Single DNA conformations and biological function

From a nanoscience perspective, cellular processes and their reduced in vitro imitations provide extraordinary examples for highly robust few or single molecule reaction pathways. A prime example are biochemical reactions involving DNA molecules, and the coupling of these reactions to the physical conformations of DNA. In this review, we summarise recent results on the following phenomena: We investigate the biophysical properties of DNA-looping and the equilibrium configurations of DNA-knots, whose relevance to biological processes are increasingly appreciated. We discuss how random DNA-looping may be related to the efficiency of the target search process of proteins for their specific binding site on the DNA molecule. And we dwell on the spontaneous formation of intermittent DNA nanobubbles and their importance for biological processes, such as transcription initiation. The physical properties of DNA may indeed turn out to be particularly suitable for the use of DNA in nanosensing applications.Comment: 53 pages, 45 figures. Slightly revised version of a review article, that is going to appear in the J. Comput. Theoret. Nanoscience; some typos correcte

Geometric modeling, simulation, and visualization methods for plasmid DNA molecules

Plasmid DNA molecules are a special type of DNA molecules that are used, among other applications, in DNA vaccination and gene therapy. These molecules are characterized by, when in their natural state, presenting a closed-circular conformation and by being supercoiled. The production of plasmid DNA using bacteria as hosts implies a purification step where the plasmid DNA molecules are separated from the DNA of the host and other contaminants. This purification process, and all the physical and chemical variations involved, such as temperature changes, may affect the plasmid DNA molecules conformation by uncoiling or even by open them, which makes them useless for therapeutic applications. Because of that, researchers are always searching for new purification techniques that maximize the amount of supercoiled plasmid DNA that is produced. Computer simulations and 3D visualization of plasmid DNA can bring many advantages because they allow researchers to actually see what can happen to the molecules under certain conditions. In this sense, it was necessary to develop reliable and accurate geometric models specific for plasmid DNA simulations. This dissertation presents a new assembling algorithm for B-DNA specifically developed for plasmid DNA assembling. This new assembling algorithm is completely adaptive in the sense that it allows researchers to assemble any plasmid DNA base-pair sequence along any arbitrary conformation that fits the length of the plasmid DNA molecule. This is specially suitable for plasmid DNA simulations, where conformations are generated by simulation procedures and there is the need to assemble the given base-pair sequence over that conformation, what can not be done by conventional predictive DNA assembling methods. Unlike traditional molecular visualization methods that are based on the atomic structure, this new assembling algorithm uses color coded 3D molecular surfaces of the nucleotides as the building blocks for DNA assembling. This new approach, not only reduces the amount of graphical objects and, consequently, makes the rendering faster, but also makes it easier to visually identify the nucleotides in the DNA strands. The algorithm used to triangulate the molecular surfaces of the nucleotides building blocks is also a novelty presented as part of this dissertation. This new triangulation algorithm for Gaussian molecular surfaces introduces a new mechanism that divides the atomic structure of molecules into boxes and spheres. This new space division method is faster because it confines the local calculation of the molecular surface to a specific region of influence of the atomic structure, not taking into account atoms that do not influence the triangulation of the molecular surface in that region. This new method also guarantees the continuity of the molecular surface. Having in mind that the aim of this dissertation is to present a complete set of methods for plasmid DNA visualization and simulation, it is also proposed a new deformation algorithm to be used for plasmid DNA Monte Carlo simulations. This new deformation algorithm uses a 3D polyline to represent the plasmid DNA conformation and performs small deformations on that polyline, keeping the segments length and connectivity. Experiments have been performed in order to compare this new deformation method with deformation methods traditionally used by Monte Carlo plasmid DNA simulations These experiments shown that the new method is more efficient in the sense that its trial acceptance ratio is higher and it converges sooner and faster to the elastic energy equilibrium state of the plasmid DNA molecule. In sum, this dissertation successfully presents an end-to-end set of models and algorithms for plasmid DNA geometric modelling, visualization and simulation

Protein 3D Structure Computed from Evolutionary Sequence Variation

The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing

Bending rigidity, supercoiling and knotting of ring polymers: models and simulations

The first part of the thesis was focussed on the interplay between knotting propensity and bending rigidity of equilibrated rings polymers. We found a surprising result: the equilibrium incidence of knots has a strongly non- monotonic dependence on bending, with a maximum at intermediate flexural rigidities. We next provided a quantitative framework, based on the balance of bending energy and configurational entropy, that allowed for rationalizing this counter-intuitive effect. We next extended the investigation to rings of much larger number of beads, via an heuristic model mapping between our semiflexible rings of beads and self-avoiding rings of cylinder. By the mapping, we not only confirmed the unimodal knotting profile for chains of 1,000 beads, but further found that chains of > 20,000 beads are expected to feature a bi-modal profile. We believe it would be most interesting to direct future efforts to confirm this transition from uni- to bi-modality using advanced sampling techniques for very long polymer rings. The second part of the thesis focused on the interplay of DNA knots and su- percoiling which are typically simultaneously present in vivo. We first studied this interplay by using oxDNA, an accurate mesoscopic DNA model and using it to study ings of thousands of base pairs tied in complex knots and with or without negative supercoiling (as appropriate for bacterial plasmids). By monitoring the dynamics of the DNA rings we found that the simultaneous presence of knots and supercoiling, and only their simultaneous presence, leads to a dramatic slowing down of the system reconfiguration dynamics. In particular, the essential tangles in the knotted region acquire a very long-lived character that, we speculate, could aid their recognition and simplification by topoisomerase. Finally, motivated by the recent experimental breakthrough that detected knots in eukaryotic DNA, we investigated the relationship between the compactness, writhe and knotting probability. The model was tuned to capture some of the salient properties of yeast minichromosomes, which were shown experimentally to become transiently highly knotted during transcription