Figure 4.

Locking and unlocking a target complex to prohibit or allow Rep-X unwinding. (a) Schematic of the “lock–key” multistrand system. When the lock strand is annealed to foundation:n*output_2, Rep-X is unable to bind to foundation:n*output_2 (where n = 1,2,3, or 4 output_2 strands) since the 13-nucleotide 3′ overhang is no longer available. After adding the key, the lock and key strand hybridize, exposing the 3′ overhang of the foundation strand within foundation:n*output_2. Rep-X can bind to and unwind foundation:n*output_2, allowing free output_2 to react with the reporter complex. (b) Reporting scheme for measuring reaction dynamics. Free output_2 from the reaction in (a) can react with the reporter to form R2:output_2, which causes an increase in Cy3 fluorescence. (c) Predicted final R2:output_2 concentration based on the amount of lock annealed to foundation:n*output_2. (d) Schematic of the predicted R2:output_2 concentration based on the amount of key added to a solution containing lock and lock/foundation:n*output_2. (e) Measured concentrations of reacted reporter complex after different concentrations of lock are annealed with foundation:n*output_2. Each sample contained 100 nM Rep-X, 100 nM reporter, 1 mM ATP, and 100 nM foundation:n*output_2; 0, 20, 50, 100, 150, 200, and 500 nM locks were annealed with foundation:n*output_2 in the samples, as labeled. Higher concentrations of lock lead to less output_2 release. The amount of output_2 released plateaus around the 150 nM lock. (f) Average concentrations of R2:output_2 complexes formed after the experiments in (e). In the absence of side reactions, the concentration of R2:output_2 should plateau at 100 nM. (g) Concentrations of reporter complex formed using increasing concentrations of key on “locked” foundation:n*output_2. 100 nM Rep-X, 100 nM reporter, and 1 mM ATP were present in each experiment. 150 nM lock was annealed with 100 nM foundation:n*output_2 for each sample. The samples included 0, 15, 75, 150, and 300 nM key, as labeled. As the concentration of key is increased to 75 or 150 nM, large concentrations of R2:output_2 complexes are produced. A decrease in R2:output_2 is observed once the amount of key exceeds a 1:1 ratio of lock/key. (h) Average concentrations of R2:output_2 complexes formed using different concentrations of key after the experiments in (g). In the absence of side reactions, the concentration of R2:output_2 should plateau at 150 nM. Further details on this nonmonotonic behavior are shown in Figure S4, which includes higher concentrations of key.