Stability and structural features of DNA intercalation with ethidium bromide, acridine orange and methylene blue

Ethidium bromide (EB), acridine orange (AO), methylene blue (MB) and other fluorescent compounds are often used to probe DNA structure in drug–DNA and protein–DNA interactions. They bind nucleic acids via intercalative mode and cause major changes to DNA and RNA structures. The aim of this study was to examine the stability and structural features of calf-thymus DNA complexes with EB, AO and MB in aqueous solution, using constant DNA concentration (12.5 mM) and various pigment/DNA(P) ratios of 1/40, 1/20, 1/10, 1/4 and 1/2. FTIR, UV–visible spectroscopy and isothermal titration calorimetry (ITC) are used to determine the ligand intercalation and external binding modes, the binding constant and the stability of pigment–DNA complexes in aqueous solution. Structural analysis showed major intercalation of EB, AO and MB into polynucleotides G–C and A–T base pairs with minor external binding and overall binding constants of KEB = 6.58 × 104 M−1, KAO = 2.69 × 104 M−1 and KMB = 2.13 ×104 M−1. The overall binding constants estimated by UV–visible spectroscopy are consistent with thermodynamic data obtained by ITC, which showed ΔH(EB) = −13.58 kJ/mol, ΔH(AO) = −14.63 kJ/mol and ΔH(MB) = −13.87 kJ/mol with dissociation constants of KEB = 15 μM, KAO = 36 μM and KMB = 46 μM.