Nanomechanics of DNA and DNA-ligand interactions: focus on structural polymorphism and DNA condensation

In the last few decades, the constant development of novel microscopy techniques have created the basis for a new paradigm in the field of biophysics. Single-molecule techniques enabled to carry out experiments providing new information: the nanomanipulation of individual biomolecules revealed unknown insights into the elasticity and mechanics of molecules, improving the understanding of the fundamental relation between structural properties and biological functions. In particular, an AFM and mostly a MT setup were used during this thesis work, both located in biophysics laboratory of Prof. Francesco Mantegazza, at the University of Milano-Bicocca. Similar issues were encountered at the cellular level, because bulk experiments of conventional microscopy techniques provide information on average only, without taking into account the intrinsic biological heterogeneity. Recent developments in microfluidics enabled to follow individual cells over a long time and under controlled conditions. During the last part of this thesis project I used one of these microfluidic devices to perform time-lapse microscopy experiments at the single-cell level. These experiments were carried out during a visiting period of seven months in Prof. Pietro Cicuta’s laboratory, in Cavendish laboratory at University of Cambridge. In this thesis I dealt with three main research topics: • DNA structural polymorphism • nanomechanics of DNA-ligand interactions • the dual role of H-NS protein: DNA condensation and gene regulation The study of the conformational changes of DNA, namely the property of structural polymorphism, is addressed during two projects: one about the nanomechanics of a DNA analogue and another concerning the behavior of DNA at high supercoiling. The study of a DNA analogue enables to observe how a chemical modification of nucleotides can induce structural re- arrangements of the double-helix, biasing towards an A-like-form of DNA. The regimes of high supercoiling, both positive and negative supercoiling, show instead how an applied torsion at a certain forces can promote the formation of plectonemes or denaturation bubbles, which are conditions that favor particular structural transitions. The second major theme concerns the analysis of the nanomechanics of DNA-ligand complexes, particularly the interactions of DNA with anticancer drugs or with the H-NS protein and the crowding agent PEG. The project about the interactions between DNA and drugs clearly shows how the mechanical properties and the stability of DNA change due to the binding with compounds commonly used in clinics to treat tumors. On the other hand, the H-NS protein forms relatively stable DNA loops and influences the stability of the double helix, as well as the crowding agent. The protein binding mechanism has a preference for some DNA sequences and an unexpected concentration-dependent behavior. The analysis of the the DNA-H-NS interactions also enables, particularly in crowding conditions, to better understand the mechanism of DNA condensation inside the cell, one of the biological roles of H-NS. The second important function of this NAP is the gene regulation. To investigate the dual role of H-NS in great detail two complementary techniques have been combined. The nanoma- nipulation technique is employed to observe the structural role of H-NS and its combined activity with a crowding agent leading to a clear and abrupt compaction of DNA. Time-lapse fluorescence microscopy is instead used to study the regulatory role of the protein, more precisely the gene silencing mechanism, at the single-cell level. This activity has also a strong influence in the cell physiology, by significantly changing the growth rate of bacteria.