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Molecular beacons are hairpin-shaped, single-stranded oligonucleotides constituting sensitive fluorescent DNA probes widely used to report the presence of specific nucleic acids. In its closed form the stem of the hairpin holds the fluorophore covalently attached to one end, close to the quencher, which is covalently attached to the other end. Here we report that in the closed form the fluorophore and the quencher form a ground state intramolecular heterodimer whose spectral properties can be described by exciton theory. Formation of the heterodimers was found to be poorly sensitive to the stem sequence, the respective positions of the dyes and the nature of the nucleic acid (DNA or RNA). The heterodimer allows strong coupling between the transition dipoles of the two chromophores, leading to dramatic changes in the absorption spectrum that are not compatible with a Förster-type fluorescence resonance energy transfer (FRET) mechanism. The excitonic heterodimer and its associated absorption spectrum are extremely sensitive to the orientation of and distance between the dyes. Accordingly, the application of molecular beacons can be extended to monitoring short range modifications of the stem structure. Moreover, the excitonic interaction was also found to operate for doubly end-labeled duplexes.

Exciton interaction in molecular beacons: a sensitive sensor for short range modifications of the nucleic acid structure