Anal. Biochem. 2008, in press
Miquel del Toro, R. Gargallo, R. Eritja, J. Jaumot
The G-quadruplex DNA structure has been suggested to be a potential target for anticancer therapies. Therefore, there is an increasing interest in the development of drugs which could modulate the stability of G-quadruplex structures. In the present work, the interaction between the Thrombin Binding Aptamer (TBA, 5'-GGT TGG TGT GGT TGG-3'), which can form an intramolecular G-quadruplex structure, and the porphyrin 5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)-21,23H-porphyrin tetratosylate (TmPyP4) has been studied. The application of an HPLC-PDA-based method to study this kind of interactions has been tested. Molecular absorption data recorded along the chromatographic runs have been analyzed by means of multivariate data analysis methods. Moreover, Biospecific Interaction Analysis (BIA) by Surface Plasmon Resonance (SPR) and melting and mole-ratio experiments monitored by UV-VIS molecular absorption and circular dichroism spectroscopies have been applied in order to confirm and expand the chromatographic studies. The results have shown the formation of an interaction complex with a stoichiometry 1:1 (TmPyP4:TBA) and logarithm of the equilibrium constant equal to 5.7 ± 0.2. Melting and circular dichroism data reflect that the initial G-quadruplex structure of TBA is stabilized in the interaction complex, being slightly distorted by the presence of the ligand.
Keywords
G-Quadruplex, porphyrins, DNA-drug interaction, chromatography, chemometrics
a) HPLC-PDA data obtained for a sample with CTMPyP4:CTBA ratio = 2. b) Calculated concentration profiles from the MCR-ALS analysis of the whole set of HPLC-PDA data recorded for the free TBA, free TmPyP4 and for the mixtures. Dotted points, MCR-ALS resolved points; solid-line, interpolated concentration profiles with the Curve Fitting Toolbox of the MATLAB software. C) Chromatograms at 422 nm for each one of the proposed species. Blue: TBA; green: TmPyP4; red: interaction complex.