Interactions of the DNA intercalator acridine orange, with itself, with caffeine, and with double stranded DNA Original Research Article

Caffeine (CAF) inhibits the intercalation of acridine orange (AO) into cellular DNA. Optical absorption and fluorescence spectroscopy were employed to determine the molecular interactions of AO with itself, with CAF, and with double stranded herring sperm DNA (dsDNA). AO dimerization was observed at concentrations >2 μmol. The sharp increase in fluorescence (λem=530 nm) at 5 μmol of AO was attributed to AO multimer formation. From 0.5 to 5.0 μmol, the AO self-association binding constant (Kassoc) was determined to be 38 620 mol−1, however, the presence of 150 mmol NaCl increased Kassoc to 118 000 mol−1 attributed to the charge neutralization. The Kassoc for AO with CAF was confirmed to be 256 mol−1. Kassoc for the binding of AO with 20 μmol DNA ranged from, 32 000 mol−1 at 2 μmol AO, to approximately 3700 mol−1 at 10 μmol AO, in the absence of NaCl. This AO concentration dependency of Kassoc value with DNA was attributed to AO intercalation into dsDNA at high dsDNA/AO ratios, and electrostatic binding of AO to dsDNA at low AO ratios. The findings provide information used to explain fluorescence intensity values at λem at 530 nm from studies that combine AO, caffeine, and dsDNA.