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Simplified, open‐source analysis of DNA‐binding proteins

The structure, regulation, and maintenance of DNA is critical to cellular function. Errors in these underlying mechanisms play a major role in diseases such as cancer. As a result, insights into the molecular details are needed to further our understanding, improve biological models, and develop novel therapeutics. Single‐molecule techniques have emerged as useful tools for uncovering the activity and function of DNA‐binding proteins in real time. Furthermore, the data from single‐molecule techniques allows for an accurate quantification of many molecular properties, such as binding location, kinetics, etc. For many researchers, however, developing an analysis pipeline to quantify single‐molecule data is challenging due to the required time, resources, and expertise. To address these obstacles, we developed an automated, open‐source image processing and data quantification toolbox. We show that our toolbox allows for easy, rapid, and intuitive processing of single‐molecule data. Using systems like the gene editing CRISPR/Cas9 system, we demonstrate that our toolbox 1) reports DNA binding specificity at the base‐pair level, 2) measures binding constants (kon/koff) from the direct visualization of individual complexes interacting with DNA, and 3) allows for the accurate quantification of enzymatic processivity and protein diffusion. Overall, we show that our simplified, open‐source data analysis platform allows for automated processing of single‐molecule data and rapid characterization of virtually any DNA‐binding protein or polymerase. This toolbox is accessible to all researchers and will enhance efforts to understand a wide range of normal and pathological DNA processes.