Nicking-Assisted Reactant Recycle to Implement Entropy-Driven DNA Circuit

Synthetic catalytic DNA circuits are important signal amplification tools for molecular programming due to their robust and modular properties. In catalytic circuits, the reactant recycling operation is essential to facilitate continuous processes. Therefore, it is desirable to develop new methods for the recycling of reactants and to improve the recyclability in entropy-driven DNA circuit reactions. Here, we describe the implementation of a nicking-assisted recycling strategy for reactants in entropy-driven DNA circuits, in which duplex DNA waste products are able to revert into active components that could participate in the next reaction cycle. Both a single-layered circuit and multiple two-layered circuits of different designs were constructed and analyzed. During the reaction, the single-layered catalytic circuit can consume excess fuel DNA strands without depleting the gate components. The recycling of the two-layered circuits occurs during the fuel DNA digestion but not during the release of the downstream trigger. This strategy provides a simple yet versatile method for creating more efficient entropy-driven DNA circuits for molecular programming and synthetic biology.