SEQUENCE-SPECIFIC INTERACTIONS OF METHYLENE-BLUE WITH POLYNUCLEOTIDESAND DNA - A SPECTROSCOPIC STUDY

The modes of binding of the phenothiazinium dye methylene blue (1) to alternating and nonalternating polynucleotides and to calf thymus (CT) DNA have been characterized using linear dichroism (LD) and circular dichroism (CD) spectroscopy. With the polynucleotide [poly(dG-dC)](2) the interaction at low binding ratios is shown to be purely intercalat ive and the binding mode is insensitive to changes in ionic strength. The observed CD spectrum is bisignate, which may be due to intercalati on at the different base-pair steps (5'G-C3' and 5'C-G3'), giving rise to CD signals of different sign and shape, By contrast, a single inte rcalative binding mode with the alternating AT polynucleotide [poly(dA -dT)](2) is likely only at very low ionic strength; at high ionic stre ngth (200 mM phosphate, pH 6.9), a second binding mode is also manifes t which is attributed to groove binding of the dye. The absorption and linear dichroism spectroscopic features of the methylene blue/CT-DNA (42% GC) complex reflect those of complexes of the dye with both [poly (dA-dT)](2) and [poly(dG-dC)](2); the circular dichroism spectrum of t he methylene blue/CT-DNA complex and its variation with ionic strength reflect the complexity of even this simple system where numerous poss ible binding sites exist. Comparative binding to the nonalternating po lynucleotides poly(dA).poly(dT) and poly(da).poly(dC), which each poss ess only one base-pair step, was also examined. On the basis of the co mbined LD and CD evidence, it is proposed that the dye is loosely boun d with poly(dA).poly(dT), probably in the major groove, and intercalat ed with poly(dG).poly(dC).