Hybridization kinetics and thermodynamics of molecular beacons

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quanti®cation. The stem±loop structure of molecular beacons provides a competing reaction for probe±target hybridization that serves to increase probe speci®city, which is particularly use-ful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular bea-con structure±function relationship. Both probe and stem lengths are shown to have a signi®cant impact on the binding speci®city and hybridization kinetic rates of molecular beacons. Speci®cally, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon±target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate con-stants, and decreased speci®city. Molecular bea-cons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have signi®cant impli-cations for the design of molecular beacons for various applications