Linker Engineering of Ligand‐Decorated DNA Origami Nanostructures Affects Biological Activity

News from an old acquaintance: The streptavidin (STV)‐biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono‐ or bi‐dentate linker architectures on DON with a novel high‐speed atomic force microscope (HS‐AFM) enabling acquisition times as short as 50 ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub‐100 millisecond range. The linker systems are also used to present biotinylated epidermal growth factor on DON to study the activation of the epidermal growth factor receptor signaling cascade in HeLa cells. The studies confirm that cellular activation correlated with the binding properties of linker‐specific STV/DON interactions observed by HS‐AFM. This work sheds more light on the commonly used STV/DON system and will help to further standardize the use of DNA nanostructures for the study of biological processes. DNA origami nanostructures (DON) decorated with bioactive ligands via streptavidin‐biotin bridges exhibit surprisingly high binding dynamics that are influenced by the underlying linker architecture. The results of binding studies obtained by high‐speed atomic force microscopy correlate with biological activity.