Hybridization kinetics and thermodynamics of molecular beacons.
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.
Tsourkas
Andrew
A
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Behlke
Mark A
MA
Rose
Scott D
SD
Bao
Gang
G
D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D013696:Temperature; D013816:Thermodynamics
D015342:DNA Probes