Fine-tuning the tRNA anticodon arm for multiple/consecutive incorporations of -amino acids and analogs

Ribosomal incorporation of beta-amino acids into nascent peptides is much less efficient than that of the canonical alpha-amino acids. To overcome this, we have engineered a tRNA chimera bearing T-stem of tRNAGlu and D-arm of tRNAPro1, referred to as tRNAPro1E2, which efficiently recruits EF-Tu and EF-P. Using tRNAPro1E2 indeed improved beta-amino acid incorporation. However, multiple/consecutive incorporations of beta-amino acids are still detrimentally poor. Here, we attempted fine-tuning of the anticodon arm of tRNAPro1E2 aiming at further enhancement of beta-amino acid incorporation. By screening various mutations introduced into tRNAPro1E2, C31G39/C28G42 mutation showed an approximately 3-fold enhancement of two consecutive incorporation of beta-homophenylglycine (beta Phg) at CCG codons. The use of this tRNA made it possible for the first time to elongate up to ten consecutive beta Phg's. Since the enhancement effect of anticodon arm mutations differs depending on the codon used for beta-amino acid incorporation, we optimized anticodon arm sequences for five codons (CCG, CAU, CAG, ACU and UGG). Combination of the five optimal tRNAs for these codons made it possible to introduce five different kinds of beta-amino acids and analogs simultaneously into model peptides, including a macrocyclic scaffold. This strategy would enable ribosomal synthesis of libraries of macrocyclic peptides containing multiple beta-amino acids. Graphical Abstract