Genetic Code Expansion and a Photo-Cross-Linking Reaction Facilitate Ribosome Display Selections for Identifying a...

Area of Science:

• Biotechnology
• Molecular Biology
• Synthetic Biology

Background:

• Cell-free molecular display techniques are used for peptide selection but face challenges with translational termination and non-specific peptide amplification.
• These limitations hinder the effective selection of low-affinity peptides, crucial for various applications.

Purpose of the Study:

• To develop an advanced ribosome display method for selecting peptide epitopes that bind to monoclonal antibodies.
• To overcome limitations in peptide selection by employing genetic code expansion with synthetic amino acids.

Main Methods:

• Established a ribosome display scheme incorporating genetic code expansion via synthetic X-tRNA^UAG (X = Tyr, Trp, or p-benzoyl-l-phenylalanine (pBzo-Phe)) to reprogram UAG codons.
• Assessed in vitro translation efficiency with X-tRNA^UAG and performed selections using Trp-tRNA^UAG and pBzo-Phe-tRNA^UAG with photo-irradiation.

Main Results:

• Demonstrated efficient identification of affinity peptides irrespective of UAG codons in coding sequences using Trp-tRNA^UAG.
• Successfully selected sub-micromolar low-affinity peptide epitopes using pBzo-Phe-tRNA^UAG and photo-induced covalent bond formation with antibodies.

Conclusions:

• The developed ribosome display technique with genetic code expansion effectively selects diverse affinity peptides, including low-affinity ones.
• This method overcomes traditional limitations and can advance peptide-based research and antibody discovery.