Target-Navigated CBT-Cys "Stapling" Coupled with CRISPR/Cas12a Amplification for the Photoelectrochemical Nucleic...

Area of Science:

• Biochemistry
• Molecular Biology
• Nanotechnology

Background:

• Traditional rolling circle amplification (RCA) relies on enzyme-linked reactions.
• Efficient and rapid DNA end-joining methods are crucial for molecular biology applications.
• Click chemistry offers a versatile platform for bioconjugation.

Purpose of the Study:

• To develop a rapid, enzyme-free method for joining DNA molecules using click chemistry.
• To integrate this novel linking strategy with rolling circle amplification (RCA) and CRISPR/Cas12a for enhanced detection.
• To establish a sensitive photoelectrochemical (PEC) detection system for amplified nucleic acid products.

Main Methods:

• DNA ends were modified with 2-cyano-6-aminobenzothiazole (CBT) and cystine (Cys-Cys).
• Glutathione facilitated enzyme-free linkage between CBT and Cys under target navigation.
• Rolling circle amplification (RCA) with phi29 polymerase amplified the linked DNA, followed by CRISPR/Cas12a cleavage and Sb@Co(OH)F nanorod-based PEC detection.

Main Results:

• The enzyme-free CBT-Cys linking reaction demonstrated a high rate constant (23.79 M⁻¹·s⁻¹).
• The integrated system successfully amplified and detected target DNA via RCA and CRISPR/Cas12a cleavage.
• Photoelectrochemical (PEC) signals correlated with the amount of amplified product, confirming system sensitivity.

Conclusions:

• The developed enzyme-free DNA linking system offers a faster and more robust alternative to traditional enzymatic methods.
• This approach significantly enhances DNA amplification and cleavage processes for sensitive molecular detection.
• The system shows great potential for applications in DNA modification, nucleic acid-protein interactions, and nonhomologous end joining.