Figure 2. Temperature-dependent DNA hybridization and corresponding data analysis.

(a) Time traces of 300-s length are recorded for complementary DNA target at a concentration of 100 nM. Overlaid raw real-time data (black) and idealized fits (red) for a temperature series from 30 to 60 °C. The rate of bi-stable activity increases with temperature. (b) A representative survival probability curve for the 50 °C trace from a, with an event count of 763. The fit quality is characterized by R²=0.999 and R²=0.998 for τ_low→high and τ_high→low, respectively. (c) Comparison of nearest-neighbour calculated and single-molecule measured melting curves for a 100 nM complementary target showing the same temperature dependence as a ‘traditional' melting curve exhibiting a lower hybridized fraction with increasing temperature and a T_m of 52.99 °C compared with a calculated T_m=49.66 °C. (d–f) Arrhenius plots for 100, 10 and 1 nM target concentrations, showing the temperature dependence of the melting and the hybridization rates. Error bars are calculated from five different 60-s intervals at each temperature.