Abstract
Complexes of the TAT peptide with plasmid DNA (pDNA) show unusually high transfection efficiency when condensed via "soft" calcium cross links. In this study, we characterize the structure of pDNA within TAT complexes and compare it with that of branched polyethylenimine (PEI, 25 kDa) complexes. Dynamic light scattering (DLS), second derivative ultraviolet absorption spectroscopy, extrinsic dye fluorescence, Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) were employed to access various aspects of the structure of the pDNA. TAT complexes showed the highest transfection efficiency at an N/P ratio of 25 in A549 cells. FTIR and CD spectra of complexes demonstrated that the pDNA remained in the B conformation when associated with TAT or PEI. DSC showed that both TAT and PEI stabilized all forms of pDNA, with TAT increasing the melting temperature of pDNA compared to PEI complexes. Second derivative ultraviolet spectroscopy of TAT complexes showed a substantial reduction in the absorbance and an increase in the wavelengths of the peaks of pDNA at N/P>13; however, a clear correlation between pDNA structure and transfection efficiency was not readily apparent.
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