Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism

The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5′-, but not 3′-ends of biotinylated DNA.

Citation

Mueller, S. H., Fitschen, L. J., Shirbini, A., Hamdan, S. M., Spenkelink, L. M., & van Oijen, A. M. (2022). Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism. Nucleic Acids Research. https://doi.org/10.1093/nar/gkac949

Acknowledgements

Australian Research Council [DP150100956, DP180100858 to A.M.v.O.]; Australian Laureate Fellowship [FL140100027 to A.M.v.O.]; National Health and Medical Research Council [NHMRC Investigator grant 2007778 to L.M.S.]; Australian Government Research Training Program Scholarship (to S.H.M.). Funding for open access charge: Australian Research Council. The authors thank Dr Jacob Lewis (University of Wollongong) and Prof. Michael O’Donnell (Rockefeller University) for contributing reagents.