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Selecting aminoacyl-tRNA synthetase/tRNA pairs for efficient genetic encoding of noncanonical amino acids into proteins

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Abstract

A critical component of genetic code expansion applications is an aminoacyl-tRNA synthetase (RS)/tRNA pair that faithfully encodes a noncanonical amino acid (ncAA) in response to a specific codon. Here we detail a procedure to select an ncAA-specific RS from a publicly available 3.2-million-member Methanomethylophilus alvus pyrrolysyl-RS (MaPylRS) active site mutant library. Four main parts of the procedure are: (1) preparing the library for use and creating needed cell lines; (2) life and death selections that, respectively, select for functional RSs and select against RSs that incorporate canonical amino acids; (3) three fluorescence-based status checks that provide information about the efficiency and fidelity of the surviving RSs in incorporating the target ncAA; and (4) characterizing top hits to find the best ones for use in applications. The resulting RS/tRNA pairs can be used in either bacterial or eukaryotic cells to study proteins of interest. Additionally, the stability of the MaPylRSs makes them useful in cell-free ncAA-protein expression and amenable to structural and other in vitro characterizations. This Protocol is usable by those with basic molecular biology expertise and features a reliable positive control scheme for selections, status checks at different stages to interpret the level of success and a robust procedure to characterize newly engineered tRNA–RS pairs. Users of this Protocol can expect to select ncAA-specific RS/tRNA pairs from the library within about 30–50 d depending on preparation needs.

Key points

  • This Protocol outlines the steps to isolate aminoacyl-tRNA synthetase/tRNA pairs that incorporate noncanonical amino acids into proteins. RSs are selected from a publicly available 3.2-million-member MaPylRS active site mutant library and involves a series of life and death selections and fluorescence-based enrichment and evaluation.

  • Aminoacyl-tRNA synthetase hits with high fidelity and efficiency in incorporating the desired noncanonical amino acids are suitable for use in prokaryotic and eukaryotic cells and in cell-free protein expression systems.

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Fig. 1: Illustration of the three main stages for generating new high efficiency RS/tRNA pairs.
Fig. 2: Diversity of ncAA structures.
Fig. 3: Schematic flow chart for the four stages of this protocol.
Fig. 4: Assessment of library coverage.
Fig. 5: Sony SH800S sort plots of events (individual cells).
Fig. 6: Workflow for RS library propagation.
Fig. 7: Workflow for competent cell preparation.
Fig. 8: A dry ice ethanol bath using a plastic tub for flash-freezing library-ready electrocompetent cells.
Fig. 9: Example results for ncAA toxicity tests.
Fig. 10: Assessment of library coverage achieved during a transformation.
Fig. 11: Example agar plates and a DNA separation gel from the positive selection.
Fig. 12: Example agar plates and a DNA separation gel from the negative selection.
Fig. 13: Example of a FACS histogram for cells encoding nitroTyr into sfGFPNY using the functional ‘NY-F5’ MaPylRS/tRNA pair.
Fig. 14: Example results of level 1 status checks with strong, weak and no evidence for selection success after one round of positive and negative selections.
Fig. 15: In-cell fluorescence evaluation of individual hits expressing sfGFP150 with and without ncAA after a positive and negative selection.
Fig. 16: Level 3 status check results for the MaPylRS variant F4.
Fig. 17: UP50 assessment of three MaPylRS hits obtained from the nitroTyr selection.
Fig. 18: Example data of a permissivity screen to identify nitroTyr RS variants that can encode structurally analogous ncAAs.
Fig. 19: Positive selection plates during a second round of positive selection for nitroTyr under stringent conditions using increased CAM.
Fig. 20: Status check 2 of MaPylRSs isolated after two rounds of positive selection.

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Data availability

The data used to illustrate procedural outcomes of this protocol are available as Source data within the article or reported by supporting primary research papers23. Additional relevant data may be available upon request to the authors. Source data are provided with this paper.

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Acknowledgements

This work was supported by the GCE4All Biomedical Technology Optimization and Dissemination Center supported by National Institute of General Medical Science (grant no. RM1-GM144227 to R.A.M.). Graphpad Prism 10.1.1 was used to generate the figures to illustrate efficiency/fidelity and UP50 data.

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Author notes

  1. These authors contributed equally: Nathan D. Alexander, Yogesh M. Gangarde.

Authors and Affiliations

  1. GCE4All Research Center, Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA

    Nathan D. Alexander, Yogesh M. Gangarde, Riley M. Bednar, P. Andrew Karplus, Richard B. Cooley & Ryan A. Mehl

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  1. Nathan D. Alexander
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  2. Yogesh M. Gangarde
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Contributions

R.C. and R.M. conceived the protocol. N.A., Y.G., R.C. and R.M. developed the protocol, designed the experiments and analyzed the data. N.A. and Y.G. performed the experiments. N.A., Y.G., R.B. and R.C. generated figures. R.C. contributed data. N.A., Y.G., R.B., P.A.K., R.C. and R.M. wrote, provided input into and edited the manuscript.

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Correspondence to Ryan A. Mehl.

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Nature Protocols thanks Abhishek Chatterjee, Nediljko Budisa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Avila-Crump et al. ACS Chem Biol. 17, 3458–3469 (2022): https://doi.org/10.1021/acschembio.2c00639

Galles et al. Nat Commun. 14, 59 (2023): https://doi.org/10.1038/s41467-022-35761-w

Gottfried-Lee et al. ACS Chem Biol. 17, 3470–3477 (2022): https://doi.org/10.1021/acschembio.2c00640

Supplementary information

Reporting Summary

Supplementary Data

Sequences for Supplementary Tables 11 and 12.

Source data

Source Data Figs. 5, 9, 13, 14, 15, 16, 17, 18 and 20

Tab:Figure5 raw FACS data, Tab:Figure9 raw fluorescence and cell density, Tab:Figure13 raw FACS data, Tab:Figure14 raw FACS data, Tab:Figure15 nRFU data, Tab:Figure16 raw MS data, Tab:Figure17 raw fluorescence and cell density, Tab:Figure18 raw fluorescence and cell density, Tab:Figure20 nRFU data.

Source Data Fig. 11b

Uncropped gel.

Source Data Fig. 12b

Uncropped gel.

Source Data Figure 16b

Uncropped gel.

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Alexander, N.D., Gangarde, Y.M., Bednar, R.M. et al. Selecting aminoacyl-tRNA synthetase/tRNA pairs for efficient genetic encoding of noncanonical amino acids into proteins. Nat Protoc (2025). https://doi.org/10.1038/s41596-025-01241-w

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