DNA-DYE FLUORESCENCE ENHANCEMENT BASED ON SHIFTING THE DIMER-MONOMER EQUILIBRIUM OF FLUORESCENT DYE

In this paper, the investigation of DNA-dye fluorescence enhancement b ased on shifting the dimer-monomer equilibrium of a fluorescent dye, a cridine orange (AO), is reported. Formation of a virtually nonfluoresc ent 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 studi ed. The results showed that a nearly 1000-fold fluorescence enhancemen t was observed upon addition of calf thymus DNA (CT DNA). The fluoresc ence enhancement effect of DNA was thought to be based on the DNA modu lated shift of the dimer-monomer equilibrium of AO in the anionic surf actant solution. Intercalation of the monomer in DNA caused the dissoc iation of AOAO and led to a very high fluorescence enhancement. It see med that the dimeric dye molecules acted as a source of monomer molecu les ready for interacting with nucleic acids and, at the same time, de creased the inherent fluorescence of monomer molecules, which proved t o be unfavorable to the detection of fluorescence enhancement. A linea r dependence of fluorescence intensity on CT DNA concentration over a range from 7.8 ng/mL to 10.0 mu g/mL, in the presence of AO at a conce ntration of 1.65 x 10(-6) mol/L and of SDS at a concentration of 8.0 x 10(-4) mol/L, allowed sensitive quantitation of CT DNA in a conventio nal fluorometer. Calibration graphs for yeast RNA and polynucleotides, such as poly A, poly U, and poly I, were also obtained.