Fluorescence versus time curves were performed in 1.4 mL quartz cuvettes by a Horiba Fluorologmax spectrofluorometer with a 4-sample changer. Kinetic fluorescence data were collected every 60 s with an external temperature water bath to control the temperature. Prior to experiments, all cuvettes were successively washed with deionized water 5 times, 70% ethanol 3 times, and deionized water 5 times. Substrate complexes were added to three cuvettes for each strand displacement to reach the final concentration of 10 nM. After the fluorescence stabilized, the cuvettes were removed from the instrument, and the one with the lowest fluorescence was placed in position 1, the one with the middle fluorescence was placed in position 2, and the one with the highest fluorescence was placed in position 3. After recording the fluorescence data 5 times, 40, 30, and 20 nM invading strands were added to each cuvette successively, with a mixing time of approximately 60 s. Kinetics fluorescence experiments for each strand displacement were measured between 4000 and 9000 seconds.