Hitoshi Fujita

Quantitative determination of single-stranded sections in DNA using the fluorescent probe acridine orange

Abstract A method for the quantitative determination of single-stranded sections in DNA was developed. Fractions of denatured sections can be estimated using a suitable calibration curve which is obtained on the basis that acridine orange bound to denatured DNA emits a weaker fluorescence at 530 nm and a stronger one at 640 nm than does the dye bound to native DNA. The most suitable conditions, where dye is bound to both types of DNA with the same efficiency, were determined to be an ionic strength of 0.01 and a DNA phosphate to bound dye ratio of 7.5. Under these conditions, the fluorescence intensity at 530 nm of the dye added to the mixture of native and heat-denatured DNA decreased linearly as the fraction of denatured DNA increased. In relation to making a calibration curve, the conformation of heat-denatured calf thymus DNA at an ionic strength of 0.01 was studied. When heated DNA was chilled, only a few percent of total nucleotides reformed the double helical structure in spite of the considerable decrease in hyperchromicity.

The nature of strong binding between acridine orange and deoxyribonucleic acid as revealed by equilibrium dialysis and thermal renaturation.

Abstract The strong binding of acridine orange with DNA was studied by means of equilibrium dialysis and thermal renaturation of the complex. Thermodynamic parameters for the strong binding with native and denatured DNA were calculated from the data of equilibrium dialysis. The association constant and the change of free energy were nearly equal for both types of DNA, but the changes of enthalpy and entropy were quite different. Binding to both types of DNA may be strong binding in nature, but the accompanying effects on the secondary structures of DNA would be different. It was found that the presence of acridine orange inhibits the completion of renaturation, presumably due to a stabilizing effect of the bound dye on the “wrong” base pairs as they are formed. The results support the notion that binding of acridine orange with the double helix is an intercalation into adjacent base pairs, while the binding with single strand is a partial intercalation into adjacent bases.