Evolution of Translation Machinery for High Efficiency Multi-site Incorporation of Alkyne Bearing Unnatural Amino Acids

Site-specific modification of proteins is a powerful means for investigation and manipulation of the properties of proteins. However, traditional modification through natural reactive residues often results with heterogeneous product due to the presence of these residues at multiple sites at the protein, and may also disturb proper folding and function of the protein.

Site specific incorporation of bio-orthogonal unnatural amino acids (uAAs) with unique reactivity into proteins, enables selective and site-specific modification of the protein, while the orthogonal-functional group of the uAA is unreactive towards canonical amino acids and metabolites in the cell, causing minimal disturbance .

Incorporation of alkyne-bearing uAA into proteins enables conjugation of the protein to azide-bearing molecules, through the copper catalyzed azide-alkyne cycloaddition (CuAAC), also known as “click” chemistry. In this study, we evolved and characterized an orthogonal translation system for high efficiency multi-site incorporation of an alkyne-bearing uAA, by directed evolution of the aminoacyl-tRNA synthetase. The evolved system exhibits high incorporation efficiencies and is capable of incorporating up to 30-instances of the uAA into proteins, with complete control of the number and positions of the alkyne modifications along the biopolymer chain. We further show that alkyne-bearing proteins can beconjugated to azide-bearing molecules via “click” chemistry both in-vitro and in-vivo.

Such semi-synthetic biomaterials can be used to produce costumized materials and therapeutics, with precise and tunable properties.