AFM-based single-molecule observation of the conformational changes of DNA structures.

Abstract

Direct visualization of the biomolecules of interest is a straightforward way to elucidate the physical properties of individual molecules and their reaction processes. Atomic force microscopy (AFM) enables direct imaging of biomolecules in suitable solution conditions. As AFM visualizes the molecules at a nanometer-scale spatial resolution, a versatile observation platform is required for precise imaging of the molecules in action. The DNA origami technology allows precise placement of target molecules in a designed nanostructure, enabling their detection at the single-molecule level. We used DNA origami technology for visualizing the detailed movement of target molecules in reactions using high-speed AFM (HS-AFM), which enables the analysis of dynamic movement of biomolecules with a subsecond time resolution. By combining the DNA origami system and HS-AFM, DNA conformational changes, including G-quadruplex formation and disruption and B-Z transition, were visualized. In addition, enzyme-based reactions such as DNA recombination were also visualized at the single-molecule level using this combined observation system. Moreover, the enzyme-based reaction could be directly regulated in the DNA origami frame by imposing structural stress on the substrate DNAs to elucidate the reaction mechanism. These target-orientated observation systems should contribute to a detailed analysis of biomolecular motions in real time at molecular resolution.