Development and demonstration of a multiplexed magnetic tweezers assay
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This dissertation is concerned with the methods and applications of single molecule force spectroscopy. In the introduction, the traditional single molecule force spectroscopy instruments are introduced and the advantages and drawbacks of each are discussed. The first chapter is a review of methods to ensure that biomolecular bond lifetime parameter estimations are not contaminated by multiple bond data. This review culminates in an examination of the literature on the strength of the bond between biotin and streptavidin and finds that by filtering the numerous publications for those that clearly demonstrate specific single bond behavior, there is a consensus of the bond strength and kinetic parameters. The second chapter of the dissertation discusses the capabilities of a magnetic tweezer assay, which combines massive multiplexing, precision bead tracking, and bi-directional force control into a flexible and stabile platform for examining single molecule behavior. Using a novel method for increasing the precision of force estimations on heterogeneous paramagnetic beads, I demonstrate the instrument by examining the force dependence of uncoiling and recoiling velocity of type 1 fimbriae from Eschericia coli (E. coli) bacteria, and see similar results to previous studies. Chapter 3 is a study of the lifetime of the activated FimH-mannose bond under various force conditions using the previously described magnetic tweezer. The bond is found to be extremely long-lived at forces less than 30 pN, with an average lifetime > 1000 times longer than the biotin-streptavidin bond, making it one of the strongest non-covalent interactions known in nature. Furthermore, the average lifetime of the bond is similar between 9 and 30 pN of force, suggesting a force range at which the lifetime is force-independent, demonstrating ideal bond behavior for the first time in a natural system. It is hypothesized that the long lifetime and ideal behavior is due to a gateway that locks mannose into the binding pocket and opens at a rate independent of force. This study elucidates a mechanism for very strong biological binding, and provides insight into approaches for developing novel antiadhesive therapies. This dissertation is concluded with a review of the body of work in chapters 1-3 and a discussion of the future directions for this research.
- Mechanical engineering