DNA in the cell is tangled and twisted, adopts complex topologies, is decorated with a myriad of DNA binding proteins and is frequently maintained under superhelical stress (1). To process DNA, biology has evolved DNA packaging machines, such as eukaryotic histones and prokaryotic gyrases, that maintain order in this topological landscape. Unlike the crystalline, linear DNA that led to the discovery of its double helical nature (2), there is now a growing appreciation that the context of DNA in the wider genome is vital to its function (3). Understanding how DNA behaves in its cellular environment is currently as challenge of complexity, which can be enhanced by a better understanding of the fundamental properties of DNA. This includes how DNA responds to supercoiling and stress (4) to interact with other oligonucleotides and proteins. Here, we use a combination of high-resolution AFM (5) and atomistic MD simulations (6) to provide unparalleled single molecule insight into the structure of DNA under superhelical stress down to the base pair level (Figure 1). We use DNA minicircles, only twice the persistence length of DNA, to probe the structure and function of supercoiled DNA(7). We observe that though the discrete, quantised nature of these minicircles may imply a homogenous population, the innate flexibility of DNA under superhelical stress results in a large heterogenous population. Furthermore, we show that DNA at this length scale is highly dynamic, and able to change its conformation even whilst tethered to a surface. We believe that it is this dynamic conformational heterogeneity which allows for supercoiled DNA to bind with a diverse range of substrates. We demonstrate that two distinct binding modalities, triplex formation and repressor factor binding, which either rigidify or bend DNA, are both able to be accommodated within the large conformational population of DNA minicircles. Through combining high resolution microscopy and atomistic simulations, we provide a toolkit to understand the structure and dynamics of supercoiled DNA. We observe conformational changes in DNA and associated binding proteins at sub-nanometre spatial resolution and sub second temporal resolution. These results improve our understanding of DNA structure, and how this may influence the interaction of DNA with itself, diagnostic therapeutics, and innate proteins - key in regulating gene expression. Figure 1: High resolution AFM and atomistic molecular dynamics simulations reveal the structure of supercoiled DNA to the base pair level. (UNABLE TO SHOW ON THIS WEBSITE) 1. Bates AD, Maxwell A. DNA Topology. Oxford University Press; 2005. 2. Franklin RE, Gosling RG. Evidence for 2-Chain Helix in Crystalline Structure of Sodium Deoxyribonucleate. Nature. 1953 Jul;172(4):156–7. 3. Fogg JM, Randall GL, Pettitt BM, Sumners DWL, Harris SA, Zechiedrich L. Bullied no more: when and how DNA shoves proteins around. Quarterly Reviews of Biophysics 4. Irobalieva RN, Fogg JM, Catanese DJ, Sutthibutpong T, Chen M, Barker AK, et al. Structural diversity of supercoiled DNA. Nat Comms. 2015 Oct 12;6:8440. 5. Pyne A, Thompson R, Leung C, Roy D, Hoogenboom BW. Single-Molecule Reconstruction of Oligonucleotide Secondary Structure by Atomic Force Microscopy. Small. 2014 Apr 17;10(16):3257–61. 6. Fogg JM, Kolmakova N, Rees I, Magonov S, Hansma H, Perona JJ, et al. Exploring writhe in supercoiled minicircle DNA. J Phys: Condens Matter. 2006 Apr 12;18(14):S145–59.