doi: 10.1093/nar/gkg212.
Hybridization kinetics and thermodynamics of molecular beacons
Affiliations
- PMID: 12582252
- PMCID: PMC150230
- DOI: 10.1093/nar/gkg212
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Hybridization kinetics and thermodynamics of molecular beacons
Nucleic Acids Res.
.
Abstract
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.
Figures
(A) The design and structural parameters of molecular beacons considered in the study. (B) Molecular beacons in solution can have three phases: bound to target, closed and random coil.
Determination of the changes in enthalpy (slope of the fitted line) and entropy (y-intercept) describing the transition between molecular beacons bound to wild-type target and free in the stem–loop conformation for molecular beacons with stem lengths of five bases and probe lengths of 17, 18 and 19 bases.
Determination of the changes in enthalpy (slope of the fitted line) and entropy (y-intercept) describing the transition between molecular beacons bound to wild-type target and free in the stem–loop conformation for molecular beacons with probe lengths of 17 bases and stem lengths of 4, 5 and 6 bases.
Determination of the changes in enthalpy (slope of the fitted line) and entropy (y-intercept) describing the transition between molecular beacons bound to target and free in the stem–loop conformation for molecular beacons with a stem length of five bases and a probe length 18 bases in the presence of wild-type (WT) and mutant targets (target A – D).
Melting temperatures for molecular beacons with various structures in the presence of wild-type target. The melting temperature was determined for samples containing 200 nM of molecular beacons and 400 nM of targets.
(A) Melting curves for molecular beacons hybridizing to wild-type target (solid line) and target B (dashed line) and (B) the difference in the fraction of molecular beacons bound to wild-type target and that to mutant target B. The molecular beacons have a probe length of 17 bases and stem lengths of 4, 5 and 6 bases.
(A) Melting curves for molecular beacons hybridizing to wild-type target (solid line) and target B (dashed line) and (B) the difference in the fraction of molecular beacons bound to wild-type target and that to mutant target B. The molecular beacons have a probe length of 17 bases and stem lengths of 4, 5 and 6 bases.
The difference in the fraction of molecular beacons bound to wild-type target and that to mutant target B for molecular beacons with a stem length of four bases and probe lengths of 17, 18 and 19 bases.
A comparison between the difference in the fraction of molecular beacons and that of linear probes bound to wild-type target and mutant target B.
Hybridization kinetics of molecular beacons in the presence of wild-type targets. (A) The normalized restoration in fluorescence that accompanies target hybridization for molecular beacons with probe lengths of 17 bases and stem lengths of 4, 5 and 6 bases. (B) The rate of hybridization k1 (on-rate) for molecular beacons with various probe and stem lengths hybridized to their complementary targets.
Hybridization kinetics of molecular beacons in the presence of wild-type targets. (A) The normalized restoration in fluorescence that accompanies target hybridization for molecular beacons with probe lengths of 17 bases and stem lengths of 4, 5 and 6 bases. (B) The rate of hybridization k1 (on-rate) for molecular beacons with various probe and stem lengths hybridized to their complementary targets.
Normalized background fluorescence as a function of temperature for molecular beacons with a probe length of 17 bases and stem lengths of 4, 5 and 6 bases. Similar results were obtained for molecular beacons with probe lengths of 18 and 19 bases (data not shown).
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