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Hochrein, Marion (2005): From Single DNA Molecules to an Entire Virus: an Investigation with Quantitative Fluorescence Microscopy and X-Ray Reflectivity. Dissertation, LMU München: Faculty of Physics
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Abstract

This PhD thesis presents a fluorescence microscopy study about deoxyribose nucleic acid (DNA) in confined geometries. DNA serves as an established model polymer well described by polymer physics. In a first project, the dynamics of the DNA ejection through the tight viral shaft from the highly packed phage capsid was investigated. In a second project DNA was manipulated by linear substrate structures. During phage assembly the phage DNA is very tightly packaged into the phage capsid. The question arises if the internal pressure resulting from the high DNA density inside the capsid is responsible for the complete or at least the partial DNA transfer into the host during infection. This was studied for the first time on single T5 phages whose DNA release can be triggered by the receptor protein FhuA in vitro. After T5 phages had adsorbed onto a microfluidic chamber, the ejected DNA was fluorescently stained and stretched in a hydrodynamic flow. The length could thereby be measured continuously. With this setup, we succeeded for the first time in visualizing the dynamics of the DNA ejection in real time. The DNA release is not an all-or-none process but occurs in a stepwise fashion and at a rate reaching 75 000 bp/s. The pauses in between steps can last for over half an hour. The length distribution of the ejected DNA was analyzed and characteristic peaks were found at positions that coincided with the position of single-stranded interruptions (nicks) of the phage genome. DNA molecules adsorbed onto cationic fluid lipid membranes remain laterally diffusive. During this thesis rectangular grooves of a periodicity of 1000 – 1200nm and a depth of 40 – 120nm were imprinted into the thermoplastic “cyclic olefin copolymer” (COC). This substrate was coated with a lipid membrane. The influence of such a support on the conformational behavior of DNA is investigated here. The periodically structured membranes revealed the ability to stretch long DNA molecules. During a collaboration, L. Golubovic provided us with a theory explaining the observed process. The DNA stretching phenomenon can be elucidated in terms of a curvature dependent potential energy attained by the adsorbed DNA molecules. The properties of lipid membranes supported by the hydrophobic solid polymer COC are investigated. Lipid layers were prepared by vesicle fusion and solvent exchange from varying amounts of 1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP, cationic) and 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC, neutral). The influence of the membrane charge and also of the preparation method were investigated. All lipid mixtures coated the COC surface homogeneously forming a fluid membrane as verified by quantitative fluorescence microscopy, the method of Continuous Bleaching and Fluorescence Recovery After Photobleaching (FRAP). The average diffusion constant was found to be 0.8 µm2/s which is higher by a factor of 5 than the typical membrane diffusion constant for hydrophilic supports. The vertical electron density profile of the supported membranes was determined by synchrotron reflectivity experiments using a special microfluidic chamber adapted to reflectivity experiments. The x-ray data can not be fitted as expected with a model assuming a monolayer. They are however in agreement with a compressed and less densely packed bilayer. The head-to-head distance is 29˚A, the density of the headgroups is ρh = 0.34 e−/°A3 and of the tails ρt = 0.26 e−/°A3.