Thermodynamic basis of the enhanced specificity of structured DNA probes

TLDR

Molecular beacons are stem‑loop DNA probes with an internally quenched fluorophore that fluoresce upon binding complementary nucleic acids, enabling highly specific detection even for single‑nucleotide differences. Our thermodynamic analysis shows that molecular beacons exist in bound, hairpin, and random‑coil states, and that conformational constraint universally enhances probe specificity.

Abstract

Molecular beacons are DNA probes that form a stem-and-loop structure and possess an internally quenched fluorophore. When they bind to complementary nucleic acids, they undergo a conformational transition that switches on their fluorescence. These probes recognize their targets with higher specificity than probes that cannot form a hairpin stem, and they easily discriminate targets that differ from one another by only a single nucleotide. Our results 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 conformationally constrained probes.