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Ordered DNA origami arrays have the potential to compartmentalize space into distinct periodic domains that can incorporate a variety of nanoscale objects. Herein, we used the cavities of a preassembled 2D DNA origami framework to incorporate square-shaped DNA origami structures (SQ-origamis). The framework was self-assembled on a lipid bilayer membrane from cross-shaped DNA origami structures (CR-origamis) and subsequently exposed to the SQ-origamis. High-speed AFM revealed the dynamic adsorption/desorption behavior of the SQ-origamis, which resulted in continuous changing of their arrangements in the framework. These dynamic SQ-origamis were trapped in the cavities by increasing the Mg2+ concentration or by introducing sticky-ended cohesions between extended staples, both from the SQ- and CR-origamis, which enabled the directed docking of the SQ-origamis. Our study offers a platform to create supramolecular structures or systems consisting of multiple DNA origami components.

A framed photo of DNA: A pair of photoresponsive oligonucleotides containing azobenzene moieties was introduced into double-stranded DNA within the cavity of a DNA nanostructure (see scheme). The two dsDNAs, in contact at the center, were dissociated using UV irradiation and hybridized with visible light; this was directly observed using high-speed atomic force microscopy.

In den letzten beiden Jahrzehnten wurden verschiedene Methoden entwickelt, die den Nachweis und die Manipulation einzelner Molekule ermoglichen. Ein grundlegendes Verstandnis biochemischer Reaktionen wurde mit solchen Methoden ebenso erreicht wie Biomolekulfaltung und Wirkstoff-Screenings. Einzelmolekulanalysen wurden auch in der DNA-Nanotechnologie angewendet. Bis vor Kurzem konnten mit den verbreitetsten Verfahren Strukturen von 10 bis 20 nm erhalten werden, was allerdings fur viele Anwendungen nicht ausreicht. Die aktuelle Entwicklung eines “gerustgebundenen DNA-Origami” ermoglicht die Konstruktion groserer definierter Anordnungen. Einer der Hauptvorteile des Origami-Verfahrens ist die prazise Umsetzbarkeit der gewunschten Strukturen: Jeder Basenstapel kann als Anheftungspunkt verschiedener Arten von Nanoobjekten wirken. Daher ist die Methode geeignet, um verschiedene Funktionalitaten prazise zu positionieren und viele chemische und biochemische Prozesse auf Einzelmolekulebene zu untersuchen. Hier fassen wir die neuesten Erkenntnisse bei der Einzelmolekulanalyse mit DNA-Origami zusammen und diskutieren die kunftigen Aufgaben fur die Forschung.

Direct observation of enzymes interacting with DNA should be one of the ultimate technologies for investigating the mechanical behavior of the enzymes during the reactions. Atomic force microscopy (AFM) enables observation of biomolecules at a nanoscale spatial resolution; however, for a stable analysis, a scaffold to observe the reaction should be explored. DNA origami has recently been developed for the construction of a wide variety of multidimensional nanostructures, which can be used as scaffolds to incorporate various functionalities at specific positions. We intended to construct an AFM-based analysis system for DNA repair using a DNA origami scaffold carrying various substrate double-stranded DNAs (dsDNA). We employed DNA baseexcision repair (BER) enzymes 8-oxoguanine glycosylase (hOgg1) and T4 pyrimidine dimer glycosylase (PDG) to analyze the reaction on the DNA scaffold (Figure 1a). In the repair process, hOgg1 removes 8-oxoguanine (oxoG) to prevent G:C!T:A transversion during replication by DNA glycosylase activity and endonuclease activity for apurine– apyrimidine (AP) sites (AP-lyase activity) (Figure 1a). PDG removes photodamaged pyrimidine dimer including cis–syn cyclobutane thymine dimer (T T) by DNA glycosylase/AP-lyase activity. BER enzymes often require structural changes of the target DNA strands, such as DNA bending, for the reaction to proceed. The enzyme hOgg1 bends double-helix DNA by about 708 with flipping out of the oxoG for procession of the excision. The enzyme PDG bends double-helix DNA by 608 with flipping out of the 3’-side of A in the opposite strand of T T. The glycosylase/AP-lyase activity of these enzymes leads to single-strand scission at the damaged nucleotide (Figure 1b). In addition, the reaction intermediates of both reactions form a covalent bond with the enzyme by reduction with NaBH4. [5,10] We recently developed a framelike DNA origami scaffold to incorporate two different substrate dsDNAs. If the above-mentioned enzymatic and chemical reactions