Exploring the mechanics of DNA origami nanopores via DNA PAINT imaging

Abstract

Nanopores are narrow channels in cell membranes that control the passage of small molecules. In recent years, they have been repurposed for diverse applications, including single molecule sensing and drug delivery. Due to the small diameters of conventional protein-based, current research efforts are directed to designing nanopores from other materials to achieve larger diameters. DNA origami has emerged as a promising method for the precise fabrication of nanoscale structures. Using advances in structurally-adaptable DNA origami nanotechnology, here we investigate DNA-based nanoactuators with size-adjustable diameters, which can potentially reach diameters of up to 100 nm, that could be used for macromolecules translocation. The focus of this thesis is the study of the mechanical states of these nanoactuators, which can be triggered to change shape in response to a specific molecular trigger. Given the nanoscopic dimensions of our actuators, we select DNA PAINT for imaging, which is a type of super resolution technique, that can achieve a resolution of 10 nm, beating the optical diffraction limit (~200 nm) of conventional light microscopy. DNA PAINT experiments are performed in combination with total internal reflection fluorescence (TIRF) microscopy to characterize the behavior of DNA origami nanopores in physiological conditions. By testing various parameters sample related and imaging software ones we identify optimal conditions suggesting 5mM Mg^(2+) ions in buffer solution and 1nM DNA nanopores. Laser power (40mW), exposure time (400ms), waiting time between frames (300ms), and image duration (50s) are optimized, resulting in the expected fluorescent blinking behavior which enables us to perform single-molecule localization. The individual corners of the nanopores were, however, not resolved with this technique due to limitations in the resolution of the imaging system. We recommend that future work could exploit the even better resolution of a modified DNA PAINT approach i.e. Exchange PAINT, which has been proven to achieve Angstrom level resolution for imaging of DNA nanostructures.