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Proc Natl Acad Sci U S A. 1999 May 25;96(11):6171-6.

Thermodynamic basis of the enhanced specificity of structured DNA probes.

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  • 1Center for Studies in Physics and Biology, The Rockefeller University, New York, NY 10021, USA.


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.

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