DNA-Paint Imaging of DNA-Origami Rings Mimicking Biologically Relevant Structures

Single-molecule localization microscopy methods such as DNA-PAINT (point accumulation for imaging in nanoscale topography), utilize successive localizations of single molecules over time to reveal of biological structures with photon-limited resolution1,2. However, visualization of sub-diffracted discrete bimolecular assemblies still remains a challenge. “DNA origami” has emerged as powerful nano-templating technique capable of generating stable self-assembled scaffold for positioning of molecular species of interest with nanometer precision3,4. Recent studies of DNA-PAINT have focused on imaging different molecular species, such as fluorophores and proteins, positioned on synthetic grid-like DNA nanostructures1,4. These grids are well suited for determining the resolution limit of sparsely labelled points (e.g. one fluorophore per 5x5 nm2). However, biological structures often have a much higher local density and complex structure, making imaging more challenging. In this study, as a proof-of-concept, we explored imaging of circular “ring-like” DNA-origami nanostructures of dimension mimicking small bio-molecular structures (25-50 nm inner diameter), like membrane receptors, ion channels etc. Using a TIRF-based imaging technique, we have optimized the fluorophores density and its positioning on DNA-origami rings to reconstruct “doughnut” shaped images and have been able to resolve inner diameter of ∼25 to 50 nm. We have carried out these analyses on glass surfaces and also on supported bilayers. Our study utilizing DNA rings provides challenging targets for DNA-PAINT technology and underscores the use of size-templated nanostructures, as approximates for bio-molecular complexes, to optimize imaging and analysis protocols independent of biological sample preparation.1. Dai et al. (2016) Nat. Nanotechnol. 11:798-807.2. Baddeley, D. (2015) Nat. Methods 12:1019-1020.3. Yang et al. (2016) Nat. Chem. 8:476-483.4. Schlichthaerle et al. (2016) Curr. Opin. Biotechnol. 39: 41-47.