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Keywords:

  • directed evolution;
  • genetic code expansion;
  • pyrrolysine;
  • small-intelligent library;
  • unnatural amino acids
Thumbnail image of graphical abstract

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.