Unraveling the layers of DNA with the DNA-PAINT brush

Abstract

DNA is both the repository of genetic information and a versatile nanoscale material whose stability and function are governed by base pairing and base-stacking interactions. Despite their fundamental role in DNA structure, base-stacking energetics have been difficult to quantify under equilibrium conditions at the single-molecule level. In this thesis, I introduce a DNA nanotechnology–based assay that leverages DNA-PAINT to systematically measure the energetics of all 16 possible dinucleotide base-stacking interactions under equilibrium conditions. Using patterned DNA origami nanostructures and multiplexed imaging, we record hundreds of thousands of transient hybridization events, enabling robust kinetic analysis and extraction of absolute stacking energies with an accuracy of ±0.1 kcal/mol. These measurements reveal dinucleotide-dependent enhancements in binding dwell times spanning nearly two orders of magnitude and establish a quantitative framework for understanding sequence-dependent DNA stability.

Building on these insights, I demonstrate how base-stacking interactions can be repurposed as a functional design element in super-resolution microscopy. I introduce Stack-pPAINT, a novel proximity-sensing DNA-PAINT probe that detects protein-protein with nanometer-scale resolution by enabling imager binding only when two target-bound strands are sufficiently close. This approach overcomes key limitations of FRET and proximity ligation assays, enabling multiplexed, super-resolution mapping of heterodimeric protein interactions in cells, as demonstrated for the α- and β-tubulin network.

To further expand the capabilities of DNA-PAINT, I develop and characterize a new set of speed-optimized imager sequences compatible with existing multiplexing strategies. Applying these tools to the nucleus, I achieve simultaneous super-resolution imaging of multiple chromatin-associated targets within single cells and establish analytical pipelines to extract spatial organization patterns that fingerprint cellular states. Together, this work advances DNA-PAINT from a powerful imaging modality to a quantitative platform for probing nucleic acid energetics, protein interactomes, and chromatin architecture, laying the groundwork for scalable, high-resolution mapping of cellular organization and early detection of cellular abnormalities.