System leakage critically confines the development of cascade DNA systems that need to be implemented in a strict order-by-order manner. In principle, ternary DNA reactants, composed of three single-strand DNA (ssDNA) with a strict equimolar ratio (1:1:1), have been indispensable for successfully cascading upstream entropy-driven DNA circuit (EDC) with downstream circuits, and system leakage will occur with any unbalance of the molar ratio. In this work, we proposed “splitting-reconstruction” and “protection-release” strategies on the potential downstream circuit initiator derived from upstream EDC to guide the construction of EDC-involved cascade systems independent of system leakage derived from unpurified reactants. Both the reconstructed and released downstream circuit initiators were in compliance with the principle of the cascade AND logic gate. Using these two strategies, two cascade systemsEDC2-4WJ-TMSDR and EDC3-HCRwere developed to carry out the designed order, which did not require that the ratio of 1:1:1 be maintained. Furthermore, the inherent property of the upstream EDC could transfer into the downstream circuit, endowing the developed cascade systems with a more powerful signal amplification ability for the sensitive detection of the corresponding initiator strand. These two strategies may provide new insights into the process of constructing EDC-like circuit-involved high-order DNA networks.
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