High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases
文献类型：期刊论文
| 作者 | Xiao, Senhao2,3,4; Guo, Siqi2,6; Han, Jie2; Sun, Yanli2,7,8; Wang, Mingchen2,3,4; Chen, Yantao2; Fang, Xueyu2,3,4; Yang, Feng2; Mu, Yajuan9; Zhang, Liang9 |
| 刊名 | NUCLEIC ACIDS RESEARCH
 |
| 出版日期 | 2022-01-25 |
| 卷号 | 50期号:2页码:14 |
| ISSN号 | 0305-1048 |
| DOI | 10.1093/nar/gkab989 |
| 通讯作者 | Chen, Shijie(shijiechen@simm.ac.cn) |
| 英文摘要 | 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. |
| WOS关键词 | DNA METHYLATION ; RNA DEMETHYLASE ; STRUCTURAL BASIS ; METTL3-METTL14 COMPLEX ; ENZYMATIC-PROPERTIES ; GENE-EXPRESSION ; SELF-RENEWAL ; NUCLEAR-RNA ; STEM-CELLS ; INHIBITOR |
| 资助项目 | National Natural Science Foundation of China[81703415] ; National Natural Science Foundation of China[91853205] ; National Natural Science Foundation of China[81625022] ; National Natural Science Foundation of China[81821005] ; National Natural Science Foundation of China[21820102008] ; National Natural Science Foundation of China[81728020] ; K.C. Wong Education ; Science and Technology Commission of Shanghai Municipality[21ZR1474700] ; Science and Technology Commission of Shanghai Municipality[19XD1404700] ; Science and Technology Commission of Shanghai Municipality[18431907100] ; Taishan Scholar |
| WOS研究方向 | Biochemistry & Molecular Biology |
| 语种 | 英语 |
| 出版者 | OXFORD UNIV PRESS |
| WOS记录号 | WOS:000763001100003 |
| 源URL | [http://119.78.100.183/handle/2S10ELR8/300212]  |
| 专题 | 新药研究国家重点实验室 |
| 通讯作者 | Chen, Shijie |
| 作者单位 | 1.Chinese Acad Sci, Shanghai Inst Mat Med, Drug Discovery & Design Ctr, Ctr Chem Biol, Zuchongzhi Rd 555, Shanghai 201203, Peoples R China 2.Chinese Acad Sci, Shanghai Inst Mat Med, State Key Lab Drug Res, Ctr Chem Biol,Drug Discovery & Design Ctr, Shanghai 201203, Peoples R China 3.ShanghaiTech Univ, Sch Life Sci & Technol, Shanghai 201210, Peoples R China 4.Univ Chinese Acad Sci, 19A Yuquan Rd, Beijing 100049, Peoples R China 5.Chinese Acad Sci, Analyt Res Ctr Organ & Biol Mol, Shanghai Inst Mat Med, Shanghai 201203, Peoples R China 6.Nanchang Univ, Sch Pharm, Nanchang 330006, Jiangxi, Peoples R China 7.Yantai Univ, Sch Pharm, Key Lab Mol Pharmacol & Drug Evaluat, Minist Educ, Yantai 264005, Peoples R China 8.Yantai Univ, Collaborat Innovat Ctr Adv Drug Delivery Syst & B, Yantai 264005, Peoples R China 9.Shanghai Jiao Tong Univ, Dept Pharmacol & Chem Biol, State Key Lab Oncogenes & Related Genes, Sch Med, Shanghai 200025, Peoples R China 10.UCAS, Hangzhou Inst Adv Study, Sch Pharmaceut Sci & Technol, Hangzhou 310024, Peoples R China |
| 推荐引用方式 GB/T 7714 | Xiao, Senhao,Guo, Siqi,Han, Jie,et al. High-Throughput-Methyl-Reading (HTMR) assay: a solution based on nucleotide methyl-binding proteins enables large-scale screening for DNA/RNA methyltransferases and demethylases[J]. NUCLEIC ACIDS RESEARCH,2022,50(2):14. |
| APA | Xiao, Senhao.,Guo, Siqi.,Han, Jie.,Sun, Yanli.,Wang, Mingchen.,...&Chen, Shijie.(2022).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 RESEARCH,50(2),14. |
| MLA | Xiao, Senhao,et al."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 RESEARCH 50.2(2022):14. |
入库方式： OAI收割
来源：上海药物研究所
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