Nucleic acid structure refers to the structure of nucleic acids such as DNA and RNA. Chemically speaking, DNA and RNA are very similar. Nucleic acid structure is often divided into four different levels: primary, secondary, tertiary, and quaternary. Primary structure Nucleic acid sequence Primary structure consists of a linear sequence of nucleotides that are linked together by phosphodiester bond. It is this linear sequence of nucleotides that make up the primary structure of DNA or RNA. Nucleotides consist of 3 components:

Nitrogenous base

Adenine

Guanine

Cytosine

Thymine (present in DNA only)

Uracil (present in RNA only)

5-carbon sugar which is called deoxyribose (found in DNA) and ribose (found in RNA).

One or more phosphate groups.

The nitrogen bases adenine and guanine are purine in structure and form a glycosidic bond between their 9 nitrogen and the 1' -OH group of the deoxyribose. Cytosine, thymine, and uracil are pyrimidines, hence the glycos

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

Synthesis of Glycomimetics as Potential Anti-Cancer Agents

Annabelle Gillig

Cancer cells exhibit different properties that make them dangerous to the host, such as the ability to invade other tissues and the abnormal response to the control mechanisms that regulate the division of normal cells, which allow them to divide in a relatively uncontrolled fashion. Moreover, cancer cells require fewer protein growth factors than normal cells and can go on dividing indefinitely, in contrast with normal. Thus, malignant tumors cells defy not only the normal controls on their proliferation, but also the normal controls on their position. The more widely such cells spread in the body, the harder they become to eradicate surgically. Therefore, cancer research has the difficult task of analyzing malignant transformation and metastatic diffusion in order to develop new therapies. In this context, we report in the present work the development of new chemical tools related to cancer research. The first part relies on the salvage pathway of nicotinamide adenine dinucleotide NAD+ by the enzyme nicotinamide phosphoribosyl transferase. The latter enzyme is over-expressed in cancer cells and a synthetic inhibitor, called FK866, shows efficient anti-cancer activity while having little toxicity to normal cells. However, FK866 has a poor bioavailability, which highlights the need to develop new drugs. In order to optimize the bioavailability and the interactions in the active site, we report in the present work the strategy for the design of a new class of potential nicotinamide phosphoribosyl transferase inhibitors. An efficient synthetic pathway involving the preparation of an iminoribitol, its substitution on the position 1 through a three stepone pot reaction, a cross-metathesis and an ester-amide exchange has been set up. A library of new glycomimetics analogous to FK866 has been prepared and their encouraging biological evaluation on cancer cells led to the selection of eight candidates for further biological studies. The second part of the project is based on the development of cancer vaccines. Carcinomas express a multitude of different carbohydrate antigens, such as the Thomsen-Friedenreich antigen, on plasma membrane. Interestingly, the changes of the glycopatterns during the malignant transformation generally result in shorter carbohydrate chains. In usual tissue, the Thomsen-Friedenreich antigen is masked and inaccessible to the immune system, but in various tumors, it is exposed and immunoreactive. A cluster of Thomsen-Friedenreich antigen administered as vaccines has proven to be a target for the immune system. Nevertheless, due to hydrolysis catalysed by glycosidases in vivo, these disaccharides conjugates are relatively short-lived in the blood stream. Our strategy to avoid hydrolytic degradation is the use of C-linked- glycosides. Thus, we report the synthesis of non-hydrolysable clusters of C-linked disaccharides analogous to the TF-antigen through the coupling of two glycosides, the construction of disaccharides analogous to the Thomsen-Friedenreich epitope and their clustering through a peptide synthesis strategy. Conjugation of these clusters to a carrier protein such as KLH will provide the expected cancer vaccines. The last part refers to an outstanding strategy in human therapy, which use oligonucleotide analogues to inhibit the genes expression at the level of translation. This strategy, called the antisense approach, is based on the introduction of an antisense oligonucleotide, complementary to the mRNA of interest, into the cell. Base-pairing with the targeted mRNA thus inhibits its translation into a protein. RNAs tend to fold into a variety of secondary structure such as hairpin loops. Therefore, the antisense oligonucleotide should have a restricted conformation in order to bind efficiently, and with high specificity, to the targeted nucleic acid structure. Thus, we report the synthesis of a conformationally restricted dinucleoside through the application of cross-metathesis in nucleic acid chemistry.

EPFL2010

Fine DNA structure revealed by constant height frequency modulation AFM imaging

Andrea Cerreta, Giovanni Dietler, Dusan Vobornik

Few examples of sub nanometer resolution on biomolecules can be found in literature. In particular, AFM experiments on DNA mostly fail to show the double helical groove periodicity, and so far such a feat was only achieved in liquid. Here we describe a method that produces highly resolved images of DNA where contrast can be assigned to submolecular features such as DNA helix's grooves. Two types of AFM experiments are presented: (1) We started by imaging DNA with Frequency Modulation (FM) AFM, where a shift in resonance frequency is used as a feedback signal and maintained constant during imaging. With FM-AFM we performed experiments in the non-contact (NC) mode, with z-feedback on, where only gentle (few tens of pN) non compressive force is applied between the tip and the sample. (2) Then we switched off the z-feedback and scanned the DNA sample at constant height measuring frequency modulation in order to acquire frequency shift maps of the sample. This was done at several separations including the distances where the frequency feedback is not stable due to the non-monotonicity of the frequency shift curve. At this distance sub-molecular resolution was reached and a calculation of the tip-sample van der Wools interaction at constant height, as well as the periodicity of the observed features that ranges from 3 to 5 nm, suggest that these correspond to the DNA helix grooves. This is the first experimental study where submolecular features of single DNA molecules are observed in dry environment. The knowledge of DNA structure when dried and deposited on flat substrates is important for proper understanding of DNA's electronic behavior and its eventual nanotechnology applications. (C) 2013 Elsevier Ltd. All rights reserved.