Gene therapy consists of the delivery of foreign DNA to cells. Cell penetrating compounds, such as Tat peptide, have been shown to facilitate DNA delivery through the formation of a Tat/DNA complex. Different complex +/- ratios were tested using an electrophoretic mobility shift assay with the goal of finding the stoichiometric ratio at which Tat fully binds plasmid DNA. These results were confirmed and interaction thermodynamics were determined by isothermal titration calorimetry (ITC). Complex hydrodynamic diameter was determined in solvents of increasing ionic strength using dynamic light scattering. Positively charged Tat was found to interact electrostatically with negatively charged cell surface glycosaminoglycans (GAGs). A competitive displacement assay was conducted to find the concentration of GAG needed to completely displace DNA from the Tat/DNA complex. Results support Tat-GAG affinities measured by ITC and lend insight into potential cell uptake mechanisms of these complexes.

Tat peptide-mediated gene delivery: Complex formation and interaction with cell-surface glycosaminoglycans

245th National Meeting (2013)

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