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

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.

The catalytic mechanism of the RNA methyltransferase METTL3

“…These considerations are congruent with the observation that the computed DFTB3/MM free energy profile without N 6 deprotonation is possible within the reported experimental kinetics. 58,64,65 The difference in the methylation energetics of adenosine in different protonation states shown in Table 3 is consistent with the pKa difference of adenosine before and after methylation. Thus, the large difference suggests that N 6 becomes much more acidic following methylation, which is consistent with literature estimates of the pKa values of N 6 -protonated adenosine derivatives in the range of -3 to -10.…”

“…These considerations are congruent with the observation that the computed DFTB3/MM free energy profile without N 6 deprotonation is possible within the reported experimental kinetics. 58,64,65…”

A review on recent advances in assays for DNMT1: a promising diagnostic biomarker for multiple human cancers

“…134 (E) A fluorescence polarization-based high-throughputmethyl-reading (HTMR) assay for the homogeneous screening of DNA methyltransferases. 136 methylated molecules (HM) and unmethylated molecular substrates. The B-Z transition occurring in the methylated DNA can be detected by the single-molecule FRET (smFRET) experiment, which is an ideal technique for detecting the B-Z transition in Mg 2+ -rich solutions.…”

“…8E). 136 The substrate is first fluorescently labeled, and after enzyme-catalyzed methylation, the DNA methyl-binding protein MBD1 is added for binding with methylated products. Consequently, a strong fluorescence polarization (FP) signal value is detected to characterize the activity of DNA methyltransferase in the system.…”

Ghost authors revealed: The structure and function of human N6‐methyladenosine RNA methyltransferases

“…Interest in developing small molecules to target MTases, particularly METTL3 (see reference Fiorentino et al, 2023 for review), for therapeutic purposes is growing. Artificial intelligence and high‐throughput screening methods, such as the high‐throughput‐methyl‐reading assay, will enable fast and easy means of testing libraries of compounds for potential activation/inhibition of MTases (Xiao et al, 2022) in addition to virtual high‐throughput screening methods that have yielded promising hits for METTL3 (Selberg et al, 2019) and METTL16 (Mitra et al, 2023). Inhibitors of METTL3 would be therapeutically useful because inhibition limits the growth of AML (Barbieri et al, 2017).…”