An allosteric DNA switch-mediated catalytic DNA circuit for ratiometric and sensitive nucleic acid detection

Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing system was based on two DNA hybrid probes, each of which was constructed by annealing a reconfigurable DNA hairpin with single-stranded DNA. Upon target recognition by the first DNA hybrid probe, a reconfigurable DNA switch was liberated, triggering a toehold-mediated strand displacement reaction (TSDR) with the second DNA hybrid probe, which was accompanied by the release of another reconfigurable DNA switch. This released allosteric DNA switch could further interact with the first hybrid DNA probe via the TSDR strategy to form a reciprocal strand displacement network between the two DNA hybrid probes. Theoretically, this reciprocal strand displacement reaction would continue till the complete consumption of the reaction substrates. Thus, it provides a new signal amplification method leading toward target recognition. More interestingly, it creates a ratiometric signal response mode for target recognition, which involves the fluorescence increment of one fluorophore (Cy5) and concurrent decrement of another fluorophore (Cy3) accompanied by the target-triggered reciprocal strand displacement reaction. This process could achieve a low detection limit of about 0.1 pM toward the target nucleic acid and selective discrimination toward different mismatched targets. It could also be applied for detection in a serum sample. Thus, the developed catalytic DNA circuit reaction strategy together with ratiometric signal readout provides a new avenue for programmable, reliable and sensitive detection of nucleic acids and might also pave the way for developing more advanced DNA circuits or biosensors.