Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells.

Delilah Jewel, Rachel E Kelemen, Rachel L Huang, Zeyu Zhu, Bharathi Sundaresh, Xiaofu Cao, Kaitlin Malley, Zeyi Huang, Muhammad Pasha, Jon Anthony, Tim van Opijnen, Abhishek Chatterjee

Nature methods 2023 Jan

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Site-specific incorporation of unnatural amino acids (Uaas) in living cells relies on engineered aminoacyl-transfer RNA synthetase-tRNA pairs borrowed from a distant domain of life. Such heterologous suppressor tRNAs often have poor intrinsic activity, presumably due to suboptimal interaction with a non-native translation system. This limitation can be addressed in Escherichia coli using directed evolution. However, no suitable selection system is currently available to do the same in mammalian cells. Here we report virus-assisted directed evolution of tRNAs (VADER) in mammalian cells, which uses a double-sieve selection scheme to facilitate single-step enrichment of active yet orthogonal tRNA mutants from naive libraries. Using VADER we developed improved mutants of Methanosarcina mazei pyrrolysyl-tRNA, as well as a bacterial tyrosyl-tRNA. We also show that the higher activity of the most efficient mutant pyrrolysyl-tRNA is specific for mammalian cells, alluding to an improved interaction with the unique mammalian translation apparatus. © 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.

Citation

Delilah Jewel, Rachel E Kelemen, Rachel L Huang, Zeyu Zhu, Bharathi Sundaresh, Xiaofu Cao, Kaitlin Malley, Zeyi Huang, Muhammad Pasha, Jon Anthony, Tim van Opijnen, Abhishek Chatterjee. Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells. Nature methods. 2023 Jan;20(1):95-103