Single-molecule AFM characterization of individual chemically tagged DNA tetrahedra

Single-molecule characterization is essential for ascertaining the structural and functional properties of bottom-up DNA nanostructures. Here we enlist three atomic force microscopy (AFM) techniques to examine tetrahedron-shaped DNA nanostructures that are functionally enhanced with small chemical tags. In line with their application for biomolecule immobilization in biosensing and biophysics, the tetrahedra feature three disulfide-modified vertices to achieve directed attachment to gold surfaces. The remaining corner carries a single bioligand that can capture and present individual cargo biomolecules at defined lateral nanoscale spacing. High-resolution AFM topographic imaging confirmed the directional surface attachment as well as the highly effective binding of individual receptor molecules to the exposed bioligands. Insight into the binding behavior at the single-molecule level was gained using molecular recognition force spectroscopy using an AFM cantilever tip with a tethered molecular receptor. Finally, simultaneous topographic and recognition imaging demonstrated the specific receptor-ligand interactions on individual tetrahedra. In summary, AFM characterization verified that the rationally designed DNA nanostructures feature characteristics to serve as valuable immobilization agents in biosensing, biophysics, and cell biology.