Orthogonal enzyme-driven timers for DNA strand displacement reactions

Here we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand-displacement reactions. The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables the strand displacement reaction. The kinetics of the blocker enzymatic degradation thus controls the time at which the strand displacement reaction starts. By varying the concentration of the blocker strand and the concentration of the enzyme we show that we can finely tune and modulate the delayed onset of the strand displacement reaction. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed strand displacement reactions using RNase H and the two DNA repair enzymes Fpg and UDG and corresponding blockers. The achieved temporal delay can be programmed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show that the delayed strand displacement reactions can be coupled to downstream processes and used to control the onset of ligand release from a DNA nanodevice as well as protein inhibition by a DNA aptamer.