Persistence length and scaling properties of single-stranded DNA adsorbed on modiﬁed graphite

We have characterized the polymer physics of single-stranded DNA (ssDNA) using atomic force microscopy. The persistence length l(p) of circular ssDNA adsorbed on a modified graphite surface was determined independently of secondary structure. At a very low ionic strength we obtained l(p)=9.1 nm from the bond correlation function. Increasing the salt concentration lead to a decrease in l(p); at 1 mM NaCl we found l(p)=6.7 nm, while at 10 mM NaCl a value l(p)=4.6 nm was obtained. The persistence length was also extracted from the root-mean-square end-to-end distance and the end-to-end distance distribution function. Finally, we have investigated the scaling behavior using the two latter quantities, and found that on long length scales ssDNA behaves as a two-dimensional self-avoiding walk.

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.

EPFL2008

The promotion of secondary structures in single-stranded DNA by drugs that bind to duplex DNA: an atomic force microscopy study

Giovanni Dietler, Kristian Rechendorff, Guillaume Witz

We study the behavior of single-stranded DNA (ssDNA) in the presence of well-known drugs with either an intercalating binding mode, such as daunorubicin, actinomycin D, and chloroquine, or a minor groove binding mode, such as netropsin and berenil, by atomic force microscopy (AFM). At very low salt conditions, ssDNA molecules adopt an unstructured conformation without secondary structures. We observe that under these conditions additions of drugs that bind to double-stranded DNA ( dsDNA) promote the formation of secondary structures in ssDNA. Furthermore, with an increase of concentration of the drugs, the extension as well as the thermal stabilization of these hairpins was observed.