This is part of our end-to-end viral RNA modification workflow, click the image for more details

m6A Demethylase & Methylase Assays
to Study Viral Infection and Virus-Host Interaction

N6-Methyladenosine, or m6A, is formed when a methyl group is chemically added at the nitrogen-6 position of adenosine residues. Often referred to as “the fifth RNA base”, m6A is the most common and abundant eukaryotic RNA modification, accounting for over 80% of all RNA methylation. It can be found mainly in mRNA, but it is also observed in non-coding species like tRNA, rRNA, and miRNA.

Through interactions with various binding proteins called “readers”, m6A affects virtually every facet of ribonucleic acid biology: structure, splicing, localization, translation, stability, and turnover.¹  Aside from this central role in RNA metabolism, m6A plays a part in other physiological processes such as cell differentiation, immunity, inflammation, and the circadian clock.²  Abnormal m6A methylation has been implicated in diabetes, obesity, neurodegeneration, cancer, and other pathologies.  The formation of m6A RNA appears to be a co-transcriptional event occurring early on in the RNA lifecycle and is mediated by a multi-protein methyltransferase complex.  The recent discoveries of these methylase “writers” (METTL3/14, WTAP, RBM15/15B, KIAA1429) and their associated demethylase “erasers” (FTO, ALKBH5, TET-like enzymes) in mammals have uncovered the reversibility of the m6A modification, revealing potential therapeutic targets for m6A dysregulation-related diseases.

Epigenetic modifications are also known to influence the life cycles of RNA viruses like human coronavirus.  Modified adenosines like m6A are reported to affect the viability of specific RNA viruses by modulating viral cap structures, viral replication, innate sensing pathways, and the innate immune response.³  Interestingly, members of the coronaviruses encode their own methyltransferases for self-methylating adenosine residues and promoting immune evasion.  Thus, examining the effects of m6A methylase/demethylase activity on the epigenome and epitranscriptome of coronaviruses and other RNA viruses, as well as their targets, may provide valuable insights into the impact of viral and host-derived m6A RNA modifications on infection so as to guide remedial courses of action.

m6A Methylase & Demethylase Activity Assays

EpiGentek offers convenient ELISA-based colorimetric assays to rapidly measure activity levels of m6A methylases and demethylases from cell/tissue nuclear extracts or purified enzymes in a high-throughput format.  A unique m6A substrate is provided with each kit, which stably binds to the assay wells.  Bioactive enzymes from input samples will transfer methyl groups to, or remove them from, the bound substrate, depending on the kit.  Substrate m6A methylation is subsequently detected by a high-affinity antibody specific for this modification.  Quantitative measurements of methylase/demethylase activity and inhibition (if testing enzyme inhibitors) can be obtained within 4-5 hours.  EpiGentek also offers a nuclear extraction kit designed to preserve enzymatic activity of extracted nuclear proteins and optimized for use with these ELISA assays

Kit Measures	Kit Details
Demethylase Activity/Inhibition	Click image to see more details	Epigenase m6A Demethylase Activity/Inhibition Assay Kit (Colorimetric) ELISA-based measurement of the activity/inhibition of total m6A demethylases using nuclear extracts or purified m6A demethylases like FTO and ALKBH5. Fast: Easy-to-follow, 5 hour procedure High Sensitivity: Detection from as low as 2 µg of nuclear extracts or 50 ng of purified enzymes with minimal background levels Convenient: Includes assay standard for specific measurement of m6A demethylase activity Flexible: Strip-well microplate format for manual or high throughput analysis Sizes: 48 & 96 Assays
Methylase Activity/Inhibition	Click image to see more details	Epigenase m6A Methylase Activity/Inhibition Assay Kit (Colorimetric) ELISA-based measurement of the activity/inhibition of total m6A methylases (methyltransferases) using nuclear extracts or purified m6A methylases like METTL3/METTL14. Fast: Easy-to-follow, 4 hour procedure High Sensitivity: Detection from as low as 2 µg of nuclear extracts or 50 ng of purified enzymes with minimal background levels Convenient: Includes assay standard for specific measurement of m6A demethylase activity Flexible: Strip-well microplate format for manual or high throughput analysis Sizes: 48 & 96 Assays

If you would like to order multiple RNA kits, we also make it convenient by allowing you to purchase via a workflow. This way, you can get the assay kits you need while viewing other related kits that may fit your study as well, including nuclear extraction kits.

Have questions about which viral RNA kit is right for your particular needs? Feel free to give us a call at (631) 755-0888 or send us a message through our contact form. One of our representatives will be happy to field any questions and get you the right kits for your current projects.

Also, be sure to check out our complete suite of SARS-CoV-2 Related Antibodies, Proteins & Assays.

References

1. Zaccara, S., Ries, R.J. & Jaffrey, S.R. Reading, writing and erasing mRNA methylation. Nat Rev Mol Cell Biol 20, 608–624 (2019). https://doi.org/10.1038/s41580-019-0168-5
2. Hastings MH. m(6)A mRNA methylation: a new circadian pacesetter. Cell. 2013;155(4):740‐741. doi:10.1016/j.cell.2013.10.028
3. Gonzales-van Horn SR, Sarnow P. Making the Mark: The Role of Adenosine Modifications in the Life Cycle of RNA Viruses. Cell Host Microbe. 2017;21(6):661‐669. doi:10.1016/j.chom.2017.05.008