Pyrene-labeled DNA probes for homogeneous detection of complementary DNA sequences: poly(C) model system

DNA and RNA probes are important analytical reagents in molecular biology and in the detection of infectious and genetic diseases. However, the present polynucleotide probe technology is complex and labor-intensive. We have been investigating the possibility of using fluorescent-labeled DNA probes to develop assays which do not require the separation of free from hybridized probe (homogeneous assays). Such assays are possible if the fluorescence efficiency or fluorescence anisotropy of the fluorescent label changes upon hybridization of probe with target DNA. In this article we examine pyrene as a fluorescent label for DNA or RNA probes. Experiments were performed using a model system in which poly(C) and poly(I) are respectively the probe and target sequences. A small fraction of the nucleotide bases of poly(C) was randomly labeled with pyrene using the bisulfite-catalyzed diamine reaction. The results show that the uncorrected emission spectrum of pyrene-poly(C) decreases by a factor of 4 and shifts toward longer wavelengths upon hybridization with poly(I) at saturating concentrations. The average lifetime changes from 10.78 to 4 ns. These fluorescence changes occur in a wide range of chemical environments, including the high salt concentrations normally used to increase the velocity of the hybridization reaction in clinical assays. The pyrene label can thus be used to readily detect the amount of poly(I) in an unknown sample without having to separate free and bound labeled probe. To unravel the mechanism responsible for the observed changes in fluorescence intensity upon hybridization, we have performed polarized fluorescence intensity measurements and analyzed the results by approximate steady-state expressions that allow evaluation of the relative contributions of changes in lifetimes (fluorescence efficiency) and rotational motions to the changes in fluorescence intensity. The results indicate that the latter changes are due chiefly to changes in lifetime or fluorescence efficiency and that these changes seem to be due to the movement of the pyrene label to a more hydrophilic environment upon hybridization.