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

ABSTRACT: 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 fundamen

SUBMITTER: Mueller SH 

PROVIDER: S-EPMC9841422 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature