Fig. 3: ATP-fueled transient DSD cascades using the dual invasion strategy.

a Schematic representation of ATP-fueled transient DSD cascades. b Time-dependent FI at different amount of Input 2. Substrate 3 has a ratio of fluorophore strand to quencher strand of 1:2. Shaded areas correspond to the standard deviation of duplicate measurements. c Time-dependent FI of the system via Substrate 3 with a ratio of fluorophore strand to quencher strand of 1:1. Shaded area corresponds to the standard deviation of duplicate measurements. The lower increase and the slower kinetics are due to low efficiency of Substrate 3 formation at equimolar ratio. d Multiple activation of the system by repeatedly adding ATP. Shaded area corresponds to the standard deviation of duplicate measurements. e, f Programmable lifetimes for the systems running at 37 °C seen by time-dependent FIs at different ATP concentrations. Shaded areas and error bars correspond to the standard deviation of duplicate measurements. Conditions: b 25 °C, 20 μM Complex 1, 5 μM Substrate 1, 1 μM Substrate 3 (fluorophore strand to quencher strand ratio, 1:2), 10 μM Input 1, 5 or 10 μM Input 2, 0.8 WU μL⁻¹ T4 DNA ligase, 1.5 U μL⁻¹ BsaI, and 40 μM ATP. c The same as b except that Substrate 3 with one equivalent amount of quencher strand and 10 μM Input 2 were used. d The same as b but using 10 μM Input 2, and the system was repeatedly fueled with 20 μM ATP for four transient lifecycles. e 37 °C, 20 μM Complex 1, 5 μM Substrate 4, 1 μM Substrate 5 (fluorophore strand to quencher strand ratio, 1:2), 10 μM Input 3, 10 μM Input 4, 0.8 WU μL⁻¹ T4 DNA ligase, 1.5 U μL⁻¹ BsaI, and varied concentration of ATP.