Structural and functional study of a simple, rapid, and label-free DNAzyme-based DNA biosensor for optimization activity

Peroxidase-mimicking DNAzyme has a potential to self-assemble into a G-quadruplex and shows peroxidase activity. In comparison to proteins, peroxidase-mimicking DNAzyme is less expensive and more stable. Herein, it is used in fabricating non-labeling biosensors. This paper investigates the structural and functional properties of a DNA biosensor based on split DNAzyme with a detection limit in nM range (9.48 nM). Two halves of DNAzyme were linked by a complementary sequence of DNA target. Hybridization of the DNA target pulled two DNAzyme halves apart and peroxidase activity decreased. This study can be divided into 3 stages. First, the characteristics of DNAzyme were studied by Circular Dichroism technique and UV-Vis spectroscopy to find out DNAzyme's optimum activity. It is worth to note that some divalent cations were used to form G-quadruplex, in addition to common monovalent cations. Furthermore, the hemin incubation was also optimized. Secondly, the structural and functional properties of two types of split DNAzyme were compared with DNAzyme. Thirdly, the hybridization of DNA target was monitored. The results revealed that peroxidase activities of split types decreased by half without any specific conformational changes. Interestingly, the catalytic activities of split DNAzymes could be promoted by adding Mg²⁺ . Besides, it was demonstrated that the structure, peroxidation reaction, and DNA target hybridization of 2:2 and 3:1 split modes were almost alike. It was also illustrated that magnesium promoted the possibility of hybridization.