Atomistic Molecular Dynamics Simulations of DNA in Complex 3D Arrangements for Comparison with Lower Resolution Structural Experiments.

Atomic-level computer simulations are a very useful tool for describing the structure and dynamics of complex biomolecules such as DNA and for providing detail at a resolution where experimental techniques cannot arrive. Molecular dynamics (MD) simulations of mechanically distorted DNA caused by agents like supercoiling and protein binding are computationally challenging due to the large size of the associated systems and timescales. However, nowadays they are achievable thanks to the efficient usage of GPU and to the improvements of continuum solvation models. This together with the concurrent improvements in the resolution of single-molecule experiments, such as atomic force microscopy (AFM), makes possible the convergence between the two. Here we present detailed protocols for doing so: for performing molecular dynamics (MD) simulations of DNA adopting complex three-dimensional arrangements and for comparing the outcome of the calculations with single-molecule experimental data with a lower resolution than atomic.

Integration host factor bends and bridges DNA in a multiplicity of binding modes with varying specificity.

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.

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides.

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.

SerraNA: a program to determine nucleic acids elasticity from simulation data.

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 elastic constants emerge from local fluctuations is less understood. Here, we present SerraNA, which is an open software that calculates elastic parameters of double-stranded nucleic acids from dinucleotide length up to the whole molecule using ensembles from numerical simulations. The program reveals that global bendability emerge from local periodic bending angles in phase with the DNA helicoidal shape. We apply SerraNA to the whole set of 136 tetra-bp combinations and we observe a high degree of sequence-dependence with differences over 200% for all elastic parameters. Tetramers with TA and CA base-pair steps are especially flexible, while the ones containing AA and AT tend to be the most rigid. Thus, AT-rich motifs can generate extreme mechanical properties, which are critical for creating strong global bends when phased properly. Our results also indicate base mismatches would make DNA more flexible, while protein binding would make it more rigid. SerraNA is a tool to be applied in the next generation of interdisciplinary investigations to further understand what determines the elasticity of DNA.