title: Single-molecule imaging of dynamic motions of biomolecules in DNA origami nanostructures using high-speed atomic force microscopy.
abstract: CONSPECTUS: Direct imaging of molecular motions is one of the most fundamental issues for elucidating the physical properties of individual molecules and their reaction mechanisms. Atomic force microscopy (AFM) enables direct molecular imaging, especially for biomolecules in the physiological environment. Because AFM can visualize the molecules at nanometer-scale spatial resolution, a versatile observation scaffold is needed for the precise imaging of molecule interactions in the reactions. The emergence of DNA origami technology allows the precise placement of desired molecules in the designed nanostructures and enables molecules to be detected at the single-molecule level. In our study, the DNA origami system was applied to visualize the detailed motions of target molecules in reactions using high-speed AFM (HS-AFM), which enables the analysis of dynamic motions of biomolecules in a subsecond time resolution. In this system, biochemical properties such as the placement of various double-stranded DNAs (dsDNAs) containing unrestricted DNA sequences, modified nucleosides, and chemical functions can be incorporated. From a physical point of view, the tension and rotation of dsDNAs can be controlled by placement into the DNA nanostructures. From a topological point of view, the orientations of dsDNAs and various shapes of dsDNAs including Holliday junctions can be incorporated for studies on reaction mechanisms. In this Account, we describe the combination of the DNA origami system and HS-AFM for imaging various biochemical reactions including enzymatic reactions and DNA structural changes. To observe the behaviors and reactions of DNA methyltransferase and DNA repair enzymes, the substrate dsDNAs were incorporated into the cavity of the DNA frame, and the enzymes that bound to the target dsDNA were observed using HS-AFM. DNA recombination was also observed using the recombination substrates and Holliday junction intermediates placed in the DNA frame, and the direction of the reactions was controlled by introducing structural stress to the substrates. In addition, the movement of RNA polymerase and its reaction were visualized using a template dsDNA attached to the origami structure. To observe DNA structural changes, G-quadruplex formation and disruption, the switching behaviors of photoresponsive oligonucleotides, and B-Z transition were visualized using the DNA frame observation system. For the formation and disruption of G-quadruplex and double-helix DNA, the two dsDNA chains incorporated into the DNA frame could amplify the small structural change to the global structural change, which enabled the visualization of their association and dissociation by HS-AFM. The dynamic motion of the helical rotation induced by the B-Z transition was also directly imaged in the DNA frame. Furthermore, the stepwise motions of mobile DNA along the DNA track were visualized on the DNA origami surface. These target-orientated observation systems should contribute to the detailed analysis of biomolecule motions in real time and at molecular resolution.
authors.affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University , Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Endo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sugiyama
mesh_terms: D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D004260:DNA Repair; D004798:Enzymes / Q000032:analysis / Q000737:chemistry*; D019426:Integrases / Q000737:chemistry; D018625:Microscopy, Atomic Force / Q000379:methods*; D057054:Molecular Imaging / Q000379:methods*; D049329:Nanostructures / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry
chemical_list: D004798:Enzymes; D009692:Nucleic Acid Heteroduplexes; D009841:Oligonucleotides; D004247:DNA; C020098:DNA modification methylase EcoRI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C045073:Cre recombinase; D019426:Integrases