Poster: Case Study for PRMT5, MLL4, METTL3/14, and NSP14 Using the AptaFluor® Assay

METTL3/METTL14 Assay - An AptaFluor SAH Assay Application

The AptaFluor Methyltransferase Assay measures METTL3/METTL14 (methyltransferase-like 3/methyltransferase-like 14) activity via direct detection of SAH produced during methylation of SAM. The AptaFluor Assay relies on a  SAH-sensing RNA Aptamer that provides the ultra-sensitivity required to measure METTL3/METTL14 activity at physiological KM concentrations.

The kit comes complete with the detection reagents required to measure activity. METTL3/METTL14 enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme. This assay is designed to be used with purified enzyme preparations. AptaFluor assays are not validated with cell lysates, blood, serum, or other biological samples.

Application Note - Detection of Methyltransferase-like 3 Activity with the AptaFluor SAH Methyltransferase Assay

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. 

Application Note Preview Image

How Does This METTL3/METTL14 Assay Work?

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. 

Direct Detection of SAH to Measure METTL3/METTL14 Enzymatic Activity

Time-Resolved Förster Resonance Energy Transfer (TR-FRET)

Schematic of METTL3-METTL14 Assay


  • Measure enzymatic activity of METTL3/METTL14
  • Screen Compound Libraries for METTL3/METTL14 Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling


  • Use SAM concentrations as low as 100 nM and as high as 5 μM
  • Direct detection of SAH
  • Simple, mix-and-read format ideal for HTS.
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method

Easy-to-Use, Mix-&-Read, HTS-Ready

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.

AptaFluor Methyltransferase Assay Protocol Outline

Robust Assay Amenable to HTS

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.

Z' Data for METTL3-METTL14

Detection of SAH Under METTL3/METTL14 Initial Velocity Conditions

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. 

METLL3/METTL14 Enzyme Titration

METTL3-METTL14 Assay Enzyme Titration Data

Linear Response

Linear Response for METTL3-METTL14 Activity

Screen for Inhibitors & Perform SAR

The AptaFluor Assay is designed for screening compound libraries in a high throughput format. Follow-up SAR can also be performed using the assay to determine the inhibitor potency with ease. Here, we used inhibitors UZH2 and STM257.

Dose Response with UZH2

Dose Response Curve with UZH2 using METTL3-METTL14 Assay

IC50 = 0.01587 μM

Dose Response with STM2457

STM2457 DRC with METTL3-METTL14 Assay

IC50 = 0.01715 μM

METTL3/METTL14 Assay Services

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.

Assay Development Services

Lead Discovery Services Include:

  • Inhibitor Screening – To identify or confirm activity with the target.
  • Inhibitor Potency Profiling – Dose-response with target and/or related proteins. Fast IC50 results.
  • Residence Time Measurements – Determination of koff using ‘jump dilution’ enzymatic assay method.
  • Mechanism of Action Studies – Kinetic analysis to define the mode of inhibition.

Contact Us to Learn More

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.

Far-Red TR-FRET Readouts Validated on Major Multimode Readers

SupplierInstrumentTR-FRET Assays
TriStar2S LB 942Click Me ArrowValidated
Tristar 5In Review
Mithras2 LB 943Click Me ArrowValidated
CytationTM 5Validated
CytationTM 3Validated
CytationTM 1Validated
SynergyTM H1Validated
SynergyTM 2/H4/4Click Me ArrowValidated
SynergyTM HTXNot Capable
SynergyTM Neo 2Validated
POLARstar® OmegaClick Me ArrowValidated
FLUOstar® OmegaClick Me ArrowValidated
PHERAstar® FSXClick Me ArrowValidated
PHERAstar® Plus/FSClick Me ArrowValidated
CLARIOstar® /PlusClick Me ArrowValidated
VANTAstarTMClick Me ArrowValidated
SenseIn Review
Analyst® GT/HTClick Me ArrowValidated
Gemini® XPS/EMNot Capable
SpectraMax® M2/M2eNot Capable
SpectraMax® M5/M5e/FlexStation® 3Click Me ArrowValidated
SpectraMax® ParadigmClick Me ArrowContact Us
SpectraMax® iD3/iD5Click Me ArrowValidated
SpectraMax® i3xClick Me ArrowValidated
EnVision®/EnVision® XciteClick Me ArrowValidated
Infinite® M1000/M1000Pro/Safire2TMClick Me ArrowValidated
Infinite® M200Not Capable
Infinite® F500Click Me ArrowValidated
Infinite® F200/UltraevolutionClick Me ArrowContact Us
SparkTM 10MClick Me ArrowValidated

What's Included

What You Will Need

AptaFluor SAH Methyltransferase Assay Kits - For METTL3/METTL14 Activity Assays

* For custom or bulk orders (over 10,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

The Role of METTL3/METTL14 As a Therapeutic Target

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.