Single-Molecule Kinetics and Super-Resolution Microscopy by Fluorescence Imaging of Transient Binding on DNA Origami

TLDR

DNA origami enables programmable assembly of nanoscale molecular structures, and real‑time kinetic studies with high spatial resolution are increasingly important for their use as functional biomaterials. The authors present a single‑molecule assay to study binding and unbinding kinetics on DNA origami. Using reversible specific binding of labeled oligonucleotides, the assay implements PAINT imaging with sub‑30‑nm resolution on DNA nanostructures. The method reveals that hybridization kinetics to single‑stranded extensions on DNA origami are similar to isolated DNA with slight position dependence, and it successfully images both flat monomeric and multimeric ribbon‑like structures.

Abstract

DNA origami is a powerful method for the programmable assembly of nanoscale molecular structures. For applications of these structures as functional biomaterials, the study of reaction kinetics and dynamic processes in real time and with high spatial resolution becomes increasingly important. We present a single-molecule assay for the study of binding and unbinding kinetics on DNA origami. We find that the kinetics of hybridization to single-stranded extensions on DNA origami is similar to isolated substrate-immobilized DNA with a slight position dependence on the origami. On the basis of the knowledge of the kinetics, we exploit reversible specific binding of labeled oligonucleotides to DNA nanostructures for PAINT (points accumulation for imaging in nanoscale topography) imaging with <30 nm resolution. The method is demonstrated for flat monomeric DNA structures as well as multimeric, ribbon-like DNA structures.