Interfacing DNA nanotechnology and single-molecule microscopy to stabilize functionality on the nanoscale

Scheckenbach, Jürgen Michael (2025): Interfacing DNA nanotechnology and single-molecule microscopy to stabilize functionality on the nanoscale. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

DNA nanotechnology and, in particular, the introduction of DNA origami nanostructures (DONs), have enabled the easy design and synthesis of a plethora of highly sophisticated, functional nanodevices with applications in various fields such as biosensing, biocomputing, or nanorobotics. Simultaneously, advancing single-molecule microscopy has become an essential tool to investigate and readout DNA based nanodevices with high spatiotemporal resolution at a single-device level, breaking even the diffraction limit of light. While an unprecedent level of functionality has been reached in the design of DONs, their widescale applicability is still hampered by their intrinsic low structural and functional stability in application specific conditions. Single-molecule microscopy, on the other hand, suffers from photobleaching of fluorescent labels and photoinduced sample damage, limiting the observation time of DONs at the nanoscale. Multiple strategies have been developed to increase the stability of DONs and their fluorescent labels, but most of these approaches only slow down the degradation under wear and tear conditions and are incapable to reverse an occurring damage. This calls for novel stabilization strategies, which either ensure an improved static stability or provide dynamic stability by the repair of damaged building blocks within a functional DON, while preserving its functionality. In this thesis, I used single-molecule microscopy, the super-resolution imaging technique DNA points accumulation for imaging in nanoscale topography (DNA PAINT), and atomic force microscopy (AFM) to investigate the stability of functional DONs and demonstrated novel strategies to slow down or reverse the degradation under wear and tear conditions. First, I applied protective coatings to DONs and investigated their improved stability, using either AFM, DNA PAINT, or a single-molecule reporter sensitive to the coating process. Here, I showed that designed DNA docking sites stay functional and addressable during the coating process with silica and that the structural integrity of the coated DONs can be probed using DNA PAINT. Additionally, I employed a molecular reporter for probing the coating process of DONs by reading out the fluorescence lifetime shift of an environment-sensitive cyanine dye. I applied the sensor to track the coating of DONs with silica and with commonly used poly-L-lysine polyethylene glycol (PLL-PEG) and studied the stability of the coated nanostructures under degrading conditions. Next, I implemented dynamic, self-repairing strategies into DONs by exploiting the self-assembling nature of DNA. Using single-molecule microscopy and DNA PAINT, I showed, that the dynamic exchange of damaged building blocks with intact analogues from an excessive pool helps overcoming the irreversible bleaching of fluorescent labels, as well as structurally stabilizing DONs in blood serum. Finally, I designed and implemented a novel strategy to improve the longevity of fluorescence labels in single-molecule and super-resolution imaging. It relies on directing a photostabilizing agent to the location of the fluorescent label via specific DNA interactions, thereby enabling long-term imaging without the addition of high amounts of chemical agents. After I applied this approach to single-molecule and DNA PAINT imaging on DONs, it was subsequently employed for photostabilized super-resolution imaging in cells even under aerobic conditions. In summary, this thesis adds new approaches and characterization methods to the toolbox of stabilization strategies of functional DONs and fluorescent labels used in single-molecule and super-resolution microscopy. DNA PAINT and the molecular cyanine reporter help to understand the coating process of DONs at a single-molecule level, providing new insights into its homogeneity and integrity. The ability of DONs to repair damaged building blocks opens up new design possibilities, especially for future applications such as artificial cells, in which a constant turnover of building blocks will be needed to mimic the behavior and properties of natural cells. The modularity of the DNA mediated photostabilization approach makes it an affordable and attractive tool for future single-molecule applications, such as biosensing or minimally-invasive long-term super-resolution imaging of biological samples.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Single-molecule microscopy, DNA nanotechnology, super-resolution microscopy, stability, functionality
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 28. Februar 2025
• 1. Bericht­erstatter:in: Tinnefeld, Philip