Single Molecule Localization and Discrimination of DNA–Protein Complexes by Controlled Translocation Through Nanocapillaries

Through the use of optical tweezers we performed controlled translocations of DNA-protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on lambda-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the non equilibrium work done during the translocation of a DNA-protein complex and used it to obtain an estimate of the effective charge of the complex. In combination with conductivity measurements, we provided a proof of concept for discrimination between different DNA protein complexes simultaneous to the localization of their binding sites.

Relevance of the Drag Force during Controlled Translocation of a DNA–Protein Complex through a Glass Nanocapillary

Roman Bulushev, Sanjin Marion, Aleksandra Radenovic

Combination of glass nanocapillaries with optical tweezers allowed us to detect DNA-protein complexes in physiological conditions. In this system, a protein bound to DNA is characterized by a simultaneous change of the force and ionic current signals from the level observed for the bare DNA. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA, and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries. Force profiles obtained for DNA-RecA in our system were different than those in the system with nanopores in membranes and optical tweezers. We suggest that such behavior is due to the dominant impact of the drag force comparing to the electrostatic force acting on a DNA-protein complex inside nanocapillaries. We explained our results using a stochastic model taking into account the conical shape of glass nanocapillaries.

American Chemical Society2015

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

Modeling and analysis of parts and devices of genetic regulation

Yves Berset

DNA encrypts the composition of the cellular material that is synthesized through transcription and translation. Never-theless, gene regulation mechanisms determine the final amount of transcribed and translated material. Transcription factors (TF) are a class of proteins that bind to DNA motifs and can either facilitate or prevent the RNA polymerase to transcribe a strand of DNA into an mRNA. These mechanisms allow the cell to sense the environment and adapt to different environmental conditions, e.g. presence of toxic compounds, oxidative stress or absence of nutrients. TF interactions with DNA are depicted by networks of molecular interactions. Some TFs bind to very specific DNA sites, and others have a broad range of binding sites. Moreover, TFs often interact each other, e.g. through heterodimerization prior to bind to DNA, leading to changes in their binding specificities. However, these effects remain poorly understood. By combining detailed mathematical modeling and high-throughput experimental techniques for quantification of molecular interactions, we built a heterodimer-DNA specificity model with higher predictive power than a one-site model. Bioreporters are living cells that emit a signal in the presence of a chemical compound. In arsenic bioreporters, a TF triggers the detoxification response in presence of arsenic. To better understand the key mechanisms involved in the response, we built a detailed mechanistic model of the gene regulatory circuits of different bioreporters. In this study, we used mathematical modeling (ODE, SSA) to create, calibrate and analyze detailed networks of molecular interactions involved in gene regulations. We quantified the cooperative binding of transcription factors forming heterodimers on a DNA library, and optimized bioreporters for the detection of arsenic by modeling feedback, uncoupled and toggle switched-based gene regulatory circuits.