Structure of methylene blue–DNA complexes studied by linear and circular dichroism spectroscopy

Abstract Bradley et al. [(1972) Biopolymers 11 , 645–652] used electro‐optical measurements to show that methylene blue (MB), like acridine orange, in its DNA complex is oriented more or less perpendicular to the helix axis as expected if intercalated. High‐precision flow linear dichroism (LD) here confirms that MB is coplanar with the DNA bases at low dye/DNA binding ratios and low ionic strengths. CD shows two origins of induced optical activity for the transition of lowest energy (polarized parallel to the long‐axis of the dye molecule): at low binding ratios ( r < 0.05), a weak monomeric CD with the same shape as the absorption curve is observed, while at higher binding ratios, a strong exciton CD dominates due to interaction between pairs of MB ligands. The monomeric CD spectrum shows a remarkable dependence on ionic strength: it is negative in the absence of extra salt, but changes sign (at ca 20 m M NaCl or 0.15 m M MgCl 2 ) and becomes essentially the positive mirror‐image spectrum at high ionic strengths (>300 m M Na + or >0.4 m M Mg 2+ ). Nondegenerate coupled‐oscillator theory can explain the CD in terms of interactions of transition moments of the dye and the nearest nucleotide bases and indicates a change between two intercalation geometries: a Lerman type of mode, denoted γ − , and an orthogonal mode, denoted γ + . This rotation of MB in the base‐pair pocket is accomplished at Na + and Mg 2+ concentrations when the phosphates are effectively screened and the result suggests a more localized bonding of Mg 2+ than is expected from simple polyelectrolyte models. The exciton effect at high binding ratios, observed both in CD and in LD, can be interpreted in terms of an interaction between an intercalated and a nonintercalated MB. The geometry of this “accidental” dimer is consistent with a location of the nonintercalated MB in the minor groove, bridging the strands by the positively charged amino groups directed towards phosphate groups. The dihedral angle of the MB pairs, corresponding to a left‐handed helix, is opposite to that with acridine orange and proflavine on DNA, indicating that the latter ligands bind to DNA in a different way.