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.
Example analysis mapping CIRSPR/Cas9 binding to a DNA substrate for multiple samples.
