Stemless molecular beacon probes for single-molecule detection of supercoil-induced DNA denaturation.

While the formation of single-stranded regions in DNA plays important roles in processes such as replication, repair, and transcription, the underlying mechanics and kinetics of these structures are not well understood. Here, we combine single-molecule Convex Lens-induced Confinement (CLiC) microscopy with stemless molecular beacons to detect and study correlations in the formation of multiple single-stranded regions within plasmids in suspended solution conditions. We demonstrate that negative supercoiling not only increases denaturation of these sites, but also augments oligo-plasmid binding interactions. The observed denaturation disagrees with theoretical predictions stemming from equilibrium thermodynamics, suggesting the structures may form metastable states. To our knowledge, this is the first single-molecule study of plasmids in suspended solution conditions to make measurements correlating the formation of multiple single-stranded regions within the same plasmid. Beyond DNA mechanics, these methods have the potential to enable insights in a broad range of fields, such as informing the development of therapeutics sensitive to DNA and RNA structure.