In the present study, we demonstrate single-molecule imaging of triple helix formation in DNA nanostructures. The binding of the single-molecule third strand to double-stranded DNA in a DNA origami frame was examined using two different types of triplet base pairs. The target DNA strand and the third strand were incorporated into the DNA frame, and the binding of the third strand was controlled by the formation of Watson–Crick base pairing. Triple helix formation was monitored by observing the structural changes in the incorporated DNA strands. It was also examined using a photocaged third strand wherein the binding of the third strand was directly observed using high-speed atomic force microscopy during photoirradiation. We found that the binding of the third strand could be controlled by regulating duplex formation and the uncaging of the photocaged strands in the designed nanospace.
Single-molecule imaging of the triple-helix formation in a DNA frame was demonstrated. The binding of the third strand to the double-stranded DNA was examined using two different types of triplet base pairs. The triple-helix formation was also examined using photocaged third strands. Third-strand binding was observed directly by using high-speed atomic force microscopy with photoirradiation.