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.
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Data availability
Sequences of fully base-paired selected tRNAs are available in Supplementary Tables 2 and 5. Sequences of all of the primers used in the study are available in Supplementary Table 3. The NGS datasets for the tRNAPyl and tRNATyr experiments are available via Zenodo at accession codes https://doi.org/10.5281/zenodo.7186997 and https://doi.org/10.5281/zenodo.7187354, respectively.
Code availability
All scripts used in this manuscript are available on GitHub50.
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Acknowledgements
The authors thank the National Science Foundation (MCB-1817893), and National Institutes of Health (R35GM136437 to A.C. and U01 AI124302 to T.v.O.) for financial support.
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A.C., R.E.K. and D.J. designed the project; R.E.K. developed and optimized the original VADER system and performed selections on tRNAPyl; D.J. performed additional selections on tRNAPyl and selections on tRNATyr, and characterized the tRNA mutants; R.L.H. and Z.H. assisted in the characterization of tRNA mutants; K.M., M.P. and X.C. assisted in cloning; T.v.O., Z.Z., B.S. and J.A. were involved in Illumina sequencing and analyses; and A.C., R.E.K. and D.J. prepared the manuscript.
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A patent application has been submitted on the improved tRNA mutants in which A.C., D.J. and R.E.K. are listed as co-inventors. A.C. is a senior advisor at BrickBio, Inc. All other authors have no competing interests.
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Nature Methods thanks Aditya Kunjapur, Matthew Shoulders and the other, anonymous, reviewer for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.
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Supplementary Figs. 1–16, Supplementary Tables 1–5
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Jewel, D., Kelemen, R.E., Huang, R.L. et al. Virus-assisted directed evolution of enhanced suppressor tRNAs in mammalian cells. Nat Methods 20, 95–103 (2023). https://doi.org/10.1038/s41592-022-01706-w
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DOI: https://doi.org/10.1038/s41592-022-01706-w
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