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

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Metal-ion-dependent cleavage-active DNAzymes (caDz) are vital for sensing and diagnostics.
• Current methods (AM-caDz) require complex modifications and are sensitive to environmental changes.
• A need exists for simpler, more robust DNAzyme assay formats.

Purpose of the Study:

• To develop a label-free and dehybridization-free fluorescent DNAzyme (F-caDz) system.
• To utilize hypericin (Hyp) as a fluorogen that binds to the GR5 caDz catalytic core.
• To establish a turn-on fluorescence assay for detecting Pb2+.

Main Methods:

• Developed F-caDz by associating hypericin with the GR5 caDz catalytic core for turn-on fluorescence.
• Utilized Pb2+-mediated cleavage to unfold the catalytic core and release hypericin, causing fluorescence alteration.
• Monitored fluorescence changes to quantify Pb2+ concentration and assess cleavage activity.

Main Results:

• Demonstrated specific Pb2+ detection using the F-caDz system with turn-on fluorescence.
• Showed that hypericin association did not impede cleavage efficiency but altered kinetics.
• The F-caDz system operates without substrate dehybridization, enhancing robustness.

Conclusions:

• The developed F-caDz offers a sensitive, specific, and convenient method for Pb2+ detection.
• This approach is adaptable to other caDz by identifying suitable fluorogens.
• F-caDz presents a promising platform for simplified DNAzyme assays and activity regulation.