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Directed evolution of a highly efficient TNA polymerase achieved by homologous recombination

Nature Catalysis volume 7, pages 1173–1185 (2024)Cite this article

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Abstract

Reprogramming DNA polymerases to synthesize xeno-nucleic acids (XNAs) is an important challenge that tests current enzyme engineering tools. Here we describe an evolutionary campaign aimed at generating an XNA polymerase that can efficiently make α-l-threofuranosyl nucleic acid (TNA)—an artificial genetic polymer that is recalcitrant to nucleases and resistant to acid-mediated degradation. Starting from a homologous recombination library, iterative cycles of selection were performed to traverse the fitness landscape in search of neutral mutations with increased evolutionary potential. Subsequent directed evolution of focused mutagenic libraries yielded 10–92, a newly engineered TNA polymerase that functions with a catalytic rate of ∼1 nt s−1 and >99% fidelity. A crystal structure of the closed ternary complex reveals the degree of structural change required to remodel the active site pocket for improved TNA synthesis activity. Together, these data demonstrate the importance of recombination as a strategy for evolving XNA polymerases with considerable practical value for biotechnology and medicine.

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Fig. 1: Homologous recombination as a path to engineered XNA polymerases.
Fig. 2: Directed evolution of a highly efficient TNA polymerase.
Fig. 3: Functional analysis of TNA polymerases.
Fig. 4: Kinetic analysis of TNA synthesis.
Fig. 5: Enhanced functional activity.
Fig. 6: Structural topology of the 10–92 TNA polymerase.
Fig. 7: Active site of the 10–92 TNA polymerase.

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Data availability

The crystal structure of 10–92 was deposited in the Protein Data Bank [PDB:8T3X]. The repository furthermore contains PDB structures of all ensemble refinements presented in this work. Other data are available in the main text, Supplementary Information or from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by a grant to J.C.C. from the NSF (MCB 1946312).

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Author notes

  1. These authors contributed equally: Victoria A. Maola, Eric J. Yik.

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA, USA

    Victoria A. Maola, Eric J. Yik, Mohammad Hajjar, Joy J. Lee, Manuel J. Holguin, Riley N. Quijano, Kalvin K. Nguyen, Katherine L. Ho, Jenny V. Medina, Nicholas Chim & John C. Chaput

  2. Department of Chemistry, University of California, Irvine, Irvine, CA, USA

    John C. Chaput

  3. Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA

    John C. Chaput

  4. Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA

    John C. Chaput

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Contributions

J.C.C., V.A.M. and E.J.Y. conceived the project and designed the experiments. E.J.Y. and V.A.M. evolved the enzyme. E.J.Y., V.A.M. and M.H. performed the biochemical characterization. V.A.M., J.J.L., M.H., M.J.H., R.N.Q., K.K.N., K.L.H., J.V.M. and N.C. contributed to protein structure determination. J.C.C. wrote the paper. All authors reviewed and commented on the paper.

Corresponding author

Correspondence to John C. Chaput.

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Competing interests

J.C.C., V.A.M., E.J.Y. and the University of California, Irvine have filed a patent application (PCT/US24/11595) on the composition and activity of the 10–92 TNA polymerase. The other authors declare no competing interests.

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Nature Catalysis thanks Masayasu Kuwahara, Vitor Pinheiro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Methods, Tables 1–5, Figs. 1–19, and uncropped gels for Supplementary Figs. 2, 5, 8, 9, 10, 13–15.

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Source data

Source Data Fig. 3

Unprocessed Urea-PAGE gel.

Source Data Fig. 4

Kinetics raw and processed data.

Source Data Fig. 5

Unprocessed Urea-PAGE gel.

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Maola, V.A., Yik, E.J., Hajjar, M. et al. Directed evolution of a highly efficient TNA polymerase achieved by homologous recombination. Nat Catal 7, 1173–1185 (2024). https://doi.org/10.1038/s41929-024-01233-1

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