Quantitative Assessment of Tip Effects in Single‐Molecule High‐Speed Atomic Force Microscopy Using DNA Origami Substrates

• Published in: Angewandte Chemie International Edition Vol. 59; no. 34; pp. 14336 - 14341
• Main Authors: Kielar, Charlotte, Zhu, Siqi, Grundmeier, Guido, Keller, Adrian
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 17.08.2020; John Wiley and Sons Inc
• Edition: International ed. in English
• Subjects: Atomic force microscopy; Bacterial Proteins - chemistry; Binding; biomolecular interaction; Biotin; Biotin - analogs & derivatives; Biotin - chemistry; Communication; Communications; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - metabolism; DNA origami; high-speed atomic force microscopy; Ligands; Microscopes; Microscopy; Microscopy, Atomic Force - methods; Nanostructures - chemistry; protein–ligand binding; Single Molecule Imaging - methods; single-molecule studies; Streptavidin; Substrates; protein-ligand binding; high-speed atomic force microscopy; DNA origami; biomolecular interaction; single-molecule studies
• ISSN: 1433-7851; 1521-3773; 1521-3773
• DOI: 10.1002/anie.202005884

High‐speed atomic force microscopy (HS‐AFM) is widely employed in the investigation of dynamic biomolecular processes at a single‐molecule level. However, it remains an open and somewhat controversial question, how these processes are affected by the rapidly scanned AFM tip. While tip effects are commonly believed to be of minor importance in strongly binding systems, weaker interactions may significantly be disturbed. Herein, we quantitatively assess the role of tip effects in a strongly binding system using a DNA origami‐based single‐molecule assay. Despite its femtomolar dissociation constant, we find that HS‐AFM imaging can disrupt monodentate binding of streptavidin (SAv) to biotin (Bt) even under gentle scanning conditions. To a lesser extent, this is also observed for the much stronger bidentate SAv–Bt complex. The presented DNA origami‐based assay can be universally employed to quantify tip effects in strongly and weakly binding systems and to optimize the experimental settings for their reliable HS‐AFM imaging. Heed the speed: A DNA origami‐based assay for the quantitative evaluation of tip effects in high‐speed atomic force microscopy (HS‐AFM) is presented. Using streptavidin–biotin as a model system, it is shown that the rapidly scanned AFM tip may disrupt even strongly binding biomolecular complexes with dissociation constants in the femtomolar range.