A versatile "multiple fishhooks" approach for the study of ligand-receptor interactions using single-molecule atomic force microscopy.
Despite the powerfulness of atomic force microscopy (AFM)-based single-molecule force spectroscopy in the study of ligand-receptor interactions, complicated cantilever functionalization and data interpretation have often been a great hurdle for its widespread application. Here, we present a much simplified experimental scheme by using a "multiple fishhooks" approach. In this strategy, multiple ligands are labeled on a single polymer chain, which forms complexes with receptors anchored on the substrate surface. Therefore, multiple single-bond rupture events can be captured in the same force-extension curves, similar to the widely used polyprotein approach. This method also allows nonsingle-molecule events and nonspecific interactions between cantilever and surface to be readily excluded from real data pool and greatly increases the quality and quantity of single-molecule data. The biggest advantage of our approach over the previously reported one is the choice of a naturally occurring polysaccharide, hyaluronan, the conformation of which in solution can be fine-tuned by pH, as the polymer backbone of the "multiple fishhooks" handle. Furthermore, our approach greatly simplifies the chemical synthesis of the polymer handle, allowing bioactive molecules to be easily one-step labeled on the handles in aqueous solution. We validate this strategy using the widely studied streptavidin-biotin system, and our single-molecule AFM results are in good agreement with previously reported ones. We anticipate that this novel strategy can be used as a versatile tool to study other complex and challenging ligand-receptor interactions.
National Laboratory of Solid State Microstructure, Department of Physics, Nanjing University, Nanjing, Jiangsu, People's Republic of China.
Xiaotian
X
Han
Meng
M
Qin
Hai
H
Pan
Yi
Y
Cao
Wei
W
Wang