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Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs

Nature Chemistry volume 15, pages 948–959 (2023)Cite this article

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Abstract

Mutually orthogonal aminoacyl transfer RNA synthetase/transfer RNA pairs provide a foundation for encoding non-canonical amino acids into proteins, and encoded non-canonical polymer and macrocycle synthesis. Here we discover quintuply orthogonal pyrrolysyl-tRNA synthetase (PylRS)/pyrrolysyl-tRNA (tRNAPyl) pairs. We discover empirical sequence identity thresholds for mutual orthogonality and use these for agglomerative clustering of PylRS and tRNAPyl sequences; this defines numerous sequence clusters, spanning five classes of PylRS/tRNAPyl pairs (the existing classes +N, A and B, and newly defined classes C and S). Most of the PylRS clusters belong to classes that were unexplored for orthogonal pair generation. By testing pairs from distinct clusters and classes, and pyrrolysyl-tRNAs with unusual structures, we resolve 80% of the pairwise specificities required to make quintuply orthogonal PylRS/tRNAPyl pairs; we control the remaining specificities by engineering and directed evolution. Overall, we create 924 mutually orthogonal PylRS/tRNAPyl pairs, 1,324 triply orthogonal pairs, 128 quadruply orthogonal pairs and 8 quintuply orthogonal pairs. These advances may provide a key foundation for encoded polymer synthesis.

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Fig. 1: Relationship between sequence identity and cross-reactivity in previously characterized ∆N PylRSs and Pyl tRNAs.
Fig. 2: Selection of candidate PylRS and tRNAPylCUA sequences and partitioning of the Pyl system into five distinct sequence-defined classes.
Fig. 3: Activity mapping of candidate PylRS enzymes and Pyl tRNAs, and discovery of new triply orthogonal PylRS/tRNAPyl pairs.
Fig. 4: Screening of engineered Pyl tRNAs permits the control of 18 out of 20 cross-reactivities between specific members of the five Pyl classes.
Fig. 5: Unique activity patterns of S∆ PylRS enzymes enable the development of quadruply orthogonal pairs.
Fig. 6: Quintuply orthogonal PylRS/tRNAPyl pairs via directed evolution.

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Data availability

All materials generated or analysed in this study are available from the corresponding author upon reasonable request. All generated datasets are provided in the Supplementary Information. Protein and nucleotide sequences were obtained from the NCBI Protein and NCBI Nucleotide databases, respectively.

Code availability

The code for PylRS and tRNAPyl clustering and mutually orthogonal PylRS/tRNAPyl pair identification is available at https://github.com/JWChin-Lab/Quint-Pyl.

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Acknowledgements

This work was supported by the Medical Research Council (MRC), UK (MC_U105181009 and MC_UP_A024_1008) and an ERC Advanced Grant SGCR, all to J.W.C. For the purpose of Open Access, the MRC Laboratory of Molecular Biology has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. D.L.D. was supported by the Boehringer Ingelheim Fonds and Magdalene College, Cambridge.

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  1. These authors contributed equally: Adam T. Beattie, Daniel L. Dunkelmann.

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Adam T. Beattie, Daniel L. Dunkelmann & Jason W. Chin

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Contributions

D.L.D., A.T.B. and J.W.C. designed the project. A.T.B. and D.L.D. performed the experiments. A.T.B. generated the computational discovery pipeline with inputs from D.L.D. A.T.B., D.L.D and J.W.C. wrote the paper.

Corresponding author

Correspondence to Jason W. Chin.

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The authors declare the following competing financial interest: J.W.C. is a founder of the company Constructive Bio. The Medical Research Council have filed a patent application on the basis of this work.

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Nature Chemistry thanks Heinz Neumann, Jiangyun Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Structures of non-canonical amino acids used in this study.

a. Chemical structure of Nε-tert-butyloxycarbonyl-l-lysine BocK 1. b. Chemical structure of N6-((allyloxy)carbonyl)-l-lysine (AllocK) 2.

