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Rapid evolution of a highly efficient RNA polymerase by homologous recombination

Nature Chemical Biology (2026)Cite this article

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Abstract

Engineering DNA polymerases to efficiently synthesize artificial or noncognate nucleic acids remains an essential challenge in synthetic biology. Here we describe an evolutionary campaign designed to convert a family of highly selective DNA polymerases into an unnatural homolog with strong RNA synthesis activity. Starting from a homologous recombination library, a short evolutionary path was achieved using a single-cell droplet-based microfluidic selection strategy to produce C28, a newly engineered polymerase that can synthesize RNA with a rate of ~3 nt s−1 and of >99% fidelity. C28 is capable of long-range RNA synthesis, reverse transcription and chimeric DNA–RNA amplification using the PCR. Despite strong discrimination against other genetic systems, C28 readily accepts several 2′F and base-modified RNA analogs. Together, these findings highlight the power of directed evolution as an approach for reprogramming DNA polymerases with activities that could help drive future applications in biotechnology and medicine.

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Fig. 1: Library generation and selection strategy.
Fig. 2: Evolution of a DNA-dependent RNA polymerase.
Fig. 3: Functional analysis of RNA polymerase C28.
Fig. 4: Fidelity and template composition requirements of RNA polymerase C28.
Fig. 5: Modified RNA synthesis by RNA polymerase C28.
Fig. 6: Crystallographic analysis of the engineered C28 RNA polymerase.

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Data availability

The atomic coordinates and structure factors for the crystal structure of C28 have been deposited with the Research Collaboratory for Structural Bioinformatics (PDB ID: 9MYE). Other data are available in the main text and Supplementary Information section. Source data are provided with this paper.

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Acknowledgements

The authors would like to thank members of the Chaput lab for their helpful comments and suggestions. This work was supported by the CLP and GM Divisions of the National Science Foundation (CHE-2433788 to J.C.C.) and the University of California, Irvine.

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Author notes

  1. These authors contributed equally: Esau L. Medina, Victoria A. Maola.

Authors and Affiliations

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

    Esau L. Medina, Victoria A. Maola, Mohammad Hajjar, Grace K. Ko, Ethan J. Ho, Alexandria R. Horton, 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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  1. Esau L. Medina
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Contributions

J.C.C., N.C. and E.L.M. conceived the project and designed the experiments. E.L.M. and V.A.M. evolved the enzyme. E.L.M., V.A.M., M.H. and A.R.H. performed the biochemical characterization. M.H., G.K.K. and E.J.H. crystallized the enzyme. J.C.C., E.L.M., V.A.M. and M.H. wrote the manuscript. All authors reviewed and commented on the manuscript.

Corresponding author

Correspondence to John C. Chaput.

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Competing interests

J.C.C., E.L.M., V.A.M. and the University of California, Irvine, have filed a patent application on the composition and activity of the C28 RNA polymerase. The other authors declare no competing interests.

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Nature Chemical Biology thanks Jack Szostak, Yi Shi for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 C28 reversion analysis.

a, Three segments of C28 harboring acquired mutations were reverted back to the parental Tgo-QGLK to create individual reversion constructs, RC1 (triangle), RC2 (square), and RC3 (diamond). RC3 contains five mutations, three of which are not observed in the crystal structure. b, Comparison of C28 activity against RC1, RC2, and RC3. Product formation was evaluated after 15, 30, or 60 s of incubation at 55 °C in an RNA primer extension assay with decreasing substrate concentrations.

Source data

Extended Data Fig. 2 C28 activity under fidelity assay conditions.

a, Schematic depicting the standard primer extension assay using a 5’ IR680-labeled primer annealed to a DNA template. b, Schematic depicting the ligation of a 5’ IR680-labeled primer to a chemically synthesized 5’ phosphorylated RNA oligonucleotide to generate an authentic standard for the product of the primer extension reaction. c, Denaturing PAGE analysis of C28 product formation after 1-h of incubation at 55 °C along with an authentic standard of primer-extended product.

Source data

Extended Data Fig. 3 C28 extends primers despite template mismatches.

A schematic depicts the 5’ IR680-labeled primer terminating in a 3’ cytosine, paired with a DNA template in which the complementary position (X) is either a matched guanine or one of the three mismatched bases (adenine, cytosine, or thymine). Comparison of C28 product formation reveals full-length product in all primer-duplex pairs following a 1-h incubation at 55 °C.

Source data

Extended Data Fig. 4 Mapping mutations acquired by homologous recombination.

a, The crystal structure of C28 is colored according to the distance to the active site, which is defined as the 3’-hydroxyl of U12. Residues within a 10 Å radius are green, residues between 10 and 35 Å are yellow, and residues outside 35 Å are red. Spheres indicate the alpha carbons of residues in C28 that differ from Tgo-QGLK (exo-). A rotated view provides clarity and perspective. b, The distance of each residue to the active site is measured in Å and mapped over the polymerase domains. Spheres are color coordinated to mutations shown in a. Three green triangles indicate the catalytic aspartates in the active site. Data exclude three C-terminal mutations not observed in the crystal structure.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1–8 and Supplementary Figs. 1–19.

Reporting Summary

Supplementary Data 1

Raw and processed data for kinetic analysis of RNA polymerases.

Source data

Source Data Fig. 3

Uncropped gels for Fig. 3b–f.

Source Data Fig. 4

Uncropped gels for Fig. 4c,e.

Source Data Fig. 5

Uncropped gel.

Source Data Extended Data Fig. 1

Uncropped gels.

Source Data Extended Data Fig. 2

Uncropped gel.

Source Data Extended Data Fig. 3

Uncropped gels.

Source Data Extended Data Fig. 4

Distance to active site data.

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Medina, E.L., Maola, V.A., Hajjar, M. et al. Rapid evolution of a highly efficient RNA polymerase by homologous recombination. Nat Chem Biol (2026). https://doi.org/10.1038/s41589-025-02124-7

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