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

Nature Methods volume 20, pages 95–103 (2023)Cite this article

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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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Fig. 1: The VADER selection scheme.
Fig. 2: Directed evolution of tRNAPyl.
Fig. 3: Characterization of tRNAPyl-A2.1 activity.
Fig. 4: Origin of the improved activity of tRNAPyl-A2.1.
Fig. 5: Directed evolution of tRNATyr.

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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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Author notes

  1. These authors contributed equally: Delilah Jewel, Rachel E. Kelemen.

Authors and Affiliations

  1. Department of Chemistry, Boston College, Chestnut Hill, MA, USA

    Delilah Jewel, Rachel E. Kelemen, Rachel L. Huang, Xiaofu Cao, Kaitlin Malley, Zeyi Huang, Muhammad Pasha & Abhishek Chatterjee

  2. Biology Department, Boston College, Chestnut Hill, MA, USA

    Zeyu Zhu, Bharathi Sundaresh, Jon Anthony & Tim van Opijnen

  3. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Tim van Opijnen

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Contributions

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.

Corresponding author

Correspondence to Abhishek Chatterjee.

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Competing interests

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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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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