Extended Data Fig. 2 Screening class N, A and B Pyl tRNAs for orthogonality to synthetases from other classes.

a. Screening of previously reported class N Pyl tRNAs against key active PylRS enzymes from each class (and S∆ PylRS variants). Most class N Pyl tRNAs tested are highly specific to N+-MmPylRS. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2). b. Screening of previously reported A-AlvtRNAPyl mutants against key active PylRS enzymes from each class (and S∆ PylRS variants). Multiple mutants are highly specific to A∆-1R26PylRS. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2). c. Screening of previously reported B-InttRNAPyl mutants against key active PylRS enzymes from each class (and S∆ PylRS variants). The activity of B∆-Lum1PylRS with class B Pyl tRNAs is closely matched by S∆-ClosPylRS, S∆-I2PylRS, and – most problematically – C∆-NitraPylRS. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2).

Extended Data Fig. 3 Families of quadruply orthogonal pairs containing both S∆B and S∆C PylRSs.

a. Activity heatmap of the two (overlapping) quadruplet families with both class B and class C PylRS enzymes substituted by S∆ PylRS variants (labelled in two shades of blue), shown along with the most orthogonal fifth pair from the final class (class N or class S). The tRNAPyl that requires engineering or replacement to abolish unwanted cross-reactions is labelled in red, while the Pyl tRNAs that already satisfy all necessary orthogonality requirements are labelled in green. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2).b. Schematic of the interactions within the quadruplets from a (both o.c. 2.9). For both, cross-reactivity (red arrow) between class N and class S must be eliminated to yield quintuply orthogonal pairs. In addition, although the problematic cross-reaction between class C PylRS and class B tRNAPyl has been diminished by substitution of both class B and C pairs by pairs formed with a S∆ PylRS variant, the remaining cross-reactivity (yellow arrow) limits the o.c. of these sets. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2).

Extended Data Fig. 4 Directed evolution of Pyl tRNAs and resulting quadruply orthogonal pairs.

a. Heatmap showing the activity of hits obtained from positive selection of the library with S∆B-ClosPylRS followed by successive negative screening with the PylRS enzymes from classes N, A, C and S. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2). b. Heatmap showing the activity of the tRNAPyl hit S-I2tRNAPyl-S52 obtained from positive selection of the library with S+-DebPylRS followed by successive negative screening with the PylRS enzymes from the other classes. This tRNAPyl exhibits selective activity with S+-DebPylRS. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2). c. Activity heatmaps from each family of quadruply orthogonal PylRS/tRNAPyl pairs obtained following the tRNAPyl directed evolution strategy; the quadruplets with the highest o.c. are shown. Dark grey box: quadruply orthogonal families discovered in Fig. 5. Light grey box: quadruply orthogonal family discovered in Fig. 5, but which has a higher o.c. when incorporating a newly evolved tRNAPyl. Orthogonality coefficient, o.c., is shown in grey. Each heatmap value represents the average of three biological replicates. All numerical values and bar charts including error bars showing s.d. are provided (Supplementary Table 2).

Supplementary information

Supplementary Information

Supplementary Notes 1–3, Figs. 1–10, and Tables 4 and 5.

Reporting Summary

Supplementary Tables

Supplementary Tables 1 (sheets 1–4), 2 (sheets 5–11), 3 (sheets 12–14), 6 (sheets 15 and 16) and 7 (sheets 17 and 18).

Supplementary Code 1

Custom scripts used for aaRS and tRNA clustering, and identification of sets of mutually orthogonal aaRS/tRNA pairs.

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Beattie, A.T., Dunkelmann, D.L. & Chin, J.W. Quintuply orthogonal pyrrolysyl-tRNA synthetase/tRNAPyl pairs. Nat. Chem. 15, 948–959 (2023). https://doi.org/10.1038/s41557-023-01232-y

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