Super-Resolution Fingerprinting Detects Chemical Reactions and Idiosyncrasies of Single DNA Pegboards

We employ the single-particle fluorescence nanoscopy technique points accumulation for imaging in nanoscale topography (PAINT) using site-specific DNA probes to acquire two-dimensional density maps of specific features patterned on nanoscale DNA origami pegboards. We show that PAINT has a localization accuracy of ∼10 nm that is sufficient to reliably distinguish dense (>10⁴ features μm^–2) sub-100 nm patterns of oligonucleotide features. We employ two-color PAINT to follow enzyme-catalyzed modification of features on individual origami and to show that single nanopegboards exhibit stable, spatially heterogeneous probe-binding patterns, or “fingerprints.” Finally, we present experimental and modeling evidence suggesting that these fingerprints may arise from feature spacing variations that locally modulate the probe binding kinetics. Our study highlights the power of fluorescence nanoscopy to perform quality control on individual soft nanodevices that interact with and position reagents in solution