Thermodynamic basis of the enhanced specificity of structured DNA probes

Molecular beacons are DNA probes that form a stem-and-loop structure and po ssess an internally quenched fluorophore. When they bind to complementary n ucleic acids, they undergo a conformational transition that switches on the ir fluorescence. These probes recognize their targets with higher specifici ty than probes that cannot farm a hairpin stem, and they easily discriminat e targets that differ from one another by only a single nucleotide. Our res ults show that molecular beacons can exist in three different states: bound to a target, free in the form of a hairpin structure, and free in the form of a random coil. Thermodynamic analysis of the transitions between these states reveals that enhanced specificity is a general feature of conformati onally constrained probes.