Dehybridization-Free and Fluorescent Cleavage-Active DNAzyme by a Catalytic Core-Associative Fluorogen

Metal-ion-dependent cleavage-active DNAzymes (caDz) are increasingly utilized in fields of sensing, environmental monitoring, and diagnostics. Currently, screening caDz and programming its cleavage activity rely on the "catalytic beacon" approach needing covalent duplex arm modifications by fluorophores and quenchers (thus named AM-caDz). Although this approach is widely used, the tedious modifications for operation limit its utilization in ordinary laboratories. Furthermore, dehybridization of cleaved substrate strands from DNAzyme strands is needed to signal the cleavage events. Thus, the arm length must meet a compromise to keep AM-caDz at a duplex state for cleavage and ensure a dehybridization state after cleavage for signaling, which is highly susceptible to environmental fluctuation. Herein, we developed fluorescent caDz (F-caDz) that can operate in a label-free and dehybridization-free manner. A fluorogen of hypericin (Hyp) was found to be able to specifically associate with the folding catalytic core of the GR5 caDz, resulting in a turn-on fluorescence (thus named F-caDz). The Pb²⁺-mediated cleavage subsequently unfolded the catalytic core and released Hyp but without dehybridization of cleaved substrates. The resultant fluorescence alteration was used to evaluate the cleavage activity of F-caDz. Furthermore, this folding catalytic core association did not affect the final cleavage efficiency but caused a modification in the cleavage kinetics. This F-caDz provides a sensitive and specific method to detect Pb²⁺. By finding the appropriate fluorogens, this method can be applied to other caDz. We expect that F-caDz will also provide a convenient approach to regulating the cleavage behavior of caDz.