Nucleic acids and proteins are fundamental molecular units of life. To understand their properties, we need powerful tools that allow investigation at the single-molecule level. Over the past three decades, the development of single-molecule force and fluorescence techniques has provided us new knowledge that was previously unattainable through ensemble measurements. However, we lack methods that allow us to precisely measure the mechanical properties of these molecules while visually detecting multiple molecules at the same time. In this dissertation, we maximize the information obtained in single-molecule measurements by pushing the techniques to be more precise and more complex. We then utilize our instrument to directly observe the folding and unfolding of the nucleic acid G-quadruplex structure. Among the single-molecule force techniques, angstrom-resolution has been achieved by optical tweezers using the dual-trap instrument design. Dual-traps can be produced by acousto-optic (AO) devices, which have many advantages, but trap positioning inaccuracies have limited their usage at high-resolution. We have designed a method to remove the inaccuracies by randomizing the phase of the radio frequency that drives the AO device. We demonstrated that the trap inaccuracies are completely eliminated and high-resolution trapping quality is achieved. This advance allows us to perform long duration measurements with reduced drift in trap measurement over time. Next, we present instrumentation advances that combine high-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single molecule assembly. Multicolor not only allows the detection of multiple observables but also increases the flexibility in the choice of fluorophores. We demonstrated the ability to simultaneously measure angstrom-scale changes in tether extension and single fluorophore signals. The biggest challenge in integrating optical tweezers and fluorescence is the potential for greatly enhanced photobleaching which can make experiments impossible. We showed that the mean number of photons emitted before bleaching is unaffected by the trap laser when interlacing the fluorescence and optical trap lasers. We investigated the photostability of quantum dots and fluorophores. Finally, we devised computer-controlled automation to conserve the fluorophore lifetime. This advance enables us to observe multiple molecules or multiple degrees of freedom within a molecular complex while mechanically manipulate and detect them. Taking advantage of these method and instrumentation advances, we investigate the folding and unfolding of a DNA secondary structure: thrombin-binding aptamer G-quadruplex (TBA-GQ). Studying the kinetics of G-quadruplex formation is essential for understanding telomere regulation (the ends of chromosomes) and therapeutic approaches for disease. TBA-GQ is the smallest G-quadruplex. Although many experiments and simulations have been done on G-quadruplex, the small size and low stability make it very difficult to observe folding and unfolding of TBA-GQ directly. Our high-resolution optical tweezers have the sensitivity and stability to directly observe TBA-GQ at very low forces. We found that with increasing force, the folding rate decreased and the unfolding rate increased. Our work demonstrates that at a given force, the TBA-GQ formation is facilitated by metal ions and is stabilized by thrombin. It also indicates that the equilibrium force increased as KCl concentration increased. From a detailed analysis of the folding and unfolding rate constants vs applied force, we were able to detect a single transition state conserved across all conditions and identify the structure of the transition state as the G-triplex structure.
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