Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a Wide Range of Affinity Peptides.

Cell-free molecular display techniques have been utilized to select various affinity peptides from peptide libraries. However, conventional techniques have difficulties associated with the translational termination through in-frame UAG stop codons and the amplification of non-specific peptides, which hinders the desirable selection of low-affinity peptides. To overcome these problems, we established a scheme for ribosome display selection of peptide epitopes bound to monoclonal antibodies and then applied genetic code expansion with synthetic X-tRNA UAG reprogramming of the UAG codons (X = Tyr, Trp, or p -benzoyl-l-phenylalanine ( p Bzo-Phe)) to the scheme. Based on the assessment of the efficiency of in vitro translation with X-tRNA UAG , we carried out ribosome display selection with genetic code expansion using Trp-tRNA UAG , and we verified that affinity peptides could be identified efficiently regardless of the presence of UAG codons in the peptide coding sequences. Additionally, after evaluating the photo-cross-linking reactions of p Bzo-Phe-incorporated peptides, we performed ribosome display selection of low-affinity peptides in combination with genetic code expansion using p Bzo-Phe-tRNA UAG and photo-irradiation. The results demonstrated that sub-micromolar low-affinity peptide epitopes could be identified through the formation of photo-induced covalent bonds with monoclonal antibodies. Thus, the developed ribosome display techniques could contribute to the promotion of diverse peptide-based research.