Interaction of a novel red-region fluorescent probe, Nile Blue, with DNA and its application to nucleic acids assay

A novel fluorimetric method was developed for the rapid determination of DN A and RNA based on their quenching effect on the cationic red-region fluore scent dye Nile Blue (NB) In the investigation of the interaction of NE with DNA by steady-state polarization measurements, thermal denaturing study, d etermination of absorption and fluorescence characteristics, salt effect st udy and electrophoresis experiments, the results supported the suggestion t hat NE served as an intercalator to the stack base pairs of nucleic acids. Further evidence showed that the quenching could be ascribed to the static quenching mode. A binding constant of about 10(6) M-1 and a binding site si ze of about three base pairs were obtained by spectral methods. Under optim um conditions, the calibration curves for the determination of calf thymus DNA (CT DNA) and yeast RNA were linear over the ranges 3.0 ng mL(-1)-2.0 mu g mL(-1) and 27 ng mL(-1)-10 mu g mL(-1), respectively, The detection limi ts were 3.0 ng mL(-1) for CT DNA and 27 ng mL(-1) for RNA. The relative sta ndard deviation (n = 6) was within 2.1% in the middle of the linear range. Interferences from some interesting co-existing substances in the determina tion of DNA were also examined.