Discovering anomalous hybridization kinetics on DNA nanostructures using single-molecule fluorescence microscopy

DNA nanostructures are finding diverse applications as scaffolds for molecular organization. In general, components such as nucleic acids, proteins, and nanoparticles are attached to addressable DNA nanostructures via hybridization, and there is interest in exploiting hybridization for localized computation on DNA nanostructures. This report details two fluorescence microscopy methods, single-particle fluorescence resonance energy transfer (spFRET) and DNA-PAINT (points accumulation for imaging in nanoscale topography), that have been successfully used to detect anomalies of hybridization reactions on individual DNA nanostructures. We compare and contrast the two techniques, highlighting their respective strengths in studying equilibrium and non-equilibrium hybridization as well as assessing the variability of behaviors within individual nanostructures and across a population of nanostructures.