Probing the interaction of copper cofactor and azachalcone substrate with G-quadruplex of DNA based Diels-Alderase by site-specific fluorescence quenching titration.

DNAzymes have been widely used in biosensors, asymmetric synthesis and pharmaceuticals. Typically, metal cofactor and substrate interact with DNA by supramolecular interactions in DNAzyme based asymmetric catalysis. However, binding positions of cofactor and substrate with DNA scaffold are not well understood, which is an obstacle to reveal the assembly and catalysis mechanisms of DNAzyme. Herein, we report a method of site-specific fluorescence quenching titration to elucidate the assembly and catalysis processes of a G-quadruplex based Diels-Alderase DNAzyme. Titration data indicate that cofactor Cu(II)-terpyridine stacked atop 5' and 3' external G-quartets with high and low binding affinities respectively, and induced the G-quadruplex to form a hybrid-1 topology. Substrate azachalcone interacted with 3' quartet exclusively, implicating that asymmetric Diels-Alder cycloaddition may occur at 3' G-quartet. In addition, enzyme kinetic analyses show that activity and enantioselectivity of the DNAzyme were substantially preserved after attaching the fluorophores. Overall, site-specific fluorescence quenching is a concise and efficient approach to probe the assembly processes of DNAzyme.