(550f) A Tethered-Particle Method Study of DNA Hybridization

The tethered-particle method is used to probe the complex process of DNA hybridization. This experiment analyzes tethers formed between PEGylated polystyrene microspheres and PEGylated glass surfaces, to which DNA strands have been grafted. This experiment is performed near the duplex melting temperature, resulting in dynamic binding and unbinding. Single molecular tethers are analyzed using high performance particle tracking with a high-speed camera. By analyzing 2D trajectories of the particle's centroid versus time, we can detect single binding and unbinding events from multiple bridged states. Molecular rates for transitioning between different bridging states are obtained; they indicate that multiple bridged states are less stable than expected for a zero cooperativity model. We hypothesize that this instability may be due to extreme force sensitivity of individual DNA duplexes. Interaction kinetics will also be investigated in the multiple bridge limit.