The METTL3/METTL14 Assay directly detects SAH via a naturally occurring RNA Aptamer, or “riboswitch”, coupled with a TR-FRET detection format suitable for automated HTS (high-throughput screening) applications. SAH produced by the complex drives the assembly of the split aptamer allowing FRET between a Terbium chelate donor and DyLight 650 acceptor.
Current non-radioactive SAH detection methods are not sufficiently sensitive to allow detection of many histone methyltransferases (HMTs) using physiological concentrations of SAM. The exquisite affinity and selectivity of the AptaFluor riboswitch overcomes this technical gap, providing screening and profiling of virtually any methyltransferase enzyme with unparalleled sensitivity.
The METTL3/METTL14 assay is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.
This application note describes methods for using the AptaFluor SAH Methyltransferase Assay to measure METTL3/METTl14 enzymatic activity in a high-throughput (384-well) format, e.g., for inhibitor screening and/or inhibitor dose-response measurements.
Run your enzyme reaction, add quenching reagent, add AptaFluor reagents, and read your plates. The METTL3/METTL14 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized METTL3/METTL14 reaction conditions indicate a robust assay. Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z’ = 0.775.
The assay demonstrates linearity when raw data is converted to SAH using a standard curve. Here, we used 1 μM RNA substrate and 0.25 μM SAM. The enzyme buffer included 20 mM Tris-HCl (pH 7.5), 0.5 mM MgCl2, 0.01% BSA, 0.01% Tween-20, 0.02 U/μL RNAseOUT, and 1 mM DTT. The Enzyme Stop Mix included 1X Enzyme Stop Reagent and 1X SAH Detection Buffer. The SAH Detection Mix consisted of 20 nM P1-Terbium Mix, 40 nM P2-Dylight 650 nM, and 1X SAH Detection Buffer. The METTL3/14 enzyme reaction took place for 120 minutes at 30°C. The Detection Mix was added and incubated for 180 minutes at room temperature and read with a CLARIOstar plate reader.
Interested in moving your program forward, but don’t want to bring an assay in-house? Our scientists can help! BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your METTL3/METTL14 program.
Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.
| Component | Notes |
|---|---|
| SAM | SAM supplied in the kit should be used for MT enzyme reactions and to create a SAM/SAH standard curve. |
| SAH | SAH is used to create a SAM/SAH standard curve. |
| Enzyme Stop Reagent, 10X | Enzyme Stop Reagent is diluted to 1X in SAH Detection Buffer and used to quench the activity of MT enzyme components. |
| SAH Detection Buffer, 10X | This buffer is used to prepare the SAH Detection Mix and the Enzyme Stop Mix. |
| P1-Biotin | P1-Biotin is a 55-base RNA oligomer with a biotin molecule at the 5' end. It is incubated with TB-Streptavidin to produce P1-Terbium, the donor piece of the SAH aptamer. |
| P2-Dylight 650 | P2-Dylight 650 is an 18-base RNA oligomer with a Dylight 650 molecule at the 3' end. It is the acceptor piece of the SAH aptamer. |
| Tb-Streptavidin | Tb-Streptavidin is used to produce the Tb-labeled (donor) piece of the SAH aptamer. |
| Conjugation Buffer | This buffer is used for the P1-Biotin/Tb-Streptavidin conjugation reaction. |
| Component | Notes |
|---|---|
| METTL3/METTL14 Enzyme | We have successfully used recombinant METTL3/METTL14 methyltransferase enzyme from Active Motif #31570. Here, we used a concentration of 0.25 μM. |
| RNA Substrate | ssRNA Oligomer (5'-GUUGCCUGUUCGUGUUGGACUUGCCUGU-3) |
| Enzyme Buffer | 20 mM Tris-HCl (pH 7.5), 0.5 mM MgCl2, 0.01% BSA, 0.01% Tween-20, 0.02 U/μL RNAseOUT, and 1 mM DTT. Buffer components may be optimized as required. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to read TR-FRET is required for the assay. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products and RNA aptamers. Careful handling and use of ultrapure water eliminates this potential problem. |
| Laboratory Incubator | An incubator model that can maintain temperature stability at 30°C is required. |
METTL3 serves as the catalytic subunit for the methylation of mRNA resulting in N6-Methyladenosine (m6A); it plays a critical role in the modification of RNA thus impacting RNA metabolism and function. METTL14 aids METTL3 by allowing for substrate recognition through a positively charged groove within the interface of the heterodimer.
Mutations in METTL3/METTL14 correlate to many cancers, including pancreatic, colorectal, liver, acute myeloid leukemia, gastric, lung bladder, and more (Zeng et al, 2020). The search for effective METTL3/METTL14 inhibitors show great promise for cancer therapeutics. The METTL3/METTL14 assay provides an easy-to-use and robust method to screen for inhibitors in a high-throughput format.
Find the right resources to help you get to your solution faster.
Please fill out the form below. We will respond quickly to get the conversation moving and learn how we can help. We keep things discrete, confidential, and professional.
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
