Interaction analysis of DNA-binding molecules using electro-switchable biosurfaces

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Biosensors utilizing electro-switchable biosurfaces (ESB) enable the active movement of analytical molecules attached to DNA nanolevers in liquid environments, allowing for the simultaneous observation of fluorescent dye emissions at the nanolevers' ends. This sensitivity to hydrodynamic friction changes facilitates the study of protein interactions. The dissertation presents innovative ESB biosensor applications that detect structural protein changes due to post-translational modifications or thermal transitions, with measurable hydrodynamic friction alterations for both types of changes. A second experimental focus is on analyzing DNA-related interactions, yielding association and dissociation rate constants (kon and koff) and the equilibrium dissociation constant (KD) across various interaction systems. The research highlights how DNA-binding proteins, such as Smc5/6 and MutS, exhibit interaction variations based on DNA states (single-stranded or double-stranded) and the presence of basepair mismatches. The zinc finger protein PRDM9 exemplifies the ability of ESB biosensors to detect sequence-specific DNA binding in complex samples like cell lysates. Additionally, a detailed case study illustrates the development of a modular sequence screening system for assessing the DNA affinity of a bacterial transcription factor. Finally, fluorescence proximity sensing is demonstrated as a method to characterize the sequence-specific D