Single-Molecule AFM Study of DNA Damage by ¹O₂ Generated from Photoexcited C₆₀

Abstract

Light-induced oxidative damage of DNA by 1 O 2 generated from photoexcited C 60 was observed at the single-molecule level by atomic force microscopy (AFM) imaging. Two types of DNA origami with uniform morphologies were immobilized on a mica surface and used as DNA substrates. Upon visible light irradiation (528 nm) in the presence of a C 60 aqueous solution, the morphology changes of DNA origami substrates were observed by time-lapse AFM imaging at the single-molecule level by tracking a discrete DNA molecule. The origami showed nicked and flattened morphologies with relaxed features caused by the covalent cleavage of the DNA strands. The involvement of 1 O 2 in the on-surface DNA damage was clearly confirmed by AFM experiments in the presence of a 1 O 2 quencher and ESR measurements with a spintrapping agent for 1 O 2 . This study is the first example of single-molecule observation of oxidative damage of DNA by AFM with corresponding morphology changes in a photocontrolled and time-dependent manner by 1 O 2 generated catalytically from photoexcited C 60 .

Anion-specific structure and stability of guanidinium-bound DNA origami

“…Our data suggest that the supramolecular structure of a DNA origami amplifies subtle steric effects by their accumulation over the large number of linked dsDNA segments. While similar amplification effects have recently been found also in DNA origami degradation by UV irradiation [62] and reactive oxygen species [63] , this is the first time that such a behavior could be observed not only at the nanostructural but also at the molecular level. Therefore, in combination with the thermodynamic analysis, geometric, energetic, and hydration effects accompanying DNA-ligand interactions may find a novel sensitive nanoscopic and thermodynamic readout.…”

Self‐Regeneration and Self‐Healing in DNA Origami Nanostructures

“…Photobleaching still yielded dark areas ( Figure 1F under constant illumination conditions can also lead to photodamage of the DNAs caffold and the staple strands. [35] Hence,f ull recovery cannot be reached in line to what has been observed for binding site bleaching in DNAP AINT experiments. [36,37] To suppress the damage to the docking sites by ROSa nd to photostabilize the fluorescent labels,w e removed oxygen enzymatically and quenched reactive triplet and radical states by areducing and oxidizing system (ROXS) ( Figure 1G).…”

“…The intensity of the spots, however, did not recover completely but saturated at 20–60 % of the initial fluorescence intensity (see Figure 1 D, blue). Besides the fluorophore photobleaching, generation of reactive oxygen species (ROS) under constant illumination conditions can also lead to photodamage of the DNA scaffold and the staple strands [35] . Hence, full recovery cannot be reached in line to what has been observed for binding site bleaching in DNA PAINT experiments [36, 37] .…”

Selbstregeneration und Selbstheilung in DNA‐Origami‐Nanostrukturen

“…Die Intensität der Punkte erholte sich jedoch nicht vollständig, sondern sättigte bei 20–60 % der ursprünglichen Fluoreszenzintensität (siehe Abbildung 1 D, blau). Neben dem Photobleichen der Fluorophore kann die Entstehung reaktiver Sauerstoffspezies (Reactive Oxygen Species ROS) unter konstanter Belichtung ebenfalls zu Photoschäden am Scaffold‐Strang und an den Staple‐Strängen führen [35] . Daher kann in Übereinstimmung mit dem Photobleichen von Anbindestellen in DNA‐PAINT‐Experimenten keine vollständige Wiederherstellung der Helligkeit beobachtet werden [36, 37] .…”

“…Neben dem Photobleichen der Fluorophore kann die Entstehung reaktiver Sauerstoffspezies (Reactive Oxygen Species ROS) unter konstanter Belichtung ebenfalls zu Photoschäden am Scaffold-Strang und an den Staple-Strängen führen. [35] Daher kann in Übereinstimmung mit dem Photobleichen von Anbindestellen in DNA-PAINT-Experimenten keine vollständige Wiederherstellung der Helligkeit beobachtet werden. [36,37] Um eine Schädigung der Docking-Stränge durch ROS zu verhindern und die fluoreszierenden Markierungen zu photostabilisieren, haben wir den gelçsten Sauerstoff enzymatisch entfernt und reaktive Tr iplet-und Radikalzustände des Fluorophores durch ein reduzierendes und oxidierendes System (ROXS) gequencht (Abbildung 1G).…”