| Description |
The goal of this dissertation was to develop single-molecule fluorescence microscopy to investigate hybridization of surface-immobilized DNA probes with target DNA in solution. These experiments required surface chemistry that could immobilize single-stranded DNA probe sites while passivating the surrounding surface to avoid non-specific adsorption of fluorescently-labeled target DNA strands. Two different immobilization schemes were investigated: a ‘click' chemistry method employed short noncomplementary DNA for surface passivation, where the negatively-charged sugar-phosphate backbone and random base sequence reduced interactions with solution-phase target DNA. The epoxide chemistry employed back-filling the surface with negatively-charged sulfonate groups for surface passivation. While the ‘click' chemistry method produced surfaces with higher probe-site densities, sulfonate-passivated epoxide-derivatized surfaces were more repellant to nonspecific interactions with target DNA. Using epoxide-immobilized DNA probes, single-molecule fluorescence imaging was applied to measuring rates of hybridization between fluorescently-labeled target DNA and unlabeled-probes on glass surfaces. Immobilized DNA probe sites were quantified by counting individual DNA duplexes at low concentrations, and those results were used to calibrate fluorescence intensities to measure a binding isotherm. Dissociation rates were determined from interfacial-residence times of individual DNA duplexes. The kinetics of the probe-target reaction at a surface were compared with the same reaction in free-solution, and equilibrium constants, dissociation, and association rates were found to be nearly equivalent. Because the immobilized probe DNA on these surfaces was unlabeled, photobleaching of a probe label was not an issue, allowing capture substrates to be used for long periods of time or even reused in multiple experiments. Single-molecule fluorescence imaging was then applied to studies of single-nucleotide polymorphism (SNPs) on the kinetics of DNA duplex formation and dissociation. A mismatch greatly reduced the association equilibrium constant, but the position of the mismatch impacted how the on- and off-rates governed this change. It was found that a mismatch near the strand center increased the off-rate, which was offset by a faster on-rate, likely indicating nucleation of the duplex near its ends with zippering to form a hybridized duplex. These results illustrate the power of single-molecule kinetic measurements to reveal how small structural differences can significantly impact the kinetics of DNA hybridization. |
