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 lambda 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.

Automated summary

Traditional enzyme characterization methods only provide population averages, while single-molecule methods can reveal kinetics and population heterogeneity but are often complex and time-consuming. We present a high-throughput single-molecule assay for rapidly characterizing DNA metabolism-related proteins by measuring changes in fluorescence intensity. Our method, combined with automated data analysis, can provide enzymatic activity data for thousands of molecules in less than an hour. We demonstrated three different enzyme activities and discovered a previously unknown activity of the UvrD helicase.