Expanding the library and substrate diversity of the pyrrolysyl-tRNA synthetase to incorporate unnatural amino acids containing conjugated rings

Unnatural amino acids (UAAs) containing conjugated ring systems are of interest for their optical properties. Until now, such bulky and planar UAAs could not be incorporated into proteins using the pyrrolysyl tRNA/synthetase shuttling system. Using the "small-intelligent" approach to construct a highly diverse library, we evolved novel synthetases specific for two such UAAs and incorporated them into proteins in E. coli and mammalian cells.