Research in the field of single biopolymers using scanning probe microscopy

Current thesis is devoted to the investigation of biopolymers using AFM, a well established and widely used technique. To begin with, we studied processing of AFM images and its automation. Our software product, DNA Trace, permit one to quickly extract scientifically meaningful data from a huge number of AFM scans. Then, this program enabled us to perform a study on how polymers, with DNA as a model, change their conformation from three to two dimensions upon their deposition onto the surface. DNA molecules suit well for such an investigation as they are studied in detail, easy to deposit onto mica surface, have relatively large persistence length to be traced on the images and, the most important, relax from 3D to 2D, the timescale of this process being in order of tens seconds to minutes. Besides, we introduced so-called concentration function to describe numerically such transition and adapted formulas of the theory of diffusion to polymers. We depicted this process as a diffusional propagation of relaxation of short segments of polymer. The results we have obtained are very promising and hopefully will lead to the better understanding of the theory of polymers, as the latter are widely used in all areas of industry. Then, this work describes the possibility to increase the sensitivity of cantilever deflection detection. We etched a diffraction grating on the backside of cantilever and used diffractive rather than mirror reflection of a laser beam, which gave geometrical advantage. We also used position-sensitive duo-lateral diode in this setup and finally got the increase of sensitivity of about 5 - 10 times compared to common detection scheme. Finally, a big chapter of this thesis is devoted to force spectroscopy, the technique, which permit one to measure forces of interaction between biomolecules and thus obtain kinetic parameters of interaction. We studied barnase - barstar pair and then flaviviral proteins - putative cell receptors interaction. The results obtaining by the latter study will hopefully lead to the model of flaviviral - cell membrane fusion.

Study of Topoisomerase-DNA Interaction Using Atomic Force Microscopy

Livan Bladimir Alonso Sarduy

The aim of this thesis was to explore the topoisomerase-DNA interactions using atomic force microscopy (AFM) in a liquid environment. Firstly, we present a programmable microcontroller-driven injection system for the exchange of an imaging medium while using AFM. Using this low-noise system, high-resolution imaging can be performed during the process of injection without disturbance. The use of the injection system was put into practice when studying the conformational changes of DNA molecules during the injection of intercalating agents, such as daunorubicin (an anticancer drug) and ethidium bromide, into the fluid chamber. An observation of their mode of interaction with DNA might help elucidating their mechanism of action, thereby facilitating the development of more specific drugs. Part of this thesis deals with the way enzymes interact with DNA molecules. We found that human type II topoisomerases (Topo II) bind preferentially to DNA crossovers. About 50% of the DNA crossings, where the Topo II were bound to, presented an angle of a nearly perpendicular orientation, suggesting a favoured binding geometry in the Topo II recognition. Our studies with AFM also helped us visualize the dynamics of the unknotting action of Topo II in knotted DNA molecules. Additionally we investigated the interactions between Topo II and DNA by applying single molecule force spectroscopy. This study evidenced the inhibitor effect of the aclarubicin (anticancer drug) and evaluated the preferential binding of Topo II to specific DNA supercoiled forms. Finally, we explored the Topo II dynamics using a novel technique that detects motion from nano- to micro-metre sized systems. This method opens new possibilities for the study of conformational changes of single proteins, biochemical reactions, as well as drug-target inhibition.

