Master of Science / Department of Chemistry / Daniel A. Higgins / The potential-dependent reorientation dynamics of double stranded DNA (ds-DNA) covalently attached to planar glassy carbon electrode (GCE) surfaces were studied in this thesis. The orientation of ds-DNA was investigated via the distance-dependent quenching of fluorescence from a 6–carboxyfluorescein (FAM6) flurophore to the electrode surface. The fluorophore was covalently bound to the distal end of the DNA. Fluorescence microscopy was employed for optical detection of FAM6 fluorescence and hence the DNA dynamics. The variation of the fluorescence from the dye with electrode potential is attributed to distance-dependent dipole-electrode energy transfer. Application of positive potentials (i.e., +0.2 V vs. open circuit potential, OCP) to the GCE caused the ds-DNA to align approximately parallel to the surface, yielding strong FAM6-electrode energy transfer and low fluorescence intensity. With the switching of the potential towards negative values (i.e., -0.4 V vs. OCP) the ds-DNA realigned perpendicular to the GCE surface leading to a reduction in energy transfer and high fluorescence intensity. Initial DNA reorientation upon a change in electrode potential is very fast. These fast dynamics have been observed and characterized in a number of previous publications. We have observed subsequent slow dynamics that we attribute to slow orientational relaxation of the DNA. Our observations were first reported by Q. Li, et al., J. Am. Chem. Soc. 2012, 134, 14467. In this thesis, this prior work is extended to verify the reproducibility of these new dynamics and to eliminate the possibility of certain artifacts as their source. Specifically, the experiments are repeated using a new cell design and a different buffer. In the primary experiments performed in this thesis, the dependence of the DNA reorientation dynamics on surface coverage was investigated by observing the fluorescence modulation as a function of probe concentration in the functionalization bath. Concentrations of 0.25, 1.0 and 1.5 µM 35-mer ds-DNA were employed. Electrodes functionalized at these concentrations have ds-DNA surface coverages of 1.18 x 10[superscript]12, 3.24 x 10[superscript]12 and 4.26 x 10[superscript]12 cm[superscript]-2, respectively. With increasing concentration of the DNA probe, the reorientation time constant at positive applied bias (vs. OCP) increased, indicting reorientation was slowed. In contrast, the time constant decreased with the negative applied bias (vs. OCP) indicating faster orientational relaxation. The possible origins for the observed trends in the reorientation time constant are discussed.

Numerous short flapped DNA structures are created during the semi-discontinuous replication. These toxic intermediates are quickly resolved to produce a fully intact duplex of replicated DNA. Structure specific nuclease are key to resolving these structures, and show a high degree of specificity for their cognate substrate structures while being essentially insensitive to nucleotide sequence. Herein I demonstrate through confocal based single molecule experiments that the 5’ structure specific nuclease Flap Endonuclease 1 (FEN1) achieves its substrate specificity by coupling the bending of DNA substrate with structuring of the active site in a way that non-cognate structures binding is significantly destabilized and enzymatic features are incapable of structuring in the absence of particular substrate features, in particular a single nucleotide 3’ flap the FEN1 induces in nearly all DNA substrates. Debate remained over whether DNA was bound via a conformational capture or induced fit mechanism, and so I proceed to investigate the dynamics of the DNA itself in solution. Conclusions about conformational capture or induced fit remain elusive, however I did determine that DNA structures are rigidified by charge repulsion, an effect lessened by the salt concentration, which functions to shield the negative charge of DNA from itself. Additionally unstacking of the DNA in nicked structures incurs a significant free energy penalty, which FEN1 overcomes by its hydrophobic wedge motif, lending credence to an induced fit mechanism.

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Master of Science / Department of Chemistry / Jun Li / This study is focused on exploring the mechanisms of DNA dynamics at carbon electrode surfaces under a strong electric field for the development of novel DNA hybridization sensors. Oligonucleotides with FAM6 attached at the distal end are covalently tethered on the carbon electrode surface. The fluorescence emission from the FAM6 is strongly quenched in close proximity to the electrode surface. The modulation to the fluorescence intensity is correlated with the reversible reorientation of the negatively charged DNA molecules under the electric field within the electric double layer. The orientation dynamics are apparently determined by the interplay of the electropotential, salt concentration, and stiffness of the DNA molecules. We have observed that dsDNAs switch with fast dynamics (in < 0.05 second) followed by relaxation at a slower rate (in > 0.1 second) when the electric field is altered by stepping the electropotential to a more positive or negative value. The DNA reorientation exhibits strong dependence on the PBS buffer concentration and electric double layer thickness. A preliminary calculation based on dipole-surface energy transfer theory indicates that the critical distance between FAM6 and glassy carbon surface is 10.95 nm. In connection with the fluorescence study, the effect of DNA hybridization on electrochemical impedance spectroscopy (EIS) has also been investigated by two methods in an attempt to develop a fast electronic detection method. First, EIS at high AC amplitude (141 mV rms) with DNA-modified glassy carbon electrodes before and after target DNA hybridization have shown notable change at high frequencies, likely related to the DNA reorientation processes. Second, reversible EIS detection of DNA hybridization has been demonstrated with patterned regular carbon nanofiber arrays at normal AC amplitude (10 mV rms). The combination of these two methods will be explored in future studies. The effects of the electric field on surface-tethered molecular beacons (MBs) have also been studied with fluorescence spectroscopy. An increase in fluorescence at negative bias is observed accompanying the opening of the MB stem, which leads to larger separation between fluorophore and quencher. At positive bias, the rehybridization of the MB stem leads to a decrease in fluorescence intensity.

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