 amino acid/ aminoacyl-tRNA synthetase (AARS) pairs in the translation