Two-Photon Spectroscopy To Differentiate Molecule-DNA Binding Interactions
Usakoski, Kelly M.
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The objective of this study is to search for novel spectroscopic techniques to monitor molecule-DNA interactions using the 2PA cross-sections of molecules as markers. The hypothesis is that the 2PA cross-section is very sensitive to electric fields, and the DNA backbone possesses an inherent electric field that can alter the 2PA cross-sections of DNA chromophores. Molecules can interact with DNA through intercalation or minor-groove binding, altering the specified electric field. The method of molecules binding to DNA was studied using five dyes; Hoe33258, AcrO, ThT, DTI, and DTDCI. The 2PA cross-section was determined and 2PA enhancements were obtained and used as markers for DNA-molecule interactions. In addition, CD and absorption spectral changes were used to follow the binding of the molecules with DNA. The results of the 2PA cross-sections have shown that ThT, AcrO, and DTI dyes bind to DNA through intercalation, shown by a decrease or no change in the 2PA cross-section, while Hoe33258 and DTDCI dyes are minor-groove binders, shown by an enhancement of the cross-sections. These results confirm the electric field hypothesis where the electric field is parallel to the molecular dipole for minor-grove binding and perpendicular to the molecular dipole for intercalation. Monitoring the electric field of the samples could also be an efficient and cost-effective way to determine which molecule is a reliable indicator for specific DNA. Measurements were performed to understand the role of single, double and quadruple stranded DNA on molecule-DNA interactions. The research was mainly focused on obtaining better markers to study molecule-G-quadruplex DNA interactions that were implied in apoptosis. Hoe33258 was unable to differentiate between duplex and quadruplex DNA. Hoe33258 and AcrO were able to differentiate between single and quadruplex DNA. ThT was able to successfully indicate each strand, single, duplex, and quadruplex DNA.