Novel peptide probes for super-resolution microscopy and optimized DNA origami for cellular applications

Eklund, Alexandra S. (2023): Novel peptide probes for super-resolution microscopy and optimized DNA origami for cellular applications. Dissertation, LMU München: Faculty of Chemistry and Pharmacy

Abstract

Fluorescence microscopy is one of the most widely used techniques in the life sciences to observe cells, subcellular structures, single proteins and microorganisms due to the possibility of target-specific labeling and real-time imaging. However, conventional fluorescence microscopy is limited to a spatial resolution of ~200 nm due to the diffraction of light. This restrictive spatial resolution is insufficient for the observation of many biological structures with dimensions reaching only a couple of nanometers. In the last decades, fluorescence microscopy has seen a true revolution by the development of super-resolution techniques such as STED, PALM, STORM and PAINT that overcame the diffraction barrier. More recently, DNA-PAINT was developed using the repetitive binding of dye-labeled DNA-based imager strands to complementary docking strands on target molecules. DNA-PAINT has become a straightforward method for super-resolution imaging of biological targets with an achievable resolution of <5 nm and offers spectrally unlimited multiplexing. However, DNA-PAINT fails to reach its true potential when it comes to cellular imaging due to the subpar performance of labeling probes with sizes often larger than 5 nm. In the first part of this thesis, the programmability and transient binding nature of DNA-PAINT was translated to coiled coil interactions of short peptides for the development of Peptide-PAINT to address the issue related to probe sizes. The binding kinetics of different variations of one coiled coil pair was tested using a single-molecule imaging assay and the achievable spatial resolution was characterized on DNA origami structures (Publication I). In the second part, a second coiled coil pair was introduced and proven to be orthogonal to the first coiled coil pair by performing single-molecule and DNA origami orthogonality assays. The novel coiled coil pair was tested as a small direct labeling tag in fixed cells and enabled super-resolution imaging of membrane proteins at the single-receptor level in 2D and 3D using Peptide-PAINT (Publication II). In the third and final part of this thesis, the strand accessibility of disk-shaped 3D DNA origami structures was investigated. Here, the bottom side of the disk (facing the glass surface) was successfully imaged using DNA-PAINT with similar binding kinetics as the top side of the disk. Finally, all available functionalized handles of the disk were demonstrated to be accessible for DNA-PAINT imaging, despite the presence of a protective coating (Publication III).

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: super-resolution microscopy, coiled coil interactions, DNA-PAINT, DNA origami, single-molecule imaging, structure stability, handle accessibility, nanotherapeutics
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 540 Chemistry and allied sciences
• Faculties: Faculty of Chemistry and Pharmacy
• Language: English
• Date of oral examination: 6. February 2023
• 1. Referee: Tinnefeld, Philip
• Signature of the printed copy: 0001/UMC 29451