Novel Spectroscopic Tools to Differentiate Molecule-DNA Binding Interactions, Sense DNA and Track DNA Melting
Date of Award
Doctor of Philosophy
Dr. Ramakrishna Guda
Dr. Sherine Obare
Dr. Ekkehard Sinn
Dr. Charles Ide
Two-photon absorption, molecule-DNA binding, intercalation, minor groove-binding, electric field orientation, spectroscopic tools
The discovery of DNA as a genetic material and its double helical structure led to numerous studies directed at understanding molecule-DNA interactions. These studies have played an integral role in medical diagnostics, forensics, imaging and therapeutics. Typically, molecule-DNA interactions have two prominent modes: intercalation and minor groove binding. Several optical techniques are available that can monitor molecule-DNA binding interactions, but they cannot differentiate between intercalation and minor-groove binding. The major goals of the research are to develop novel optical spectroscopic tools that can differentiate molecule-DNA binding interactions, selectively and sensitively detect one form of DNA over another, and track DNA melting curves. To accomplish these goals, we developed two-photon absorption (2PA) cross-section based techniques to differentiate between molecule-DNA binding interactions. The hypothesis is that the 2PA cross-sections of molecules are sensitive to the electrostatic fields offered by the DNA backbone and that the alignment of molecular dipoles with the electric field can differentiate the mode of binding.
To test the hypothesis, investigations were carried out using two dye molecules, Hoechst 33258 (Hoe) and Acridine Orange (AcrO) binding to DNA. Hoe binds to the DNA via minor groove while AcrO intercalates with DNA. Relative 2PA cross-section studies have shown a more than 4-fold enhancement for Hoe whereas AcrO has no enhancement. These results confirmed our hypothesis that 2PA cross-sections of dye molecules are sensitive to local electric fields and that they can differentiate molecule-DNA interactions. This technique was extended to monitoring the interaction of other prominent dye molecules (like Thioflavin T and cyanine dyes) with DNA, and the technique was successful in elucidating the mode of binding in these systems. Also, one-, two-photon fluorescence sensing and relative 2PA cross-sections of dye molecules were used as markers to differentiate between single-stranded, duplex and G-quadruplex DNA structures. We have also used the power of 2PA cross-sections to track DNA melting transitions in duplex and quadruplex structures. Furthermore, a novel two-photon based induced fluorescence resonance energy transfer (2P-iFRET) technique was developed to monitor the DNA melting. Results have shown that this technique is sensitive and offers flexibility and sharper melting transitions over conventional techniques.
Alotaibi, Saad Hmoud, "Novel Spectroscopic Tools to Differentiate Molecule-DNA Binding Interactions, Sense DNA and Track DNA Melting" (2015). Dissertations. 512.