Title: Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells
Author: Jewel, Delilah, Kelemen, Rachel E., Huang, Rachel L., Zhu, Zeyu, Sundaresh, Bharathi, Cao, Xiaofu, Malley, Kaitlin, Huang, Zeyi, Pasha, Muhammad, Anthony, Jon, van Opijnen, Tim, Chatterjee, Abhishek
Issue&Volume: 2022-12-22
Abstract: 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.
DOI: 10.1038/s41592-022-01706-w
Source: https://www.nature.com/articles/s41592-022-01706-w
Nature Methods:《自然—方法学》,创刊于2004年。隶属于施普林格·自然出版集团,最新IF:47.99
官方网址:https://www.nature.com/nmeth/
投稿链接:https://mts-nmeth.nature.com/cgi-bin/main.plex