Directed Evolution of a Bacterial Leucyl tRNA in Mammalian Cells for Enhanced Noncanonical Amino Acid Mutagenesis.

ABSTRACT: The Escherichia coli leucyl-tRNA synthetase (EcLeuRS)/tRNA^EcLeu pair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNA^EcLeu. Here, we overcome this limitation by evolving tRNA^EcLeu directly in mammalian cells by using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNA^EcLeu could be simultaneously engineered, which resulted in

SUBMITTER: Huang RL 

PROVIDER: S-EPMC11789822 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature