Abstract
DNA polymerases are complex molecular machines capable of replicating genetic material using a template-driven process. While the copying function of these enzymes is well established, their ability to perform untemplated DNA synthesis is less well characterized. Here, we explore the ability of DNA polymerases to synthesize DNA fragments in the absence of a template. We use long-read nanopore sequencing, real-time fluorescence assays, and atomic force microscopy to observe the synthesis and physical structure of pools of DNA products derived from a diverse set of natural and engineered DNA polymerases across varying temperatures and buffer compositions. We detail the features of the DNA fragments generated, enrichment of select sequence motifs, and demonstrate that the sequence composition of the synthesized DNA can be altered by modifying environmental conditions. This work provides extensive data to better discern the process of untemplated DNA polymerase activity and may support its potential repurposing as a technology for the guided synthesis of DNA sequences on the kilobase-scale and beyond.
Data availability
The DNA sequencing data generated in this study have been deposited in the European Nucleotide Archive (ENA) under accession code PRJEB107084 (https://www.ebi.ac.uk/ena/browser/view/PRJEB107084). In addition, sequencing data (raw and processed), real-time PCR data and AFM images are available from Zenodo at: https://doi.org/10.5281/zenodo.17956595. Source data are provided with this paper.
Code availability
Analysis scripts are available from Zenodo at: https://doi.org/10.5281/zenodo.17956595.
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Acknowledgements
We would like to thank the anonymous reviewers for their valuable comments and suggestions that have significantly strengthened this work. This research was supported by Replay Holdings Inc., a Royal Society University Research Fellowship grant URF\R\221008 (T.E.G.), a Turing Fellowship from The Alan Turing Institute under the Engineering and Physical Sciences Research Council (EPSRC) grant EP/N510129/1 (T.E.G.), BrisEngBio, a UKRI Engineering Biology Transition Award grant BB/W013959/1 (T.E.G.), and by the Medical Research Council (MRC) as part of United Kingdom Research and Innovation (UKRI) MRC program grant MC_U105178804 (P.H.) We would also like to thank the Replay Genome Writing Team for insightful discussions and feedback on the research.
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A.W., A.H., T.E.G., L.M.O. and G.L. conceived the project. T.E.G., S.D.C., A.W. and G.L. designed the experiments. P.H. provided the 3A10 and RT521K engineered DNA polymerases for testing. S.D.C., T.C.T.I. and T.E.G. performed the sequencing experiments. S.D.C. performed the real-time fluorescence assays. L.P. performed and analyzed data from the AFM experiments. T.C.T.I. supported the AFM experiments. T.E.G. and S.D.C. carried out all the analyses of the sequencing data with input from G.L., B.T.R. and I.D.W.S. T.E.G. supervised all experiments and wrote the initial manuscript. All authors contributed to the interpretation of the results and final editing of the manuscript.
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A.W., G.L., B.T.R. and L.M.O. have been employees of Replay Holdings Inc. T.E.G., I.D.W.S., A.H. and P.H. have consulted for Replay Holdings Inc. The remaining authors declare no competing interests.
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Castle, S.D., Irvine, T.C.T., Woolfson, A. et al. Analysis and control of untemplated DNA polymerase activity for guided synthesis of kilobase-scale DNA sequences. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69915-x
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DOI: https://doi.org/10.1038/s41467-026-69915-x
