Determination of thermodynamic parameters. (*A*) The increase in fluorescence that accompanies the melting of the molecular beacon’s hairpin stem was used to determine the thermodynamic parameters that describe this transition. The slope of the fitted line is equal to the negative value of the enthalpy (−Δ*H*°_{2→3}) and the intercept is equal to the entropy (Δ*S*°_{2→3}). (*B*) The increase in the melting temperature of the probe–target duplex that results from increasing the concentration of target oligonucleotides was used to determine the thermodynamic parameters that describe the dissociation of probe–target duplexes. Separate determinations were carried out with perfectly complementary duplexes (●) and with mismatched duplexes (○). The slope of each fitted line is equal to the negative value of the enthalpy (−Δ*H*°_{1→2}) and the intercept is equal to the entropy (Δ*S*°_{1→2}). In this graph, the independent variable, *R* ln(T_{0} − 0.5B_{0}), is plotted on the ordinate and the dependent variable, 1/θ_{m}, is plotted on the abscissa, to illustrate the similarities in the manner in which the enthalpies and entropies were determined for the dissociation of probe–target duplexes and for the dissociation of hairpin stems.

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