Figure 4.

Free energy of the three phases of a solution of molecular beacons in equilibrium with target oligonucleotides. Although this phase diagram was calculated for a solution containing a particular molecular beacon at 50 nM and its target at 300 nM, the relative positions of the phase lines are generally the same for all molecular beacons under all conditions. The equation of each line is ΔG = ΔH − θ ΔS, where ΔG₃ = 0; ΔG₂ = 0.104 θ − 34 kcal/mol; ΔG_1P = 0.371 θ − 118; and ΔG_1M = 0.320 θ − 99. The difference between the melting temperatures of perfectly matched duplexes (phase 1_P) and mismatched duplexes (phase 1_M) is greater if the probe can form a structure after dissociation (Δθ = 14°C) than it is if the probe cannot form a structure (Δθ′ = 8°C).