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

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• Peroxidase-mimicking DNAzymes offer a cost-effective and stable alternative to protein-based peroxidases.
• DNAzymes can self-assemble into G-quadruplex structures exhibiting peroxidase activity.
• Non-labeling biosensors are desirable for simplified detection strategies.

Purpose of the Study:

• To investigate the structural and functional properties of a novel non-labeling biosensor based on split DNAzyme.
• To evaluate the performance of the split DNAzyme biosensor for DNA target detection.
• To optimize conditions for DNAzyme activity and biosensor fabrication.

Main Methods:

• Circular Dichroism (CD) spectroscopy and UV-Vis spectroscopy were employed to characterize DNAzyme properties.
• Optimization of DNAzyme activity involved screening of monovalent and divalent cations (including Mg2+) and hemin incubation.
• Split DNAzyme designs (2:2 and 3:1 modes) were compared to intact DNAzyme for structural and functional analysis.

Main Results:

• The split DNAzyme biosensor achieved a detection limit in the nanomolar (nM) range (9.48 nM).
• Hybridization of the DNA target with the complementary linker sequence separated the DNAzyme halves, reducing peroxidase activity.
• Catalytic activity of split DNAzymes could be enhanced by Mg2+, which also promoted DNA target hybridization.

Conclusions:

• Split DNAzyme systems can be effectively utilized for fabricating sensitive non-labeling biosensors.
• The presence of Mg2+ plays a crucial role in enhancing both the catalytic activity and hybridization efficiency of the split DNAzyme system.
• This approach offers a promising platform for the development of advanced DNA-based diagnostic tools.