Nucleic Acids Res: A high-throughput 5mC/m6A modified enzyme activity detection method

DNA methylation (5mC) and RNA methylation (m ^.
6 A) is widely found in plants, fungi, and prokaryotes, which plays an important role in a variety of life processes ^1,2
.

Modification enzymes that regulate the level of 5mC/m6A include methyltransferases DNMTs, METTL3/METTL14 and demethylases TETs, FTO, ALKBH5 and so on

In response to this problem, based on the preliminary work of the research group and the cooperative research group in nucleic acid modification research ( Nature .
2015 Nov 5;527(7576):118-22.
; J Am Chem Soc .
2012 Oct 31;134(43):17963 -71; Nucleic Acids Res .
2015 Jan;43(1):373-84; Chem Sci .
2019 Jun 27;10(31):7407-7417; Phys Chem Chem Phys.
2016 Feb 14;18(6):4728 -38; ACS Chem Biol .
2018 Mar 16;13(3):772-781; J Med Chem .
2014 Nov 13;57(21):9028-41), Chen Shijie, Deputy Chen Shijie, Shanghai Institute of Materia Medica, Chinese Academy of Sciences The researcher and Dr.
Hsiao Senhao developed a simple, efficient and high-throughput 5mC/m6A nucleic acid modification enzyme activity detection method.
The research work was published online on October 29, 2021 in Nucleic Acids Research (High-Throughput -Methyl-Reading (HTMR) Assay: a solution based on Nucleotide Methyl-Binding Proteins enables largescale screening for DNA/RNA Methyltransferases and Demethylases)
.

Figure 1.
Schematic diagram of the activity detection of 5mC/m ⁶ A nucleic acid modification enzyme

　　In this study, the researchers used a class of “chemical substances” that exist in the cell itself (methylation reader proteins: UHRF1, MBPs, YTH family proteins, etc.
) as methyl-recognition proteins, which can detect different methyl groups.
DNA/RNA in the chemical state produces different responses (the thermodynamic process of protein-substrate nucleic acid binding)
.

Subsequently, the researchers coupled this thermodynamic process with the fluorescence polarization experiment to establish a homogeneous-"High-Throughput-Methyl-Reading" (HTMR) analysis method: The substance is fluorescently labeled, and methyl binding proteins MBD1 and YTHDF1 are added after enzymatic catalysis

Figure 2.

a) Experimental operation and quality control process of HTMR method; b) Parallel methylation/demethylation HPLC quantitative experiment; c) Stability evaluation experiment of HTMR method;

　　In summary, this study provides a simple, rapid, homogeneous, low-cost, high-sensitivity, high-throughput 5mC/m6A modified enzyme activity detection method-HTMR
.

This method does not need to undergo cumbersome processing processes such as immobilization, washing, transfer, and enzyme treatment.

　　This project was completed with the assistance of researcher Luo Cheng from Shanghai Institute of Pharmacy.
Associate researcher Chen Shijie of Luo Cheng's research group is the corresponding author of the paper, and Xiao Senhao, a Ph.
D.
co-trained by Shanghai Institute of Pharmacy and Shanghai University of Science and Technology, is the first author of the paper
.

The research was also supported by Professor Yin Ping of Huazhong Agricultural University, Professor Xu Yanhui of Fudan University, Professor Zhang Liang of Shanghai Jiaotong University, Academician Chen Kaixian of Shanghai Institute of Materia Medica, Academician Jiang Hualiang, Researcher Zhou Hu, Researcher Yang Caiguang, and Researcher Zhang Naixia

Full text link:

https://academic.

references:

1 Jones, PA Functions of DNA methylation: islands, start sites, gene bodies and beyond.

2 Zaccara, S.

3 Huang, Y.

4 Li, Z.

5 Barbieri, I.

6 Huang, Y.

7 Lyko, F.
The DNA methyltransferase family: a versatile toolkit for epigenetic regulation.
Nat Rev Genet 19, 81-92, doi:10.
1038/nrg.
2017.
80 (2018).

8 Constantinides, PG, Jones, PA & Gevers, W.
Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment.
Nature 267, 364-366, doi:10.
1038/267364a0 (1977).

9 Cimmino, L.
et al.
Restoration of TET2 Function Blocks Aberrant Self-Renewal and Leukemia Progression.
Cell 170, 1079-1095 e1020, doi:10.
1016/j.
cell.
2017.
07.
032 (2017).

10 Agathocleous, M.
et al.
Ascorbate regulates haematopoietic stem cell function and leukaemogenesis.
Nature 549, 476-481, doi:10.
1038/nature23876 (2017).

11 Gros, C.
et al.
Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies.
Nucleic Acids Res 41, e185, doi:10.
1093/nar/gkt753 (2013).

12 Li, F.
et al.
A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5.
J Biomol Screen 21, 290-297, doi:10.
1177/1087057115623264 (2016).

13 Hu, L.
et al.
Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation.
Cell 155, 1545-1555, doi:10.
1016/j.
cell.
2013.
11.
020 (2013).

14 Gerken, T.
et al.
The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.
Science 318, 1469-1472, doi:10.
1126/science.
1151710 (2007).