Directing Uphill Strand Displacement with an Engineered Superhelicase

The ability to finely tune reaction rates and binding energies between components has made DNA strand displacement circuits promising candidates to replicate the complex regulatory functions of biological reaction networks. However, these circuits often lack crucial properties, such as signal turnover and the ability to transiently respond to successive input signals that require the continuous input of chemical energy. Here, we introduce a method for providing such energy to strand displacement networks in a controlled fashion: an engineered DNA helicase, Rep-X, that transiently dehybridizes specific DNA complexes, enabling the strands in the complex to participate in downstream hybridization or strand displacement reactions. We demonstrate how this process can direct the formation of specific metastable structures by design and that this dehybridization process can be controlled by DNA strand displacement reactions that effectively protect and deprotect a double-stranded complex from unwinding by Rep-X. These findings can guide the design of active DNA strand displacement regulatory networks, in which sustained dynamical behavior is fueled by helicase-regulated unwinding.


Figure S1
. Amount of output released from initial complex varied over different thawing times of Rep-X.We thawed Rep-X at room temperature directly from the -20°C freezer for 5, 10, 30, 60, 90, and 120 minutes.Each sample contained 100 nM of R1:output_1, 100 nM Rep-X, and 1 mM ATP.We observed the highest Rep-X unwinding rate or efficiency at 90 minutes with an unwound fraction of ~75%.From these results, we decided to follow a 60 to 90 minute room temperature thawing Rep-X protocol for each of the three DNA systems.

Figure S3
. Evaluating the effectiveness of the reporting scheme as measure of "lock-key" reaction dynamics.With 400 nM of free, single-stranded output_2 and 100 nM of reporter, ~60% of available reporter was unwound by the hybridization of output_2 to R2. Red shading represents standard deviation of the duplicate samples, however, there is not enough deviation between the duplicates to be easily visible.

Figure S4
. a) Concentrations of reporter complex formed using increasing concentrations of key on "locked" foundation:output_2n. 100 nM Rep-X, 100 nM of reporter, and 1 mM ATP were present in each experiment.150 nM of lock was annealed with 100 nM foundation:output_2n for each sample.The samples included 0, 15, 75, 150, 300, 750, and 1500 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.b) Average concentrations of R2:output_2 complexes formed using different concentrations of key after the experiments in (a).c) Complementarity of key:output_2 complex.There are 10 complementary bases (pink) between output_2 and key.The formation of key:output_2 complexes prevents R2:output_2 complexes from forming, leading to a decrease in fluorescence since less output_2 reacts with R2. d) NUPACK generated structure of key:output_2.Simulated in a solution of 300 nM key and 400 nM output_2, key:output_2 complexes form ~60% of the ensemble.The free energies were calculated using a reference concentration of 55.6M (water in water) since we use an aqueous solution at low concentrations of solute.

Table S1 .
DNA strand sequences.Purified strands have attached fluorophores or quenchers. 2mplementary strands are denoted by an apostrophe for reporter complexes or by matching colors.3Allstrands are listed from 5' to 3' direction.

Table S2 .
Experimental details of SI Figure 1.

Table S6 .
Experimental details of SI Figure 2.