145 Single-molecule observation of enzymes and DNA structural changes in the DNA nanostructures

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Direct observation of the movement of biomolecules including enzymes and DNAs should be one of the ultimate goals for investigating the detailed mechanical behavior of the molecules during the reactions. We designed various0020DNA nanostructures using DNA origami method for the preparation of single-molecule observation scaffolds. Using the designed DNA scaffold and high-speed atomic force microscopy (AFM), the single-molecule behaviors of the DNA-modifying enzymes, repair enzymes, and recombinases were observed in the target double-stranded DNAs (dsDNAs) placed in the DNA frame structure (Endo, Katsuda, Hidaka, & Sugiyama, 2010a, 2010b). DNA structural changes including G-quadruplex formation (Sannohe, Endo, Katsuda, Hidaka, & Sugiyama, 2010) and B-Z DNA conformational change (Rajendran, Endo, Hidaka, & Sugiyama, 2013) were also visualized. Using this system, we observed the photo-induced DNA hybridization and dissociation by detecting the global structural changes of the incorporated two dsDNAs in the DNA frame structure (Endo, Yang, Suzuki, Hidaka, & Sugiyama, 2012). A pair of azobenzene-modified oligonucleotides (ODNs) was employed, which forms duplex in the transform and dissociates in the cis-form. During UV-irradiation, hybridized azobenzene-modified ODNs at the center dissociated, and the subsequent visible-light irradiation induced the hybridization of the photoresponsive ODNs, meaning that the reversed switching behavior such as the hybridization and dissociation was directly visualized at the single-molecule level. These photoresponsive ODNs were also used for controlling assembly and disassembly of the hexagonal DNA origami structures with photoirradiation (Yang, Endo, Hidaka, & Sugiyama, 2012). The combination of the designed DNA scaffold modified with target DNA strands and high-speed AFM is valuable for visualizing and analyzing the single enzymatic and chemical reactions.