Interaction Analysis of DNA-Binding Molecules using Electro-Switchable Biosurfaces
Verein zur Förderung des Walter Schottky Instituts der Technischen Universität München (Publisher)
Published on 31. July 2019
Book
Paperback/Softback
200 pages
978-3-946379-27-0 (ISBN)
Description
Biosensors based on electro-switchable biosurfaces (ESB) can actively move analytical molecules attached to surface-grafted DNA nanolevers in a liquid environment and simultaneously report on the characteristics of this motion by observation of the emission of a fluorescent dye at the distal ends of the nanolevers. This makes ESB systems sensitive to any change of the hydrodynamic friction of the tested molecule, which is employed, for instance, to study interactions between protein molecules. Additionally, the absolute fluorescence emission of the dye can provide interaction parameters by use of fluorescence proximity sensing.
This dissertation introduces novel applications of ESB biosensors that enable the detection of structural changes of proteins caused by post-translational modifications or thermally induced molecular transitions. For the tested proteins, both post-translational modifications and temperature dependent molecular changes show up as measurable changes in hydrodynamic friction.
A second set of experiments examines the applicability of ESB biosensor systems to analyze DNA-related interactions. For this purpose, studies with a broad range of different interaction systems are performed, typically yielding association- and dissociation rate constants (kon and koff), as well as the equilibrium dissociation constant (KD).
For two DNA-binding proteins, Smc5/6 and MutS, it is shown how the DNA interaction can depend on the state of the DNA (single-stranded or double-stranded) or on the presence of a single basepair mismatch.
At the example of the zinc finger domain protein PRDM9 we demonstrate the feasibility of ESB biosensors to detect sequence-specific DNA binding in a complex sample solution (cell lysate).
Furthermore, a detailed case study demonstrates the development of a modular sequence screening system to test the DNA affinity of a bacterial transcription factor.
Finally, it is shown how fluorescence proximity sensing can be employed to characterize the sequence-specific DNA binding of small molecule polyamides.
This dissertation introduces novel applications of ESB biosensors that enable the detection of structural changes of proteins caused by post-translational modifications or thermally induced molecular transitions. For the tested proteins, both post-translational modifications and temperature dependent molecular changes show up as measurable changes in hydrodynamic friction.
A second set of experiments examines the applicability of ESB biosensor systems to analyze DNA-related interactions. For this purpose, studies with a broad range of different interaction systems are performed, typically yielding association- and dissociation rate constants (kon and koff), as well as the equilibrium dissociation constant (KD).
For two DNA-binding proteins, Smc5/6 and MutS, it is shown how the DNA interaction can depend on the state of the DNA (single-stranded or double-stranded) or on the presence of a single basepair mismatch.
At the example of the zinc finger domain protein PRDM9 we demonstrate the feasibility of ESB biosensors to detect sequence-specific DNA binding in a complex sample solution (cell lysate).
Furthermore, a detailed case study demonstrates the development of a modular sequence screening system to test the DNA affinity of a bacterial transcription factor.
Finally, it is shown how fluorescence proximity sensing can be employed to characterize the sequence-specific DNA binding of small molecule polyamides.