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Customizable NAND Logic-Gate Biosensing System Enabled by an Engineered Methylation-CRISPR/Cas12a Consensus Sequence
Jia Zhao1,2, Zhuqi Sui2, Baoqiang Chen2
1Fujian Key Laboratory of Cognitive Function and Diseases, School of Basic Medical Sciences, Fujian Medical University, Fuzhou 350122, China.
Analytical Chemistry
|January 9, 2026
Summary
This study introduces a novel NAND logic-gate biosensor for detecting DNA adenine methyltransferase (Dam MTase). The platform offers highly sensitive and selective enzyme detection, crucial for understanding epigenetic regulation and disease.
Area of Science:
- Epigenetics and Molecular Biology
- Biosensing and Diagnostics
- Biochemistry and Enzyme Activity Analysis
Background:
- DNA methyltransferases (MTases) are vital for epigenetic regulation, with aberrant activity linked to human diseases.
- Accurate detection of specific MTases, like DNA adenine methyltransferase (Dam MTase), is crucial for disease research and therapeutic development.
- Existing biosensing methods may lack the sensitivity, selectivity, or integrated logic required for complex biological samples.
Purpose of the Study:
- To develop a customizable NAND logic-gate biosensing platform for sensitive and intelligent detection of Dam MTase.
- To integrate multiple functional elements into a single recognition motif for enhanced biosensing capabilities.
- To establish a universal framework for enzyme activity analysis and inhibitor screening in biological systems.
Main Methods:
- Designed an engineered methylation-CRISPR/Cas12a consensus sequence (MCCS) incorporating Dam methylation and DpnI recognition sites.
- Utilized primer-triggered hybridization chain reaction (HCR) for signal amplification of tandem MCCS units.
- Implemented a NAND logic circuit where Dam MTase, SAM, and DpnI act as inputs, with a low-fluorescence 'OFF' output under specific conditions.
Main Results:
- Achieved an ultralow detection limit of 0.00032 U mL⁻¹ for Dam MTase with a broad linear range.
- Demonstrated outstanding selectivity against other methyltransferases and satisfactory recovery rates (98.16-100.03%) in human serum.
- Successfully evaluated Dam inhibitors, determining IC₅₀ values for 5-fluorouracil (1.75 μM) and penicillin G (11.9 μM).
Conclusions:
- The developed NAND logic-gate biosensing platform provides a highly sensitive, selective, and intelligent method for Dam MTase detection.
- This strategy offers a universal molecular computation-driven framework for enzyme activity analysis and drug discovery in complex biological contexts.
- The platform's customizable nature and robust performance in serum samples highlight its potential for clinical diagnostics and pharmaceutical research.
