Nucleic acid driven DNA machineries synthesizing Mg²⁺-dependent DNAzymes: An interplay between DNA sensing and logic-gate operations

Polymerase/nicking enzymes and nucleic-acid scaffolds are implemented as DNA machines for the development of amplified DNA-detection schemes, and for the design of logic gates. The analyte nucleic acid target acts, also, as input for the logic gates. In the presence of two DNA targets, acting as inputs, and appropriate DNA scaffolds, the polymerase-induced replication of the scaffolds, followed by the nicking of the replication products, are activated, leading to the autonomous synthesis of the Mg²⁺-dependent DNAzyme or the Mg ²⁺-dependent DNAzyme subunits. These biocatalysts cleave a fluorophore/quencher-functionalized nucleic-acid substrate, thus providing fluorescence signals for the sensing events or outputs for the logic gates. The systems are used to develop OR, AND, and Controlled-AND gates, and the DNA-analyte targets represent two nucleic acid sequences of the smallpox viral genome. Open the gate! Nucleic acid analytes (inputs) trigger polymerization/nicking DNA machines that synthesize the Mg²⁺- dependent DNAzymes. The systems allow the amplified detection of the analytes and the activation of the OR, AND, and controlled-AND logic gates by using fluorescence as output signal (see scheme).