Terminal-Optimized Aptamer-Guide-DNAzyme Triplex Boosts Clostridium butyricum Argonaute-Driven Multiplex Foodborne...

Area of Science:

• Biotechnology
• Food Science
• Molecular Biology

Background:

• Multiplexed and sensitive detection of foodborne pathogens is crucial for disease control.
• Mesophilic Clostridium butyricum Argonaute (CbAgo) has endonuclease activity for precise DNA-directed cleavage, useful for pathogen detection.
• CbAgo's activity constraints can be overcome by engineered guide DNA (gDNA) and synergistic enzymatic mechanisms.

Purpose of the Study:

• To engineer CbAgo's gDNA with a DNAzyme-functionalized fragment (cDNA) to enhance cleavage activity.
• To investigate the synergistic integration of DNAzyme and CbAgo activities for improved signal amplification.
• To develop a multiplexed biosensor for simultaneous detection of foodborne pathogenic bacteria.

Main Methods:

• Engineered gDNA by incorporating a DNAzyme-functionalized fragment (cDNA) for substrate cleavage.
• Investigated terminal modification group preferences for optimal DNAzyme-CbAgo interaction.
• Developed a DNAzyme-CbAgo synergistic fluorescence aptamer biosensor.

Main Results:

• Engineered CbAgo with cDNA demonstrated enhanced cooperative cleavage activity.
• Synergistic integration of dual enzymatic activities significantly improved signal amplification.
• The developed biosensor achieved multiplexed detection of three pathogenic bacteria with a low detection limit (69 CFU mL⁻¹).

Conclusions:

• The DNAzyme-CbAgo synergistic system offers enhanced efficiency for pathogen detection.
• The developed aptamer biosensor enables simultaneous detection of multiple foodborne pathogens.
• This platform provides a potential method for foodborne pathogen detection without DNA extraction and amplification.