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  3. High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases

High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases

  • Nucleic Acids Res. 2022 Jan 25;50(2):e9. doi: 10.1093/nar/gkab989.
Senhao Xiao 1 2 3,  Siqi Guo 1 4,  Jie Han 1,  Yanli Sun 1 5,  Mingchen Wang 1 2 3,  Yantao Chen 1,  Xueyu Fang 1 2 3,  Feng Yang 1,  Yajuan Mu 6,  Liang Zhang 6,  Yiluan Ding 3 7,  Naixia Zhang 3 7,  Hualiang Jiang 1 2 3,  Kaixian Chen 1 2 3,  Kehao Zhao 5,  Cheng Luo 1 2 3 8,  Shijie Chen 1 3 8
Affiliations

Affiliations

  • 1 The Center for Chemical Biology, Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
  • 2 School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
  • 3 University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, China.
  • 4 School of Pharmacy, Nanchang University, Nanchang 330006, Jiangxi, China.
  • 5 School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education; Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China.
  • 6 Department of Pharmacology and Chemical Biology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
  • 7 Analytical Research Center for Organic and Biological Molecules, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
  • 8 School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, UCAS, Hangzhou 310024, China.
PMID: 34718755 DOI: 10.1093/nar/gkab989
Abstract

Epigenetic therapy has significant potential for Cancer treatment. However, few small potent molecules have been identified against DNA or RNA modification regulatory proteins. Current approaches for activity detection of DNA/RNA methyltransferases and demethylases are time-consuming and labor-intensive, making it difficult to subject them to high-throughput screening. Here, we developed a fluorescence polarization-based 'High-Throughput Methyl Reading' (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases-DNMTs, TETs, ALKBH5 and METTL3/METTL14. This assay is simple to perform in a mix-and-read manner by adding the methyl-binding proteins MBD1 or YTHDF1. The proteins can be used to distinguish FAM-labelled substrates or product Oligonucleotides with different methylation statuses catalyzed by enzymes. Therefore, the extent of the enzymatic reactions can be coupled with the variation of FP binding signals. Furthermore, this assay can be effectively used to conduct a cofactor competition study. Based on the assay, we identified two Natural Products as candidate compounds for DNMT1 and ALKBH5. In summary, this study outlines a powerful homogeneous approach for high-throughput screening and evaluating enzymatic activity for DNA/RNA methyltransferases and demethylases that is cheap, easy, quick, and highly sensitive.

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