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Updated: Jan 6, 2026
Antibody-Free Assay for RNA Methyltransferase Activity Analysis
Published on: July 9, 2019
A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity
Shane M Buker1, Zachary A Gurard-Levin2, Benjamin D Wheeler3,4
1Accent Therapeutics, Lexington, MA, USA.
Abstract:
A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (m6A) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, m6A can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I S-adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC50 values of mechanism-based inhibitors S-adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.
Insights
Researchers developed a new mass spectrometry assay for METTL3/METTL14, crucial for messenger RNA (mRNA) methylation. This assay enables high-throughput screening for inhibitors of this important RNA modification enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- RNA modifications, like N6-methyladenosine (m6A), regulate gene expression.
- METTL3/METTL14 complex catalyzes m6A methylation in mRNA, impacting various RNA processes.
- No specific inhibitors for METTL3/METTL14 have been reported, hindering drug discovery.
Purpose of the Study:
- To develop a sensitive and robust assay for METTL3/METTL14 activity.
- To facilitate high-throughput screening for potential METTL3/METTL14 inhibitors.
- To enable compound characterization for RNA-modifying enzymes.
Main Methods:
- Development of a mass spectrometry-based assay utilizing self-assembled monolayer desorption/ionization (SAMDI) technology.
- Utilized an 11-nucleotide single-stranded RNA substrate for the assay.
- Compared SAMDI assay results with radiometric assays using known inhibitors like S-adenosylhomocysteine (SAH) and sinefungin (SFG).
Main Results:
- A sensitive and robust SAMDI assay for METTL3/METTL14 was successfully established.
- The assay demonstrated comparable IC50 values for inhibitors to traditional radiometric methods.
- SAMDI technology proved amenable to RNA substrates, validating its utility.
Conclusions:
- The developed SAMDI assay is suitable for high-throughput screening of METTL3/METTL14 inhibitors.
- This assay provides a valuable tool for drug discovery targeting RNA methylation.
- SAMDI technology offers a versatile platform for characterizing RNA-modifying enzymes.
