Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 20, Issue 9, Pages -Publisher
MDPI
DOI: 10.3390/ijms20092294
Keywords
aminoacyl-tRNA synthetases; noncanonical amino acids; directed evolution; genetic code expansion; synthetic biology
Funding
- US National Institutes of Health (NIH) [R35GM122560]
- Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the Department of Energy [DE-FG02-98ER20311]
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Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l--AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and -AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.
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