DNA supercoiling fundamentally constrains and regulates the storage and use of genetic information. While the equilibrium properties of supercoiled DNA are relatively well understood, the dynamics of supercoils are much harder to probe. Here we use atomic force microscopy (AFM) imaging to demonstrate that positively supercoiled DNA plasmids, in contrast to their negatively supercoiled counterparts, preserve their plectonemic geometry upon adsorption under conditions that allow for dynamics and equilibration on the surface. Our results are in quantitative agreement with a physical polymer model for supercoiled plasmids that takes into account the known mechanical properties and torque-induced melting of DNA. We directly probe supercoil dynamics using high-speed AFM imaging with subsecond time and ∼nanometer spatial resolution. From our recordings we quantify self-diffusion, branch point flexibility, and slithering dynamics and demonstrate that reconfiguration of molecular extensions is predominantly governed by the bending flexibility of plectoneme arms. We expect that our methodology can be an asset to probe protein–DNA interactions and topochemical reactions on physiological relevant DNA length and supercoiling scales by high-resolution AFM imaging.
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