Profiling demethylase activity using epigenetically inactivated DNAzyme

Juan Huang; Jiao Wang; Zhenkun Wu; Jianjun He; Jian-Hui Jiang

doi:10.1016/j.bios.2022.114186

Profiling demethylase activity using epigenetically inactivated DNAzyme

Biosens Bioelectron. 2022 Jul 1:207:114186. doi: 10.1016/j.bios.2022.114186. Epub 2022 Mar 18.

Authors

Juan Huang¹, Jiao Wang¹, Zhenkun Wu¹, Jianjun He², Jian-Hui Jiang³

Affiliations

¹ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China.
² State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China. Electronic address: jianjunh@hnu.edu.cn.
³ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biomedical Sciences, College of Chemistry and Chemical Engineering, Hunan University, Changsha, 410082, China. Electronic address: jianhuijiang@hnu.edu.cn.

PMID: 35316758
DOI: 10.1016/j.bios.2022.114186

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.

Keywords: CRISPR/Cas12a; DNAzyme; Demethylase; Epigenetic modification.

MeSH terms

Biosensing Techniques* / methods
DNA Methylation
DNA, Catalytic* / metabolism

Substances

DNA, Catalytic