Probing the Unbinding Kinetics of DNA-H-NS-DNA Protein Complexes by a High-Speed and High-Throughput Single-Molecule Pulling Assay using Atomic Force Microscopy

Abstract

We demonstrate an atomic force microscopy (AFM) pulling assay to measure the unbinding kinetics of DNA-protein interactions. Here we use this technique to investigate the interactions between DNA and the abundant, non-specific nucleoid-associated protein H-NS (Histone-like Nucleoid-Structuring protein). In particular, we probe the unbinding kinetics of the DNA-H-NS-DNA bridges under the influence of one environmental factor, Mg2+-ions. In this experiment, biotinylated DNA molecules are attached on an avidin-coated mica surface. Then thiolated DNA molecules and H-NS proteins are added to the sample solution to form DNA-H-NS-DNA complexes. Individual DNA-protein complexes are then pulled by a gold-coated AFM tip. By measuring the force-distance curve, we gain information on the strength with which H-NS forms bridges between DNA molecules. Here, we probe both single DNA-H-NS-DNA complexes and sample many complexes by fast scanning of the AFM tip over the sample's surface and performing approach/pulling cycles for each position. This experimental approach leads to high-throughput measurements with single-molecule resolution and is widely applicable to other DNA-bridging proteins and receptor-ligand interactions.