BPS2026 – Single-molecule studies of DNA structure and interactions using emerging convex lens-induced confinement-based approaches

Though DNA structures play an important role in processes such as replication, repair, and transcription, the mechanisms behind their formation are not fully understood. Single-molecule techniques are invaluable for studying these structures and related interactions at a granular level. In this work, we present recent advancements in single-molecule convex lens-induced confinement (CLiC) microscopy to the study of DNA structure and interactions. With CLiC microscopy, molecules suspended in solution can be imaged for long times and with low background through confining them within geometries of interest. First, we present an assay that combines CLiC microscopy with stemless molecular beacons to quantify correlations between the formation of multiple single-stranded structures of interest within the same DNA molecule, suitable for use at high concentrations and in complex backgrounds. We apply this assay to investigate the formation of two supercoil-induced denaturation sites as a function of DNA supercoiling and demonstrate the competitive effect of “pinning” one site into a single-stranded configuration on the melting of the second site. Second, we present the development of attolitre wells for use in conjunction with CLiC microscopy to create small reaction chambers to facilitate studying the interactions between nucleic acids and conformational dynamics. Overall, these methods lay the groundwork for future studies of nucleic acid structure and interactions in cell like conditions.