EPFL2012

From polymers to gene regulation

Aleksandre Japaridze

DNA molecule is the fundamental component of every living organism, since it encodes the hereditary information. Although the genetic code of almost 200 species has been sequenced, the direct connection between gene sequence, DNA structure and biological function remains poorly understood. The aim of this thesis was to investigate the connection between DNA function and structure. Throughout the thesis we will discuss experiments testing the link between different physical properties of DNA with its biological function. For this purpose we used atomic force microscopy (AFM), a high resolution imaging technique. The first chapter is devoted to explain the basic concepts of AFM technique, as well as the composition and structure of DNA molecules. We also describe some of the polymer physics models of DNA, defining basic quantities like the persistence and the critical exponent. In chapter 2 we discuss experiments, in which we studied changes in the physical properties of DNA molecules, when bound to the substrate surface with various methods. We show that, when DNA is strongly bound to the surface, it retains its physiological B-form. On contrary, when weakly bound, a conformational B-to-A-form transition takes place. We also discuss a novel experimental method, combining nanometer sized PDMS slits with AFM, for studying DNA in geometrical confinement. When DNA is weakly confined, DNA persistence length increases, molecules elongate and adopt more anisotropic shapes. Under stronger confinement, DNA hairpins form. In chapter 3 we study binding of staining dyes, proteins and RNA polymerase (RNAP) to DNA molecules. First we show that DNA physical properties are significantly affected when stained with dyes. Depending on the dye binding mode, either the contour length, or the persistence length or both simultaneously change. We move further and investigate the role of protein amino acids and DNA sequence in the formation of nucleoprotein complexes. On the example of EspR protein, a key virulence regulator in Mycobacterium tuberculosis (MTB), we show that single amino acid mutations, lead to lower DNA binding affinity. These mutations also impair the formation of higher order protein-DNA complexes, silencing the MTB virulence. Afterwards, on the examples of RNAP and H-NS protein, we show the importance of DNA sequence for the binding and higher order oligomerization. By introducing DNA mutations disrupting the helical phasing between RNAP binding sites in the fis promoter sequence, we significantly lower the RNAP binding affinity. The helical phasing of the binding sites somehow coordinates the cooperative binding of RNAP to the promoter. Finally on the model of H-NS protein, we show how different spatial arrangements of strong binding sites for an architectural protein in the DNA, determine the final 3D structure of the assembled nucleoprotein complex. The final chapter, is fully devoted to the study of a completely new type of DNA organisation, which we call Hyperplectonemes. Hyperplectonemes are very ordered DNA structures, formed by large supercoiled molecules, in the presence of attractive DNA-DNA potential. First, we describe their structure and its dependence on various environmental factors, than their binding to nucleoid associated proteins. We argue that this emerging DNA organisation is the basic structure of the bacterial chromatin, which is further modulated by numerous DNA binding and condensing proteins in vivo.

EPFL2015

Scaling properties of DNA knots studied by atomic force microscopy

Erika Ercolini

The main subject of the present thesis is the experimental study of the scaling properties of DNA knots of different complexities by tapping mode atomic force microscopy (AFM) in air. Homo- or heterogeneous mixture of DNA knot types were deposited onto mica in regimes of (i) strong adsorption, which induces a kinetic trapping of the molecules, and of (ii) weak adsorption, which permits relaxation on the surface. The contour of each knotted molecule was analyzed by a box counting algorithm, giving the number of boxes containing a part of the molecule, N(L), as a function of the box size, L, allowing to recover the relation N(L) ≈ L-df, where df is the fractal dimension and ν = 1/df is the scaling exponent. This relationship is complicated by the presence of a persistence length of DNA (about 50 nm) which introduces a crossover from a rigid rod behavior to a self-avoiding walk behavior. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent ν ≈ 0.58 within error. In (ii), the value ν ≈ 0.66, intermediate between the 3D and 2D (ν = 3/4) exponents, was found, indicating an incomplete 2D relaxation or a different polymer universality class. A different analysis, where the fractal dimension was determined by a customized box counting algorithm giving the knot mass as a function of the box size, yielded compatible results. In the case of weak binding conditions, AFM images show evidence of the localization of knot crossings, which is an effect theoretically predicted for knotted polymers confined in two dimensions (flat knots). Part of this thesis is dedicated to a fascinating application of AFM: the imaging of biomolecules in an aqueous buffer. In particular, images of plasmids adsorbed to mica strong enough to be visualized and loosely enough to see them moving in consecutive scans will be shown. The possibility of imaging under buffer molecules loosely anchored to the substrate opens the way to the investigation of biological processes near physiological conditions. The first step of the study of homologous recombination will be presented. We will also show images concerning our study of the activity of topoisomerase I on supercoiled plasmids. Clusters of DNA molecules and proteins were found when imaging in presence of magnesium, in agreement with recent findings by AFM in air. The research on topoisomerases and their interactions with inhibitors or poisons is a hot topic, being these enzymes targets of anti-cancer drugs. Our study paves the way to the investigation of the activity of topoisomerase II, the enzyme which removes knots from DNA. Finally, the principles of static and dynamic light scattering, and the results of dynamic light scattering on latex beads of known size will be presented. Particular emphasis will be given to the discussion of the contribution of light scattering techniques for future experiments on the study of the static and dynamic properties of DNA knotted molecules in solution. These experiments would contribute to shed light on the possible dependence of the gyration radius of knotted polymers on the knot type, and would allow testing theoretical predictions about their relaxation time.