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Updated: Sep 29, 2025
Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
Profiling demethylase activity using epigenetically inactivated DNAzyme
Juan Huang1, Jiao Wang1, Zhenkun Wu1
1State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China.
Abstract:
The level of DNA methylation has been reported to be closely associated with various carcinomas and is dynamically regulated by several demethylases. However, current demethylase detection methods are mainly antibody-based, which detect the demethylase concentrations, while the actual numbers of catalytically active demethylase remain unknown. Thus, we have developed an activity assay based on epigenetically modified DNAzymes (EMOzymes), and CRISPR/Cas12a facilitated cascade signal amplification. We have ultrasensitively quantitated the activity of O6-methylguanine-DNA-methyltransferase (MGMT), a key demethylase that contributes to the chemoresistance to alkylation agents in cancer therapy, with an estimated limit of detection of 0.054 nM. This approach opens a new avenue for sensitively profiling the activity of many disease-related demethylases.
Insights
This study introduces a novel assay to measure active demethylase levels, crucial for understanding cancer chemoresistance. The new method accurately quantifies O6-methylguanine-DNA-methyltransferase (MGMT) activity, offering a sensitive tool for disease research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- DNA methylation is linked to various carcinomas and regulated by demethylases.
- Current antibody-based methods measure demethylase concentration, not active enzyme levels.
- Understanding active demethylase levels is critical for cancer therapy and chemoresistance.
Purpose of the Study:
- To develop a novel, ultrasensitive activity assay for disease-related demethylases.
- To quantify the catalytic activity of O6-methylguanine-DNA-methyltransferase (MGMT).
- To establish a new method for profiling active demethylase levels in disease contexts.
Main Methods:
- Development of an activity assay using epigenetically modified DNAzymes (EMOzymes).
- Utilized CRISPR/Cas12a facilitated cascade signal amplification for enhanced sensitivity.
- Quantified the activity of O6-methylguanine-DNA-methyltransferase (MGMT).
Main Results:
- Achieved ultrasensitive quantification of MGMT activity.
- Established a limit of detection as low as 0.054 nM for MGMT activity.
- Demonstrated the assay's potential for profiling various disease-related demethylases.
Conclusions:
- The developed EMOzyme and CRISPR/Cas12a-based assay provides a sensitive method for measuring active demethylase levels.
- This approach overcomes limitations of antibody-based assays by quantifying enzyme activity.
- Opens new avenues for understanding the role of demethylases in cancer and developing targeted therapies.
