Fluorescence Correlation Spectroscopy Studies of DNA Binding to Catanionic Surfactant Vesicles

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Lioi, Sara Bethany
English, Douglas
DeShong, Philip
Catanionic vesicles, made from mixtures of oppositely charged surfactants, have potential in drug delivery and gene therapy applications. Fluorescence correlation spectroscopy (FCS) was utilized to study the electrostatic binding of DNA molecules to vesicles made from cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS). FCS is employed to make sensitive measurements of bilayer adsorption and compare the adsorption of two single-stranded, dye-labeled DNA molecules of different lengths. Previous experimentation had shown that small organic fluorescent dyes bind to oppositely charged vesicles, thus positively charged CTAT-rich vesicles were used in the study of DNA binding. FCS was performed on samples with a constant DNA concentration and varying surfactant concentrations in order to construct binding isotherms for a 5mer ssDNA molecule and a 40mer ssDNA molecule. The binding constant determined for 40mer ssDNA (~ 10<super>6</super>) was larger than the constant for 5mer ssDNA (~ 10<super>5</super>), and binding constants for both lengths of DNA were larger than those previously determined for small organic molecule fluorescent dyes, which were on the order of 10<super>4</super>. Additionally, 40mer ssDNA was found to probe the critical aggregation concentration, which is the lower limit at which vesicles can form in a surfactant mixture. Ordinarily it would be expected that very few vesicles would form at this surfactant concentration, however the autocorrelation curves indicate that the 40mer is bound only to vesicles. Salt studies were also done with the 40mer ssDNA to determine how the binding of cargo molecules to the exterior of the vesicle would be affected by physiological salt concentrations. Binding affinity of the 40mer ssDNA was reduced with increasing salt concentration; this was thought to be due to the tosylate ion, as it is hydrophobic and intercalates into the vesicle bilayer. Subsequent experiments using cetyltrimethylammonium bromide (CTAB) indicated that the counterion is not a factor in loss of binding affinity under normal saline conditions. Because these surfactant vesicles have been shown to have potential in both drug delivery and gene therapy, it is important that the binding of the cargo molecule be able to withstand normal saline conditions.