Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells
Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells
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DOI：
10.1038/s41592-022-01706-w
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发表时间：
2022-12-22
期刊：
影响因子：
48
通讯作者：
Chatterjee, Abhishek
中科院分区：
文献类型：
--
作者：
Jewel, Delilah;Kelemen, Rachel E. E.;Chatterjee, Abhishek
非天然氨基酸（Uaas）在活细胞中的位点特异性掺入依赖于从遥远的生命领域借来的工程化氨酰转移RNA合成酶-tRNA对。此类异源抑制性tRNA通常具有差的内在活性，推测是由于与非天然翻译系统的次优相互作用。这种限制可以在大肠杆菌中使用定向进化来解决。然而，目前没有合适的选择系统可用于在哺乳动物细胞中做同样的事情。在这里，我们报告的病毒辅助定向进化的tRNA（VADER）在哺乳动物细胞中，它使用双筛选择方案，以促进一步富集活性但正交tRNA突变体从幼稚库。使用VADER，我们开发了改进的突变体的Methanacrylcinamazei吡咯赖氨酰-tRNA，以及细菌酪氨酰-tRNA。我们还表明，最有效的突变体pyrrolysyl-tRNA的活性更高，对哺乳动物细胞是特异性的，暗示与独特的哺乳动物翻译装置的相互作用得到改善。
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 Methanosarcinamazei 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.