A time-resolved fluorescence study of 4′,6′-diamidine-2-phenylindole dihydrochloride binding to polynucleotides

Abstract

At phosphate/dye (P/D) ratios greater than 30 the quantum yield of 4',6'-diamidine-2-phenylindole dihydrochloride (DAPI)-DNA and DAPI-poly(d(A-T)) complexes was found to be 0.62 and 0.66, respectively. Contrary to earlier reports a fluorescence enhancement of DAPI-poly(d(G-C)) complexes was observed with a quantum yield of 0.22. Time-resolved fluorescence measurements of complexes with a P/D ratio of 150:1 indicate that there were three fluorescent components in DAPI-DNA complexes with lifetimes of 3.86, 1.79 and 0.13 ns. In DAPI-poly(d(A-T)) complexes the lifetimes were 3.91, 1.20 and 0.11 ns. Also, three components with lifetimes of 3.98, 0.87 and 0.12 ns were found in DAPI-poly(d(G-C)) complexes. At low P/D ratios (< 5) another binding form of DAPI was observed which was assigned to the interaction of one or more molecules of DAPI with one previously bound to DNA. It is concluded that DAPI does not exhibit A-T binding specificity and that at high P/D ratios there are two types of binding having similar binding constants.