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DNA origami based tools for the investigation of biomolecular interactions
DNA origami based tools for the investigation of biomolecular interactions
The ability to visualize and interact with nanoscale objects is of great importance for biology, as many biological phenomena can ultimately be broken down to processes on these length-scales. In this context, fluorescence microscopy and especially the distance dependent fluorescence resonance energy transfer (FRET) between two fluorescent labels have developed into valuable tools, because they allow for the elucidation of biomolecular dynamics with low invasiveness, at near physiological conditions and at the single-molecule level. Recent developments in the bottom-up self assembly of DNA nanostructures have now also made it possible to construct nanoscale components that can perform designed tasks and enabled many new applications. In this thesis, I utilized the nanoscale arranging capabilities offered by DNA nanotechnology to construct new tools for the visualization of biomolecular interactions with single molecule FRET. In the first part, I developed a new generation of DNA origami nanoantennas, which are self-assembling nanophotonic devices comprising two gold or silver nanoparticles. In the zeptoliter volume between the two nanoparticles, plasmonic effects increase the electric field intensity by orders of magnitude. The DNA origami structure here serves as a positioning device, selectively immobilizing molecular entities in this volume of highest enhancement. The increased electric field strongly increases the fluorescence intensity of fluorophores, while simultaneously making them more resistant to photobleaching. In this thesis, I made these plasmonic hotspots accessible for larger molecules such as diagnostic assays and proteins. I showed that the fluorescence intensity in single molecule FRET experiments can be increased by approximately an order of magnitude, which increases the time resolution and facilitates the observation of ultrafast processes such as the diffusive barrier crossing events between two potential energy minima, e.g. in the coupled folding and binding of two intrinsically disordered proteins. In the second part, I developed a DNA origami based tunable and modular biosensing platform. A problem that is often encountered in the field of biosensing is that on the one hand, it is difficult to determine whether a small molecule such as a metabolite has bound to a receptor, because the conformational change upon binding is too small to be directly read out with methods such as FRET. On the other hand, the sensitive concentration range of these interactions is dictated by the thermodynamics of the binding reaction and cannot easily be adjusted in the case that the concentrations of interest are not falling within this range. The platform I developed opens up new strategies to overcome these challenges by arranging the sensor components on a DNA origami scaffold. This enables the spatial decoupling of the sensing interaction from the signalling element, meaning that the signal contrast is not any longer defined by the conformational change that the ligand binding causes at its receptor binding site, but rather by the larger conformational change of the DNA origami scaffold that is a consequence of the binding. Furthermore, the DNA origami technique and its nanometer precise positioning capabilities open up the possibility to arrange several sensing elements on the same backbone, which enables the tuning of the sensors response window and even can give rise to cooperative responses. In summary, this thesis adds new tools to the repertoire of fluorescence based single-molecule biophysics and hopefully opens up new research directions for the more detailed understanding of biomolecular interactions.
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Grabenhorst, Lennart
2024
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Grabenhorst, Lennart (2024): DNA origami based tools for the investigation of biomolecular interactions. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

The ability to visualize and interact with nanoscale objects is of great importance for biology, as many biological phenomena can ultimately be broken down to processes on these length-scales. In this context, fluorescence microscopy and especially the distance dependent fluorescence resonance energy transfer (FRET) between two fluorescent labels have developed into valuable tools, because they allow for the elucidation of biomolecular dynamics with low invasiveness, at near physiological conditions and at the single-molecule level. Recent developments in the bottom-up self assembly of DNA nanostructures have now also made it possible to construct nanoscale components that can perform designed tasks and enabled many new applications. In this thesis, I utilized the nanoscale arranging capabilities offered by DNA nanotechnology to construct new tools for the visualization of biomolecular interactions with single molecule FRET. In the first part, I developed a new generation of DNA origami nanoantennas, which are self-assembling nanophotonic devices comprising two gold or silver nanoparticles. In the zeptoliter volume between the two nanoparticles, plasmonic effects increase the electric field intensity by orders of magnitude. The DNA origami structure here serves as a positioning device, selectively immobilizing molecular entities in this volume of highest enhancement. The increased electric field strongly increases the fluorescence intensity of fluorophores, while simultaneously making them more resistant to photobleaching. In this thesis, I made these plasmonic hotspots accessible for larger molecules such as diagnostic assays and proteins. I showed that the fluorescence intensity in single molecule FRET experiments can be increased by approximately an order of magnitude, which increases the time resolution and facilitates the observation of ultrafast processes such as the diffusive barrier crossing events between two potential energy minima, e.g. in the coupled folding and binding of two intrinsically disordered proteins. In the second part, I developed a DNA origami based tunable and modular biosensing platform. A problem that is often encountered in the field of biosensing is that on the one hand, it is difficult to determine whether a small molecule such as a metabolite has bound to a receptor, because the conformational change upon binding is too small to be directly read out with methods such as FRET. On the other hand, the sensitive concentration range of these interactions is dictated by the thermodynamics of the binding reaction and cannot easily be adjusted in the case that the concentrations of interest are not falling within this range. The platform I developed opens up new strategies to overcome these challenges by arranging the sensor components on a DNA origami scaffold. This enables the spatial decoupling of the sensing interaction from the signalling element, meaning that the signal contrast is not any longer defined by the conformational change that the ligand binding causes at its receptor binding site, but rather by the larger conformational change of the DNA origami scaffold that is a consequence of the binding. Furthermore, the DNA origami technique and its nanometer precise positioning capabilities open up the possibility to arrange several sensing elements on the same backbone, which enables the tuning of the sensors response window and even can give rise to cooperative responses. In summary, this thesis adds new tools to the repertoire of fluorescence based single-molecule biophysics and hopefully opens up new research directions for the more detailed understanding of biomolecular interactions.