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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Source data for the graphs and heatmaps (for Figs. 1–6 and Supplementary Figs. 5–7, 10–18 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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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.
The authors declare no competing interests.
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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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