DNA—Dye Fluorescence Enhancement Based on Shifting the Dimer—Monomer Equilibrium of Fluorescent Dye

Abstract

In this paper, the investigation of DNA–dye fluorescence enhancement based on shifting the dimer–monomer equilibrium of a fluorescent dye, acridine orange (AO), is reported. Formation of a virtually nonfluorescent dimeric dye, acridine orange homodimer (AOAO), induced by the pre-micellar aggregation of an anionic surfactant, sodium dodecyl sulfate (SDS), was observed. The possibility of using the in situ formed AOAO as a fluorescent probe for nucleic acids and polynucleotides was studied. The results showed that a nearly 1000-fold fluorescence enhancement was observed upon addition of calf thymus DNA (CT DNA). The fluorescence enhancement effect of DNA was thought to be based on the DNA modulated shift of the dimmer monomer equilibrium of AO in the anionic surfactant solution. Intercalation of the monomer in DNA caused the dissociation of AOAO and led to a very high fluorescence enhancement. It seemed that the dimeric dye molecules acted as a source of monomer molecules ready for interacting with nucleic acids and, at the same time, decreased the inherent fluorescence of monomer molecules, which proved to be unfavorable to the detection of fluorescence enhancement. A linear dependence of fluorescence intensity on CT DNA concentration over a range from 7.8 ng/mL to 10.0 g/mL, in the presence of AO at a concentration of 1.65 × 10⁻⁶mol/L and of SDS at a concentration of 8.0 × 10⁻⁴ mol/L, allowed sensitive quantitation of CT DNA in a conventional fluorometer. Calibration graphs for yeast RNA and polynucleotides, such as poly A, poly U, and poly I, were also obtained.