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Engineered triply orthogonal pyrrolysyl–tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids

Abstract

Expanding and reprogramming the genetic code of cells for the incorporation of multiple distinct non-canonical amino acids (ncAAs), and the encoded biosynthesis of non-canonical biopolymers, requires the discovery of multiple orthogonal aminoacyl–transfer RNA synthetase/tRNA pairs. These pairs must be orthogonal to both the host synthetases and tRNAs and to each other. Pyrrolysyl–tRNA synthetase (PylRS)/PyltRNA pairs are the most widely used system for genetic code expansion. Here, we reveal that the sequences of ΔNPylRS/ΔNPyltRNA pairs (which lack N-terminal domains) form two distinct classes. We show that the measured specificities of the ΔNPylRSs and ΔNPyltRNAs correlate with sequence-based clustering, and most ΔNPylRSs preferentially function with ΔNPyltRNAs from their class. We then identify 18 mutually orthogonal pairs from the 88 ΔNPylRS/ΔNPyltRNA combinations tested. Moreover, we generate a set of 12 triply orthogonal pairs, each composed of three new PylRS/PyltRNA pairs. Finally, we diverge the ncAA specificity and decoding properties of each pair, within a triply orthogonal set, and direct the incorporation of three distinct non-canonical amino acids into a single polypeptide.

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Fig. 1: Identifying two classes of ΔNPylRS/ΔNPyltRNA pairs and 18 naturally mutually orthogonal ΔNPylRS/ΔNPyltRNA pairs.
Fig. 2: The MmPylRS/MmPyltRNA pair is not orthogonal with respect to any ΔNPyltRNAs and some ΔNPylRSs.
Fig. 3: Identifying triply orthogonal and active class +N +NPyltRNAs.
Fig. 4: Identifying triply orthogonal and active class A ΔNPyltRNAs.
Fig. 5: Identifying triply orthogonal and active class B ΔNPyltRNAs.
Fig. 6: Diverging amino acid recognition and decoding properties of triply orthogonal PylRS/tRNA pairs enables the incorporation of three distinct ncAAs into a protein.

Data availability

Source data for the graphs and heatmaps (for Figs. 16 and Supplementary Figs. 57, 1018 and 21) are provided in Supplementary Table 3. Source data for the gels in Fig. 6 are provided with the paper. All other datasets and material generated or analysed in this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the UK Medical Research Council (MRC; MC_U105181009 and MC_UP_A024_1008) and an ERC Advanced Grant SGCR (all to J.W.C.). D.L.D. was supported by the Boehringer Ingelheim Fonds. We thank M. Skehel at the MRC-LMB mass spectrometry facility and K. Heesom at the proteomics facility of the University of Bristol for performing mass spectrometry.

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D.L.D., J.C.W.W. and J.W.C. designed the project. D.L.D. and J.C.W.W. performed the experiments. A.T.B. analysed and interpreted the MS/MS data. D.L.D., J.C.W.W. and J.W.C. wrote the paper with input from A.T.B.

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Correspondence to Jason W. Chin.

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Dunkelmann, D.L., Willis, J.C.W., Beattie, A.T. et al. Engineered triply orthogonal pyrrolysyl–tRNA synthetase/tRNA pairs enable the genetic encoding of three distinct non-canonical amino acids. Nat. Chem. 12, 535–544 (2020). https://doi.org/10.1038/s41557-020-0472-x

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