In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF–DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: ‘associated’ (73° of DNA bend), ‘half-wrapped’ (107°) and ‘fully-wrapped’ (147°), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.

The resistance of DNA to stretch, twist and bend is broadly well estimated by experiments and is important for gene regulation and chromosome packing. However, their sequence-dependence and how bulk...

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures.It remains unclear how this supercoiling affects the double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance 2 between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition of diverse conformational substrates.

The resistance of DNA to stretch, twist and bend is broadly well estimated by experiments and is important for gene regulation and chromosome packing. However, their sequencedependence and how bulk elastic constants emerge from local fluctuations is less understood.Here, we present SerraNA, which is an open software that infers elastic parameters of doublestranded nucleic acids from bp length up to the whole molecule using ensembles from numerical simulations. Estimations of bulk elastic constants are in general agreement with experiments, although the static persistence length and stretch modulus parameters are more challenging to calculate due to DNA chirality and vibrations at the ends. The program also reveals that a soft persistence length is built up from local periodic bending angles in phase with 1 the DNA helicoidal shape. The whole set of 136 tetra-bp combinations present big differences in all elastic constants of over 200% demonstrating the importance of sequence. SerraNA is a tool to be applied in the next generation of interdisciplinary investigations to further understand what determines the elasticity of DNA. tetranucleotide level 22,23 and, among others, (iii) an explanation of contradictory stiffness data on A-tracts. 24 On a more coarse-grained level, Monte Carlo (MC) simulations have found an average persistence length of 53 nm with a standard deviation of 4 nm, most of the variability being originated at the level of static curvature, with dynamic persistence length fluctuating around 61 ± 1 nm.Previously, we designed the Length-Dependent Elastic Model (LDEM) for describing how bulk elastic properties emerge from base-pair (bp) fluctuations using the sampling obtained by nucleic acids simulations, 25 and, from this perspective, the model was applied to test the new forcefield Parmbsc1. 26 LDEM consists of an extended version of the algorithm used on the widelyknown 3DNA program 27,28 to geometrically describe two bp separated by an increasing number of nucleotides up to several DNA turns. 25 Bending, torsion and stretch are defined via tilt/roll, twist and distance between ends, respectively, and the associated elastic constants are evaluated by the method of inverse covariance matrix. 20,29,30 Using the LDEM on linear fragments of DNA, we found that the crossover from single bp level to bulk elastic behavior occurs typically within one helical turn of DNA. 25 In terms of torsion elasticity, we observed a transition from dinucleotide values of 30-50 nm to the long-range elastic constants of 90-120 nm broadly in agreement with experimental data. 8,13,14,31 The lengthdependence of stretch modulus followed a non-monotonic behaviour with a stiffening for lengths shorter than a DNA-turn due to the strong base-stacking interactions, followed by stabilization to similar values of force-extension measurements (1100-1500 pN). 11,12 Highly soft stretch modulus measured by SAXS experiments on short oligomers 32 was observed to be caused mainly by end effects. And finally for the persistence length, we f...