Unambiguous detection of target DNAs by excimer-monomer switching molecular beacons.

A new class of molecular beacons were developed in which pyrene fluorophores were connected both at 3' and 5' ends of a single-stranded oligonucleotide. The two pyrene-based fluorophores were synthesized from the same starting material, so that the preparation of the beacons was simplified. The detection strategy of the beacons for target DNAs is based on "excimer-monomer emission switching" of the pyrene fluorophores: excimer emission of the pyrene moieties changed to monomer one when the beacons hybridized with the targets. This type of two-state mode of fluorescence allows unambiguous detection of the target DNAs because strict 1:1 correlation between the nonhybridized and the hybridized beacons can be monitored by the presence of isoemissive points of the fluorescence changes. The beacons can detect target 19-mer DNAs and can discriminate the targets from their single-nucleotide mismatches at 1 nM concentration. Advantages of the excimer-monomer switching molecular beacons were discussed in comparison with conventional ones.