Development of uniform ribosome display technology enabling easy and efficient identification of full-length proteins that interact with bioactive small and large molecules.

Identifying target proteins that interact with bioactive molecules is indispensable for understanding their mechanisms of action. In this study, we developed a uniform ribosome display technology using equal-length DNAs and mRNAs to improve molecular display principle for target identification. The equal-length DNAs were designed to contain various coding sequences for full-length proteins with molecular weights of up to 130 kDa and were used to synthesize equal-length mRNAs, which allowed the formation of full-length protein-ribosome-equal-length mRNA complexes. Uniform ribosome display selections of dihydrofolate reductase and haloalkane dehalogenase mutant were performed against methotrexate and chlorohexane, respectively. Quantitative changes of proteins after each selection indicated that the target protein-displaying ribosomal complexes were specifically selected through non-covalent or covalent interactions with the corresponding bioactive molecules. Furthermore, selection of full-length proteins interacting with methotrexate or anti-DDX46 antibody from protein pools showed that only the target proteins could be precisely identified even though the molar amounts of equal-length mRNAs encoding them were adjusted to 1/20,000 of the total equal-length mRNAs. Thus, the uniform ribosome display technology enabled efficient identification of target proteins that interact with bioactive small and large molecules through simplified operations without deep sequencing.

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-tRNAUAG reprogramming of the UAG codons (X = Tyr, Trp, or p-benzoyl-l-phenylalanine (pBzo-Phe)) to the scheme. Based on the assessment of the efficiency of in vitro translation with X-tRNAUAG, we carried out ribosome display selection with genetic code expansion using Trp-tRNAUAG, 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 pBzo-Phe-incorporated peptides, we performed ribosome display selection of low-affinity peptides in combination with genetic code expansion using pBzo-Phe-tRNAUAG 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.