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DNA modification using enzymes often requires bending specific DNA strands to facilitate the reaction. Using tense and relaxed dsDNAs incorporated in the DNA frame, relaxed dsDNA can be a better substrate for the DNA methylation enzyme EcoRI methyltransferase, which requires bending of dsDNA for the methyl‐transfer reaction [62, 64]. Methylation preferentially occurred in the relaxed dsDNA, indicating the importance of structural flexibility in the bending of dsDNA during the methyl‐transfer reaction. Therefore, DNA methylation can be regulated using tension‐controlled dsDNAs constructed in the DNA frame. DNA base excision repair enzymes, 8‐oxoguanine glycosylase [65], and T4 pyrimidine dimer glycosylase [66], also show that the relaxed substrate dsDNAs were preferable for the reactions. The DNA frame system and direct observation serve to elucidate the detailed properties of the modifying enzymes and these events.

Direct observation of DNA recombination was carried out by incorporating the substrate sequences into the DNA frame (Figure 1.9c) [61, 67, 68]. Using Cre recombinase, the Cre–DNA complex and recombinant products were clearly observed in the DNA frame, demonstrating that recombination occurred in the nanospace. Lapsed HS‐AFM images showed that the Cre–DNA complex formed first, followed by the complex dissociating into four monomers, and the simultaneous appearance of the recombinant product. In addition, the structural stress imposed on the Holliday junction (HJ) intermediates in the DNA frame can regulate the direction of recombination.

Using the HJ‐containing DNA frame and Rec U resolvase, the resolution event was visualized in the DNA frame [69]. We also visualized the binding preference and the activity of the HJ‐resolvase monokaryotic chloroplast 1 (MOC1 in Arabidopsis thaliana) using HS‐AFM [70]. The interaction of MOC1 with the center of the HJ and symmetric cleavage of the HJ structure were observed in the DNA frame. Observation of geometric arrangements of substrate dsDNAs using DNA frames is valuable for studying recombination events.

Using a DNA origami scaffold and HS‐AFM system, important DNA conformational changes including G‐quadruplex formation [59, 71], photo‐induced duplex formation [72], triple helix formation [73], G‐quadruplex/i‐motif formation [74], and B–Z transition [60] have been successfully imaged. This method can be extended to the direct observation of various enzymes and reaction events, such as DNA‐modifying enzyme [62], repair enzymes [75], recombinase [61, 76], resolvase [69, 71], Cas9 [70, 77], TET [78], DNA recognition [79, 80], and RNA interactions [81]. Using the DNA frame for the incorporation of substrates in various arrangements, the enzyme reactions can be visualized and regulated in the DNA frame to study the reaction mechanisms. The observation system can be used as a general strategy for investigating various DNA structural changes and molecular switches working at the single‐molecule level.