We also examined the influence of the temperature on DNA strand displacement rate constants. Strand displacement of above sequence sets was performed at 25°C. We found that kinetic traces of strand displacement with small rate constants were easily influenced by leaky reactions, leading to large deviations between fitted rate constants and true values. Thus, 120 sequence sets with the mean yield greater than 0.5 in 9000 seconds were selected, and these were randomly assigned to one of three different temperatures (13°C, 37°C, and 49°C) for further investigation. Fluorescence curves and kinetic parameters of 349 DNA strand displacement are elaborated in Supplementary Note 4. The best-fit values of rate constants of 240 strand displacement at different temperatures range from 2.5 orders of magnitude, with mean yields ranging from 0.26 to 0.92 (Figure ). The results show that when the temperature was lowered to 13°C, the yield of strand displacement decreased markedly, but the differences beyond 25°C were not obvious. Further analyzing rate constants of the same sequence set at different temperatures, the results show that the medians at 13, 37 and 49°C are 4.06, 5.13 and 5.18 respectively, while the medians of the same sequence set at 25°C are 4.81, 4.86 and 4.69 respectively (Figure ). The changes of rate constants under different temperatures relative to 25°C are 15.6%, 5.6% and 10.4% respectively. These findings suggests that elevated temperatures generally result in increased rate constants, and the influence of lower temperatures on rate constants is more pronounced.