Abstract
Atomic force microscopy (AFM) has proven to be a powerful tool for the study of DNA-protein interactions due to its ability to image single molecules at the nanoscale. However, the use of AFM in force spectroscopy to study DNA-protein interactions has been limited. Here we developed a high throughput, AFM based, pulling assay to measure the strength and kinetics of protein bridging of DNA molecules. As a model system, we investigated the interactions between DNA and the Histone-like Nucleoid-Structuring protein (H-NS). We confirmed that H-NS both changes DNA rigidity and forms bridges between DNA molecules. This straightforward methodology provides a high-throughput approach with single-molecule resolution which is widely applicable to study cross-substrate interactions such as DNA-bridging proteins.
Highlights
Single molecule manipulation and force spectroscopy approaches such as optical tweezers, magnetic tweezers, acoustic force spectroscopy (AFS) and atomic force microscopy (AFM) have emerged as powerful biophysical techniques[1,2,3,4,5]
The label-free ends of the DNA molecules can anchor via non-specific interaction to the Atomic force microscopy (AFM) tip by pushing the tip onto the surface (Fig. 1)
We move the tip a distance twice the DNA contour length away from the surface to ensure that the DNA molecules break off the tip after this single stretching curve
Summary
Single molecule manipulation and force spectroscopy approaches such as optical tweezers, magnetic tweezers, acoustic force spectroscopy (AFS) and atomic force microscopy (AFM) have emerged as powerful biophysical techniques[1,2,3,4,5] Through their ability to precisely and accurately measure displacements and forces, these techniques offer researchers the opportunity to directly study the interactions of biological systems. By acquiring force-distance curves, we gain information on the forces associated with H-NS induced changes in DNA rigidity and the formation of H-NS bridges between DNA molecules This simple approach allows measurements on DNA-protein interaction with single-molecule resolution and is widely applicable to other - especially divalent - DNA-binding proteins and receptor-ligand interactions
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