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Wienken, Christoph Jens (2011): Biotechnological applications of thermophoresis. Dissertation, LMU München: Faculty of Physics
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Abstract

For over 150 years it is known that particles in a temperature gradient conduct a directed movement. This is called the Soret effect or thermophoresis. Still the underlying physical principles of thermophoresis in aqueous solutions are not totally understood. In the first part of this thesis new experiments on the thermophoresis of small single-stranded DNA oligonucleotides try to elucidate the fundamental principles of the Soret effect and the results seem to support a thermodynamic description of the thermophoretic movement. With this approach the experimental results for DNA could be predicted without free fitting parameters. Assuming this theory the thermophoretic movement mainly depends on the strength of ionic shielding and on the hydration sphere of the particle. This direct influence of the water-particle interface implicates that thermophoresis is very sensitive to even slight changes of particles. Applied to biomolecules like DNA or proteins the Soret effect allows for a precise analysis of the molecule under investigation. Any binding reaction, for example, will at least result in a change of the hydration sphere of the molecule and thus, binding reactions are readily accessible with thermophoresis. This is demonstrated in the second part of this work. The experiments range from DNA aptamers binding to nucleotides or proteins over protein-protein interactions to single ion binding. Especially low molecular weight binders like small molecules or ions are notoriously difficult to measure with standard interaction analysis tools. Interestingly, in thermophoresis measurements the signal to noise ratio does not significantly depend on the molar weight ratio as it is the case for other interaction analysis techniques. High affinities in the nanomolar regime are equally well measured as low affinities in the high micromolar range. The thermophoretic method also allows monitoring interactions of biomolecules directly in biological liquids like cell lysate or blood serum. To overcome potential influences of the typically used fluorescent label on the interaction strength, intrinsic protein fluorescence is also suitable for monitoring the thermophoretic movement of proteins. This approach allows a complete label-free measurement of protein interactions directly in solution without any labeling or surface functionalizing procedure. Third, also structural changes of molecules could be analyzed with thermophoresis. This is demonstrated in the last part of this thesis with measurements on the thermal stability of nucleic acids. Most conformational changes affect at least the hydration sphere of a molecule and thus lead to a measurable readout in the thermophoresis signal. Again, the thermophoretic method shows a high sensitivity for small changes in the molecule structure and thus, allows for revealing intermediate states upon the unfolding of nucleic acids.