Single-molecule fluorescence imaging of DNA hybridization equilibria and kinetics at surfaces

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Title Single-molecule fluorescence imaging of DNA hybridization equilibria and kinetics at surfaces
Publication Type dissertation
School or College College of Science
Department Chemistry
Author Manhart, Michael William
Date 2018
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.
Type Text
Publisher University of Uta
Subject Analytical chemistry
Dissertation Name Doctor of Philosophy
Language eng
Rights Management (c) Michael William Manhart
Format application/pdf
Format Medium application/pdf
ARK ark:/87278/s6jx3rqf
Setname ir_etd
ID 1547092
Reference URL https://collections.lib.utah.edu/ark:/87278/s6jx3rqf
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