Studies of supercoiling-induced denaturation within DNA plasmids using single-molecule Convex Lens-induced Confinement microscopy

Synopsis

DNA, RNA and proteins, which drive life, have complicated, constantly changing structures. For example, DNA inside cells is supercoiled, and the amount of supercoiling is constantly under flux. This supercoiling can drive structural transitions, such as AT-rich regions in under-twisted DNA denaturing while at physiological conditions. Such denatured regions may have important functions. For example, denatured DNA can act as targets for single-stranded binding proteins which help regulate gene expression. Thus, it is important to develop tools and studies to better understand these structures. In this work, we use single-molecule Convex Lens-induced Confinement (CLiC) microscopy in conjunction with chemical footprinting to study the biophysics of these denaturation sites. First, we studied the out-of-equilibrium dynamics of such denaturation after a temperature perturbation. We found that the rate of transition of the denaturation site was dependent on the direction of the perturbation, with plasmids that were heated first exhibiting a slower relaxation than plasmids that were chilled. We hypothesized that unidentified secondary structures caused this hysteresis. Second, we developed a fluorescent probe based on a molecular beacon to improve the CLiC microscopy oligo-DNA binding assay used for detecting single-stranded DNA. The final design was a stemless molecular beacon with a fluorophore on one end and a quencher on the other. This design allowed for a 10- to 100-fold increase in probe concentration used in this assay and enabled simultaneous measurements of the states of two denaturation sites within one plasmid. Finally, we studied competition between two denaturation sites within the same plasmid. We found that at low superhelicities only one site could open, while higher superhelicities allowed both to open within the same plasmid. However, we predicted that adding a specific second denaturation site would suppress the opening of the first, which we did not observe. Overall, this work provides new insights into the behaviour of DNA secondary structures, both out-of-equilibrium and under competition, and develops new tools to better study the structural biology of DNA.