Researchers Develop HTMR Assay for DNA/RNA Methyltransferases and Demethylases Activity Detection

DNA methylation (5mC) and RNA methylation (m6A) are widely present in animals, plants, fungi, and prokaryotes, which play important roles in a variety of life processes. While there is significant potential for drug development for DNA/RNA methyltransferases and demethylases, only a few potent small molecules have been reported. This is partly due to a lack of effective high-throughput screening methods.

In a study published in Nucleic Acids Research on Oct 29, a team of researchers led by LUO Cheng from Shanghai Institute of Materia Medica of the Chinese Academy of Sciences developed a fluorescence polarization-based high-throughput methyl reading (HTMR) assay to implement large-scale compound screening for DNA/RNA methyltransferases and demethylases.

The researchers coupled the thermodynamic process of protein-substrate nucleic acid binding with the fluorescence polarization experiment and established a homogeneous assay-HTMR. By optimizing the molecular weight, concentration of the binding protein and the binding buffer, the affinity of the binding proteins to the substrate and the product was significantly different. The substrate/product would produce fluorescence polarization signals, which could characterize enzyme activities. In that way, the researchers quickly detected various activities of enzymes including DNMT1, TET2, METL3-METTL14 and ALKBH5, and implemented high-throughput screening for these enzymes. The data measured by their assay were close to that measured by HPLC, which proved the assay accuracy. Besides, this assay is highly suitable for substrate competition experiments, which showed good stability with a high Z-factor and helped eliminate some false positive compounds binding to nucleic acids.

This study provides a highly efficient high-throughput assay-HTMR for 5mC/m6A modification enzymes activity detection. The assay allows for mass screening of large and diverse compound libraries for drug discovery on DNA/RNA methyl modification regulatory enzymes.

The principle of High-Throughput-Methyl-Reading (HTMR) assay(Image by LUO Cheng 's team)





DIAO Wentong
Shanghai Institute of Materia Medica, Chinese Academy of Sciences