SEQUENCE-SPECIFIC INTERACTIONS OF METHYLENE-BLUE WITH POLYNUCLEOTIDES AND 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 intercalative and the binding mode is insensitive to changes in ionic strength. The observed CD spectrum is bisignate, which may be due to intercalation 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 intercalative binding mode with the alternating AT polynucleotide [poly(dA-dT)](2) is likely only at very low ionic strength; at high ionic strength (200 mM phosphate, pH 6.9), a second binding mode is also manifest 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 the methylene blue/CT-DNA complex and its variation with ionic strength reflect the complexity of even this simple system where numerous possible binding sites exist. Comparative binding to the nonalternating polynucleotides poly(dA).poly(dT) and poly(da).poly(dC), which each possess only one base-pair step, was also examined. On the basis of the combined LD and CD evidence, it is proposed that the dye is loosely bound with poly(dA).poly(dT), probably in the major groove, and intercalated with poly(dG).poly(dC).