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  3. A highly sensitive signal-on biosensor based on restriction enzyme-mediated molecular switch for detection of TET1

A highly sensitive signal-on biosensor based on restriction enzyme-mediated molecular switch for detection of TET1

  • Bioelectrochemistry. 2023 Apr 4;152:108433. doi: 10.1016/j.bioelechem.2023.108433.
Ying Cheng 1,  Chen Chen 2,  Fang Wang 3,  Zilin Chen 4
Affiliations

Affiliations

  • 1 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China; Department of Pharmacy, Zhongnan Hospital of Wuhan University, Wuhan 430071, China.
  • 2 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China.
  • 3 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China. Electronic address: fwang@whu.edu.cn.
  • 4 Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China. Electronic address: chenzl@whu.edu.cn.
PMID: 37031472 DOI: 10.1016/j.bioelechem.2023.108433
Abstract

Ten-eleven translocation 1 (TET1) is a member of the TET Enzyme family of dioxygenases, which plays an important role in active DNA demethylation. Therefore, the sensitive TET1 detection could help us better understand DNA methylation-demethylation in Epigenetics. Here we report a detection method that consists of electrode fabrication, TET1 modification, DNA digestion, signal-on oxidoreduction, and current peak monitoring. An exquisitely designed 5'end-G-rich oligodeoxynucleotide was synthesized bearing a methylated cytosine (5-mC), which formed into hairpin dsDNA with the MspI recognition sequence (CmCGG/GGCC). Then hairpin dsDNA was fabricated onto gold nanoparticles modified glassy carbon electrode (DNA/AuNPs/GCE) via Au-S bond. The combination uses of restriction Enzyme MspI and hemin converted fabricated-dsDNA into peroxidase-mimicking DNAzyme, thereby promoting the reduction of H2O2 with a current peak. However, the current peak was extremely decreased once TET1 and T4 β-GT were used in advance. We confirmed a delicately linear relationship matching between the current difference and TET1 activity from 0.7 to 10.5 ng μL-1 with a detection limit of 0.027 ng μL-1, which outcompeted the former methods at least one order of magnitudes. The TET1 activity evaluation in the existence of Bobcat339 was also tested as the proof of concept of inhibitors screening. Our strategy provides a novel, label-free, and sensitive electrochemical approach that enables us to complete both TET1 activity evaluation and potential TET1 inhibitors screening.

Keywords

Electrochemical biosensor; Hemin/G-quadruplex; Methylation; MspI; TET.

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