Visualizing structure-mediated interactions in supercoiled DNA molecules

Abstract

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmid's structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

Single-molecule visualization of the effects of ionic strength and crowding on structure-mediated interactions in supercoiled DNA molecules

“…When extracted from cells, topoisomers have a distribution of supercoiling values centered around σ = −0.055, though cells can further supercoil their DNA to promote unwinding (28). As shown in Scott, et al, for the system presented in this work, increasing the overall supercoiling promotes binding due to an increase in site-unwinding (13).…”

“…The pits we used are on the same size scale (3 µm diameter, 500 nm deep) as an E. coli cell (500 nm diameter, 3 µm long). In this previous study, we demonstrated how unwinding and reaction rate between the probe and plasmid increased with temperature, but only unwinding was affected by supercoiling (13).…”

“…The microscopy setup, data acquisition, and image analysis were all performed using CLiC as in Scott, et al (13). In this microscopy system, two coverslips are separated by 30 µmthick double-sided tape.…”

“…Environmental conditions within cells are particularly important when considering higher-order DNA structure created by supercoiling, the torsional stress applied to DNA. This torsional tension, caused by under-or over-twisting the DNA double helix, has been shown to drive structural transitions in DNA, such as those from B-form to Z-form, or to fully unwound (11,12,13). These transitions are important physiologically: once the DNA is in one of these alternate structures, cells may initiate important processes which require unwinding of the double-helix, such as DNA transcription and replication (14,15).…”

“…We recently reported a novel approach to study DNA unwinding in which the binding of a fluorescently-labeled oligonucleotide probe to the plasmid's known unwinding site was used to measure its open state (13). By measuring the diffusion coefficient of the fluorescently-tagged probes, it was possible to determine when they were bound to plasmid unwinding sites.…”