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Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs

Nature Chemistry volume 10, pages 831–837 (2018)Cite this article

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Abstract

Genetically encoding distinct non-canonical amino acids (ncAAs) into proteins synthesized in cells requires mutually orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs. The pyrrolysyl-tRNA synthetase/PyltRNA pair from Methanosarcina mazei (Mm) has been engineered to incorporate diverse ncAAs and is commonly considered an ideal pair for genetic code expansion. However, finding new aaRS/tRNA pairs that share the advantages of the MmPylRS/MmPyltRNA pair and are orthogonal to both endogenous aaRS/tRNA pairs and the MmPylRS/MmPyltRNA pair has proved challenging. Here we demonstrate that several ΔNPylRS/PyltRNACUA pairs, in which PylRS lacks an N-terminal domain, are active, orthogonal and efficiently incorporate ncAAs in Escherichia coli. We create new PylRS/PyltRNA pairs that are mutually orthogonal to the MmPylRS/MmPyltRNA pair and show that transplanting mutations that reprogram the ncAA specificity of MmPylRS into the new PylRS reprograms its substrate specificity. Finally, we show that distinct PylRS/PyltRNA-derived pairs can function in the same cell, decode distinct codons and incorporate distinct ncAAs.

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Fig. 1: Structures of the amino acids used in this work.
Fig. 2: Identifying ΔNPylRS/PyltRNA pairs that are active and orthogonal in E. coli.
Fig. 3: Mutual orthogonality among natural PylRS/PyltRNA pairs.
Fig. 4: Evolution of MaPyltRNA to create mutually orthogonal PylRS/PyltRNA pairs.
Fig. 5: Engineering the active site of MaPylRS for selective ncAA incorporation.
Fig. 6: Encoding distinct ncAAs using mutually orthogonal PylRS/PyltRNA pairs.

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Acknowledgements

This work was supported by the Medical Research Council, UK (MC_U105181009 and MC_UP_A024_1008), BBSRC (BB/M000842/1, for automation) and an ERC Advanced Grant (SGCR), all to J.W.C. J.C.W.W. was supported by the EPSRC Nanoscience and Nanotechnology CDT at Cambridge University.

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

    Julian C. W. Willis & Jason W. Chin

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  1. Julian C. W. Willis
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J.C.W.W. performed all experiments. J.C.W.W and J.W.C. conceived and designed experiments, analysed the data, and wrote the paper.

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Correspondence to Jason W. Chin.

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Willis, J.C.W., Chin, J.W. Mutually orthogonal pyrrolysyl-tRNA synthetase/tRNA pairs. Nature Chem 10, 831–837 (2018). https://doi.org/10.1038/s41557-018-0052-5

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