biostudies-literatureMueller SHAustralian Government Research Training Program ScholarshipNational Health and Medical Research CouncilAustralian Research Councile5https://www.ebi.ac.uk/biostudies/studies/S-EPMC9841422biostudies-literatureUnknown51(1)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 fundamenNucleic acids researchRapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism.PMC9841422DP180100858DP150100956FL1401000272007778Shirbini Avan Oijen AMFitschen LJHamdan SMSpenkelink LMMueller SHfalseRapid 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 fundamen2023-01-01T00:00:00Z2023 Jan2026-07-14T15:42:30.955Z2025-04-19T12:31:14.555ZS-EPMC98414223632165010.1093/nar/gkac949