Development and Use of Novel Transverse Magnetic Tweezers for Single-Molecule Studies of DNA-Protein Interactions
I describe several contributions to single molecule experiments. A transverse magnetic tweezers is presented that enables in-plane micromechanical manipulation of a single DNA molecule. This includes a new method for tethering DNA utilizing two labeled beads and a functionalized glass micro-rod. The attachment chemistry reported here enables rapid capture of multiple DNA tethers in parallel, overcomes the difficulties associated with bead aspiration, and preserves the ability to perform differential extension measurements from the bead centroids. Combined with micro-injection pipettes, a new sample cell design, and a buffer exchange system, the components increase the ease-of-use and experimental throughput of the magnetic tweezers device. On the software side, several unique computational methods for interrogating single molecule data are described. First, a technique that uses the diffraction pattern of beads to perform sub-pixel, ~10 nm-level localization of the bead centroids is explained. Second, a novel method for automatically detecting steps in DNA extension data is presented. This algorithm is well-suited for analyzing experiments involving binding and force-induced unbinding of DNA-protein complexes, which produce flat extension regions - steps - corresponding to the times between individual protein association or dissociation events. Finally, a new algorithm for tracking densely-populated, fast spawning, indistinguishable objects moving unidirectionally at high-velocities is developed and its performance thoroughly characterized. Together, these results should improve single molecule micromanipulation techniques by providing a hardware and software combination that can be implemented and used relatively easily, while enabling near-Brownian-noise limit force and extension measurements on DNA and DNA-protein complexes.
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