Advancing DNA origami-based nanomaterials

Since US-American researcher Nadrian Seeman laid the foundations for DNA nanotechnology in 1982 with his groundbreaking idea to use DNA as a building material on the nanoscale, the field has rapidly evolved and attracted growing interest over the past decades. Particularly the development of the DNA origami technique has led to major advancements in this reasearch area. Nowadays, the envisioned applications of nanostructures and nanodevices made from DNA span a wide range from synthetic biology via DNA-computing and information storage to optical metamaterials and nanoelectronics. In the framework of this thesis, in total four research projects will be presented that cover different aspects of DNA nanotechnology and address various challenges associated with potential future applications. To study structures at the nanoscale, a variety of biophysical methods were employed throughout the work in this thesis, ranging from state-of-the-art super-resolution light microscopy and electron microscopy to small-angle X-ray scattering. The first project focuses on the development of a large DNA origami nanopore that can undergo conformational changes in response to the presence of a stimulus. The construction of a DNA (origami) nanodevice that forms a transmembrane channel upon insertion into lipid membranes and whose lumen size can be precisely controlled even within the lipid membrane, represents a major advancement compared to the mostly static artificial nanopores published previously. Challenges regarding the fabrication of a switchable nanopore were overcome by implementing an actuator mechanism into a rhombohedral nanopore design relying on the addition or displacement of trigger DNA strands which ensures a reversible and reliable switching of the nanopore. After their efficient insertion into the lipid bilayers of GUVs, the nanopores could be used to selectively allow for the influx of molecules depending on their size. This novel nanopore type opens up new perspectives for molecular sorting or biosensing applications. In the second project, a novel strategy for the upconcentration and the long-term storage of DNA origami nanostructures is presented. While previously established methods for increasing the concentration are often prone to aggregation, the lyophilization of nanostructures with subsequent resuspension in a low volume provides a gentle alternative that ensures a well-distributed DNA origami sample. The storage of DNA origami nanostructures as a lyophilized powder also facilitates their transport and avoids the clustering of nanostructures with increasing storage time. Due to its applicability to differently shaped and even to silica-coated nanostructures, this approach holds great promise with regard to experimental protocols and techniques that require high concentrations of DNA origami, such as e.g. SAXS measurements or single-molecule analysis via cryo-EM. The third project addresses the further development of silicification strategies for DNA origami monomers and crystals. Due to the inherent instability of DNA origami e.g. in biological media containing DNA-digesting enzymes and/or only very low amounts of MgCl2, silicification has been explored as a method to protect DNA origami from detrimental influences while preserving its shape. After contributing to studies investigating the proceeding of the silicification of DNA origami monomers using in-situ SAXS and exploring the accessibility of single-stranded DNA regions after different types of silicification, the focus of the project presented in this part is laid on endowing silica coatings of nanostructures with additional functionalities, in this case fluorescence and dissolvability. This was achieved by incorporating either a customly synthesized silane carrying a fluorescein molecule or a commercially available silica precursor containing a disulfide bridge during the silicification. The findings from this study suggest great potential of these customization strategies for applications in drug delivery or material science. Finally, the fourth project deals with the incorporation of enzymes into silicified DNA origami crystal structures. Since the early days of DNA nanotechnology, there has been a high interest in crystal structures made from DNA due to the possibility for the spatial arrangement of guest molecules in the crystal cavities. Thanks to the stabilizing silica coating, the DNA origami crystals employed in this study can also host enzymes that exhibit optimal activity at conditions that would lead to the disassembly of bare DNA origami superstructures (temperatures above 55°C, low salt conditions, etc.). Such a hybrid crystal system can be of great interest for the formation of catalytic crystals and for exploring the behaviour/the activity of enzymes after their spatial arrangement with defined inter-enzyme distances. The study provides important strategies and findings with regard to the optimization of crystal quality and the incoporation of inactive and active proteins; however, certain challenges such as the quantification of protein uptake into the crystals still need to be tackled in future studies. The work in this thesis spans a bow over different areas within the broad field of DNA nanotechnology and contributes to the further development of structures on the nanoscale, paving the way for future applications e.g. in biosensing, biomedicine or beyond.