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 andMB 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 showedmajor intercalation of EB,AO andMB into polynucleotidesG–C andA–T base pairs withminor external binding and overall binding constants ofKEB = 6.58 · 10 M ,KAO = 2.69 · 10 M 1 andKMB = 2.13 ·10 M . The overall binding constants estimated byUV– visible spectroscopy are consistent with thermodynamic data obtained by ITC, which showed DH(EB) = 13.58 kJ/mol, DH(AO) = 14.63 kJ/mol andDH(MB) = 13.87 kJ/mol with dissociation constants ofKEB = 15 lM,KAO = 36 lMandKMB = 46 lM. 2006 Elsevier B.V. All rights reserved.