Hybridization kinetics and thermodynamics of molecular beacons

Nucleic Acids Res. 2003 Feb 15;31(4):1319-30. doi: 10.1093/nar/gkg212.


Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem-loop structure of molecular beacons provides a competing reaction for probe-target hybridization that serves to increase probe specificity, which is particularly useful 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 beacon structure-function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, 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 constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA Probes / chemistry*
  • DNA Probes / genetics
  • Kinetics
  • Nucleic Acid Conformation
  • Nucleic Acid Denaturation
  • Nucleic Acid Hybridization*
  • Temperature
  • Thermodynamics*


  • DNA Probes