Influence of DNA Binding Dyes on Bare DNA Structure Studied with Atomic Force Microscopy

Fluorescent dyes are widely used for staining and visualization of DNA in optical microscopy based methods. Even though for some dyes the mechanism of binding is known, how this binding affects DNA remains poorly understood. Here we present a novel experimental study of the influence of staining dyes on DNA properties. We use atomic force microscopy which allows quantification and measurement of structural properties of stained DNA with nanometer resolution. We studied the influence of dyes on the persistence length, the total contour length, and the morphology of individual DNA molecules. We tested three widely used dyes known to differently bind DNA molecules, namely PicoGreen, Dapi, and DRAQ5. Based on our measurements, when imaged at typical concentrations (manufacturer suggested concentrations used for cell imaging), PicoGreen dye showed little effect, Dapi dye decreased the DNA persistence length, and DRAQ5 decreased the persistence length and elongated the DNA. When used at high concentrations, all of the dyes induced drastic changes in the DNA morphology. Our study clearly shows that DNA-binding dyes, irrespective of their DNA binding mechanisms, strongly influence the physical properties of DNA. These changes are strongly dose and dye type dependent and therefore should be taken into consideration when conducting experiments with DNA.

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

Cryo-AFM development for high resolution DNA imaging and spectroscopy

Susana del Carmen Tobenas Borron

The thesis presents the development of a custom made atomic force microscope (AFM) conceived to image biological molecules at low temperatures in ultrahigh vacuum conditions, and its application to DNA single molecule studies. Starting from a first prototype working in contact mode, our aim was to implement non-contact AFM in order to reduce possible sample damaging due to strong tip interactions. The first chapter of the thesis discusses the technical solutions developed to improve the previous system. A complete setup for in situ exchange of samples and tips was designed and implemented. This permits a continuous operation of the microscope under the experimental conditions of UHV. Thermal instabilities and temperature differences between the tip and the sample were solved with the design of a movable shield to avoid heating by thermal radiation. The introduction of a new high voltage signal generator with variable frequency and the development of a user-friendly LabVIEW interface to communicate with this device notably improved the piezomotors driving. These piezomotors perform the micro-positioning of all the microscope parts and are driven through this interface. Finally, the previous tip-holder design was modified to incorporate a piezo actuator behind the cantilever, which provides the necessary excitation for the implementation of dynamic AFM modes. The installation of a Phase Lock Loop (PLL) circuit to lock the cantilever frequency and the replacement of old feedback electronics for a digital SPM Controller, with advanced options of frequency-shift feedback, was another requirement for the correct implementation of non-contact AFM. The second chapter deals with DNA sample preparation for high resolution imaging by AFM. Based on our studies, the advantages and drawbacks of the most commonly used techniques for DNA adsorption are discussed in detail. Our measurements in contact mode demonstrate the convenience of using hydrophobic substrates for high resolution DNA imaging. At hydrophilic surfaces, like mica, the presence of a thin water layer could be responsible of strong adhesion forces between the cantilever and the sample. In order to overcome this limitation we tested HOPG as hydrophobic substrate. HOPG, chemically modified with amphiphilic compounds, allowed us to deposit DNA without molecular damage or stress. The HOPG surface treatment was significantly improved. New protocols were developed to control the formation of dodecylamine disordered monolayers or lamella structures on the substrate. The lamella formation induces a subsequent self assembly of DNA single molecules. In the last chapter we discuss our results on DNA imaging obtained with the different operation modes implemented in our microscope: contact mode, tapping mode and frequency modulation (non-contact) AFM. We conclude that the last one provides higher signal stability and the necessary sensitivity to observe DNA details at a single-molecule level. We also present a study of frequency shift versus distance dependence (force spectroscopy experiments) performed in DNA samples. We discuss for the first time a plausible explanation to the origin of the atomic force contrast between the substrate and the DNA molecules. Force spectroscopy measurements performed along DNA molecules reveal two distinguishable types of interaction. This result suggests that the chemical composition of the minor-major groove and/or of the sugar-phosphate backbone along the molecule can be detected, opening numerous possibilities for further studies and applications of this technique.

EPFL2008

Mechanical and Functional Study of Cell Physiology at the Nanoscale

Pascal Damian Odermatt

By live-cell imaging of biological samples dynamic cellular processes can be resolved. Fluorescence microscopy (FM) and atomic force microscopy (AFM) are both capable of imaging live cells. By combining these techniques structural as well as functional information of the same biological samples are measured. While the dynamics of specific proteins can be imaged by FM, the AFM provides the structural context. To study how intracellular processes affect the mechanics, structure and kinetics of cells at the nanoscale we combined a high-speed AFM (HS-AFM) and an inverted optical microscope (OM) into one instrument. With this system cell physiological processes at different time scales are imaged. Mammalian cell migration happening at minute time scales is studied by correlated super-resolution microscopy and HS-AFM. Bacterial cell separation happening within 10s of milliseconds is characterized as well as bacterial growth over multiple generations. Additionally to imaging, the capability of the AFM to quantitatively measure mechanical properties of tissues is deployed in combination with FM on mouse corneal tissues. Different to most other rod-shaped bacteria where division happens through constriction of the cell wall at midcell, mycobacteria keep the shape of their outer cell envelope unchanged during septation. The septal wall is formed by peptidoglycan branching off from the outer cell envelop eventually splitting the cell into two sister compartments. Nanomechanical measurements show that from the initiation of septation the stiffness at the septum increases significantly. High-temporal resolution imaging of the following bacterial separation process indicated that the sibling cells separate within tens of milliseconds. The connection of the outer cell envelop at the former septum is broken apart suggesting a mechanical break. The timing of the cell separation is regulated by the tensile stress at the septum and the ultimate tensile strength of the cell wall material. The positions where these separations occur are closely associated with morphological features on the undulating cell surface. These features are formed at the poles and migrate towards midcell as the cell grows. Interestingly, sibling cell separation is restricted to the center-most wave-trough making these features the first characteristic associated with division site placement. Experiments with mutant strains show that even asymmetric divisions occur within wave-troughs emphasizing their role in division site placement. Chronic inflammation induces an increase in the stiffness of the corneal tissue and is associated with dramatic changes in the composition of the tissue. Stem cells migrating along this tissue layer of increased stiffness experience increased activation of mechanotransduction pathways. This eventually leads to induction of a skin-like rather than corneal-like character of these regenerating stem cells. It demonstrates that the stiffness of the tissue and thus pathways involved in mechanotransduction decisively influence the stem cell fate.

EPFL2016