Orthogonal Enzyme-Driven Timers for DNA Strand Displacement Reactions

Abstract

Here, we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand displacement reactions (SDRs). 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 SDR. The kinetics of the blocker enzymatic degradation thus controls the time at which the SDR 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 SDR. 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 SDRs using RNase H and two DNA repair enzymes (formamidopyrimidine DNA glycosylase and uracil-DNA glycosylase) and corresponding blockers. The achieved temporal delay can be programed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show three possible applications of the delayed SDRs to temporally control the ligand release from a DNA nanodevice, the inhibition of a target protein by a DNA aptamer, and the output signal generated by a DNA logic circuit.

Toehold-Mediated Strand Displacement in Random Sequence Pools

“…With our prediction model, a rough estimate of displacement kinetics can be made. With a more precise kinetic predictor, it would be possible to control and alter strand displacement kinetics, for example, the release of an output strand could be precisely controlled by the addition of rationally designed interfering strands, similar as previously demonstrated in the context of timer circuits. − …”

Dynamic and Reversible Decoration of DNA‐Based Scaffolds

“…[43] We envision that this approach can be employed to induce functional changes in more complex DNA structures (including origami) providing diverse clinical and sensing applications. [30,41,44] Coupling this approach with other DNA-based temporally controlled systems [45,46] could also allow for dynamic control of the decoration of structures over time and space.…”

DNA strand displacement based computational systems and their applications

“…Enzymes in DSD computational systems can be used as regulators to control their reactions. Bucci et al (2022) designed a blocker strand that can bind to the toehold domain, which prevented the DSD reactions. Then they took two steps to recover the activity of DSD.…”

“…DSD can be combined with other technologies and utilized in a wide range of applications. So far, DSD has been combined with CRISPR technology (Ishino et al, 1987;Montagud-Martínez et al, 2021), DNA origami (Rothemund, 2006;Zhang et al, 2022), enzymes (Bucci et al, 2022;Schaffter and Strychalski, 2022), proteins , etc. These combinations effectively expand the application scenarios for DSD.…”