DNA organization, regulation, and maintenance are fundamental to cellular function, and detailed molecular insights are essential for understanding disease, validating models, and developing new therapies. Real-time, single-molecule studies of protein-DNA interactions are key to this goal. Technologies such as the C-Trap enable direct observation of these processes with unprecedented resolution. In this work, we describe our efforts to advance the study of DNA-protein interactions through the integration of optical tweezers with confocal fluorescence microscopy and microfluidics. We showcase applications of these technologies in exploring DNA repair pathways, chromatin structure and stability, DNA replication, and the function of genome-editing tools such as CRISPR-Cas. We also introduce a strategy that leverages nuclear extracts in single-molecule assays, eliminating the need for protein purification and preserving post-translational modifications and native protein-protein interactions. This approach brings in vitro assays closer to the in vivo nuclear environment. Finally, we demonstrate how these innovations can be translated into accessible, user-friendly instruments, extending single-molecule technology beyond specialized biophysics labs. Together, these advances complement structural and cell biology methods, offering critical insights into essential DNA-binding processes.
Yazdi et al. (Sun,) studied this question.