Scaling properties of DNA knots studied by atomic force microscopy

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.

Investigation of Biophysical Properties of Biomolecules by Means of Atomic Force Microscopy

Jae Sun Jeong

Biomolecules are the building blocks of living organisms and perform the most important functions in a biological process. The aim of biophysics is to understand the biological functions of biomolecules in terms of their structure: structure and function of biomolecules has been an active research for several decades. Despite the evolution and emergence of new investigation techniques during the last 60 years, biological processes still present enormous challenges to our understanding due to the complex biological system spanning a wide range of spatial and temporal scales. An ideal biophysical method should have the capability to observe atomic level structures and dynamics of biological molecules in their physiological environment. It would also permit the visualization of the structures that form throughout the course of conformational changes or chemical reactions, regardless of the time scale. In this thesis, we have investigated two representative biomolecules using the well-defined biophysical methods in terms of behaviour and biophysical properties of biomolecules: Deoxyribonucleic acid (DNA) and the Amyloid-β (Aβ) peptide. Many different biophysical and biochemical tools were employed, where atomic force microscopy (AFM) was the primary instrument to observe the behaviour of the biomolecules in physiological conditions as well as the dynamic changes of structure of biomolecules. Moreover, AFM was used to measure the mechanical properties of biomolecules via direct and indirect methods. What is the behaviour of DNA molecules in an electric field? The behaviour of DNA molecules in an electric field is crucial for understanding interactions of DNA with electrically charged membrane that serves as a template for DNA based sensors and microarray applications, particularly under an electric field. Taken together, the ability to deposit a single DNA molecule on the electrode is essential to increase its specificity. To achieve this, we optimized the operational conditions in order to control a single DNA molecule adsorbing at +300 mV of potential and desorbing to/from the electrode at -25 mV of potential. Simultaneously dynamic imaging enabled us to analyse its morphological behaviour using real-time Electrochemical AFM (EC-AFM) in aqueous conditions. What is the molecular mechanism underlying Aβ42-fibrillogenesis and its role in pathogenesis? The molecular mechanism underlying Aβ42-fibrillogenesis is essential for defining the key determinant in the pathogenesis of Alzheimer’s disease. In this thesis, we carried out detailed biophysical studies to elucidate the structural changes associated with different stages of amyloid formation and provided unique perspectives on the molecular and structural basis underling the polymorphism of amyloid fibrils in Alzheimer’s disease. For the first time, we provided direct evidence of the existence of secondary-nucleation sites on the surfaces of Aβ42-fibrils and demonstrated that Aβ42-monomers play a crucial role in determining the structural properties and heterogeneity of the formed fibrils. In addition, real-time observation with in situ AFM was performed to define the mechanisms underlying oligomerization and the formation of protofibrils of Aβ42 in physiological conditions. Investigation of the elastic property of Aβ42 fibrils using two techniques based on the AFM. The self-assembly process of Aβ peptides into highly ordered amyloid fibrils is a crucial phenomenon in the cascade of pathological events that culminates in Alzheimer’s disease. Further, the assessment of the elastic properties of Aβ fibrils has recently attracted a lot of attention due to their potential biological applications. We measured the elastic property of Aβ42-fibrils immobilized on two types of substrate (positively charged mica and hydrophobic highly oriented pyrolytic graphite) and compared their properties using two techniques based on the AFM: Statistical analysis of thermal fluctuations and AFM based nanoindentation. The results from our studies show that the values of Young’s modulus of Aβ42 fibrils from the two substrates were similar (1.8 to 2.0 GPa), however they were different in the statistical analysis of thermal fluctuations (1.4 GPa, 2.3 GPa on mica and highly oriented pyrolytic graphite, respectively). These results imply that the method of thermal fluctuations was based on the shape of fibrils affected by the interaction force between the substrate and the fibrils.

EPFL2012

DNA Topology

Guillaume Witz

This thesis explores different aspects of DNA topology through experimental and numerical techniques. Topology is a vast mathematical field, that deals with the spatial properties of objects undergoing continuous deformations, but here it is restricted to the description of closed space curves such as rings, knots and catenanes. Topology is connected to practical problems in different domains including polymer physics, because a polymer chain is a type of space curve, whose statistical properties are affected by the closure of its ends; and biology, because the double-helical interwinding of the two strands formingDNA is described by topological concepts, and also because a closed DNA molecule can adopt complex topologies like knotted or catenated conformations in vivo. Both physical and biological aspects of topology are investigated in the three chapters of this thesis. Chapter 1 introduces on a general level the experimental techniques, numerical methods and topology concepts that are at the core of the presented research. Chapter 2 is an experimental study of the statistical properties of DNA rings adsorbed on surfaces, and imaged by atomic force microscopy (AFM). DNA rings of different sizes are first studied in a dilute state, allowing us to investigate the consequences of the circular topology on polymer physics of two-dimensional chains. We confirm certain theoretical results, e.g. the topological invariance of the scaling exponent in two dimensions; but we also propose new models taking into account the specificities of the problem, in particular the interplay between excluded volume effects and topology, for the description of standard polymer physics measures like the asphericity or the bond correlation function. In addition to isolated DNA rings, we study DNA rings in a semi-dilute state. Comparing the statistical properties of the DNA rings imaged by AFM with a numerical model, we formulate a simplified description of this system in terms of two dimensional pressurized vesicles. Finally, we study a last case, in which DNA rings are confined within circular boundaries in two dimensions. We show how confinement strongly affects the statistical properties of the DNA chains, and verify that our experimental system is a good model for polymer confinement by comparing the data with numerical simulations. In chapter 3, we use numerical simulations to study two other topological forms taken by closed DNA in vivo: catenanes and knots. In both cases, we analyze the conformations of these objects, in particular as a function of their torsional state, and find complex behaviors proper to these topologies: for example, the large scale supercoiling of toroidal catenanes, or the different response of left- and right-handed knots to torsion. We then focus on the activity of a class of enzymes called topoisomerases, that are able to change the topology of DNA molecules (e.g. by unknotting a DNA knot). In particular, we try to deduce how the structural properties of DNA knots and catenanes affect the activity of one of these enzymes called topo IV, and also simulate its activity. We show that our numerical model provides results that compare well with experimental data, and could therefore provide a basis for the design of new experiments. Finally, we present a new model for the simulation of the activity of another topoisomerase called gyrase, that is responsible for introducing supercoiling into DNA. This model includes a few essential features of the interaction between gyrase and DNA, which permit to efficiently reproduce experimental measurements.

EPFL2010

Conformation of Ring Polymers in 2D Constrained Environments

Giovanni Dietler, Kristian Rechendorff, Guillaume Witz

The combination of ring closure and spatial constraints has a fundamental effect on the statistics of semiflexible polymers such as DNA. However, studies of the interplay between circularity and constraints are scarce and single-molecule experimental data concerning polymer conformations are missing. By means of atomic force microscopy we probe the conformation of circular DNA molecules in two dimensions and in the concentrated regime (above the overlap concentration c*). Molecules in this regime experience a collapse, and their statistical properties agree very well with those of simulated vesicles under pressure. Some circular molecules also create confining regions in which other molecules are trapped. Thus we show further that spatially confined molecules fold into specific conformations close to those found for linear chains, and strongly dependent on the size of the confining box.