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

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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 K_M 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. Transcreener 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.

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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)

Learn More About The AptaFluor Assay

AptaFluor SAH TR-FRET Assay Tech Manual

Applications

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

Features

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.

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

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 STM257

Dose Response Curve with STM2457 using METTL3-METTL14 Assay

IC₅₀ = 0.01715 μM

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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.

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 k_off 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.

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

Supplier	Instrument	TR-FRET Assays
	TriStar²S LB 942	Validated
	Tristar 5	In Review
	Mithras² LB 943	Validated

	Cytation^TM 5	Validated
	Cytation^TM 3	Validated
	Cytation^TM 1	Validated
	Synergy^TM H1	Validated
	Synergy^TM 2/H4/4	Validated
	Synergy^TM HTX	Not Capable
	Synergy^TM Neo 2	Validated

	POLARstar^® Omega	Validated
	FLUOstar^® Omega	Validated
	PHERAstar^® FSX	Validated
	PHERAstar^® Plus/FS	Validated
	CLARIOstar^® /Plus	Validated
	VANTAstar^TM	Validated

	Sense	In Review

	Analyst^® GT/HT	Validated
	Gemini^® XPS/EM	Not Capable
	SpectraMax^® M2/M2e	Not Capable
	SpectraMax^® M5/M5e/FlexStation^® 3	Validated
	SpectraMax^® Paradigm	Contact Us
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	In Review

	EnVision^®/EnVision^® Xcite	Validated

	Infinite^® M1000/M1000Pro/Safire2^TM	Validated
	Infinite^® M200	Not Capable
	Infinite^® F500	Validated
	Infinite^® F200/Ultra^evolution	Contact Us
	Spark^TM 10M	Validated

What's Included


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.

What You Will Need


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.

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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.

Cat. No.	Product Description	Price	Add to Cart
3023-B	AptaFluor SAH Methyltransferase Assay (100 Assay, 96 Well)	$399.00	Add to Cart
3023-1K	AptaFluor SAH Methyltransferase Assay (1,000 Assay, 384 Well)	$1,299.00	Add to Cart
3023-10K	AptaFluor SAH Methyltransferase Assay (10,000 Assay, 384 Well)	$7,399.00	Add to Cart

Assay Plates for TR-FRET

Cat. No.	Product Description	Price	Add to Cart
2234	Corning 96-Well White Assay Plates (#3642), 3-Pack (200+ Assays)	$55.00	Add to Cart
2231	Corning 384-Well White Assay Plates (#4513), 3-Pack (1,000+ Assays)	$65.00	Add to Cart
2232	Corning 384-Well White Assay Plates (#4513), 30-Pack (10,000+ Assays)	$529.00	Add to Cart

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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.

Detection of METTL3/METTL14 Activity with the AptaFluor SAH Methyltransferase Assay

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Interpolating Data from a Standard Curve to Generate a Dose-Response Using GraphPad Prism

Preview of Data Analysis Application Note

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METTL3/METTL14 Assay – Application

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

Ordering Information

Assay Services

Related Resources

METTL3/METTL14 Assay - An AptaFluor SAH Assay Application

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

How Does This METTL3/METTL14 Assay Work?

Direct Detection of SAH to Measure METTL3/METTL14 Enzymatic Activity

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

Applications

Features

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

Robust Assay Amenable to HTS

Detection of SAH Under METTL3/METTL14 Initial Velocity Conditions

METLL3/METTL14 Enzyme Titration

Linear Response

Screen for Inhibitors & Perform SAR

Dose Response with UZH2

IC50 = 0.01587 μM

Dose Response with STM257

IC50 = 0.01715 μM

METTL3/METTL14 Assay Services

Lead Discovery Services Include:

Contact Us to Learn More

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

What's Included

What You Will Need

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

Assay Plates for TR-FRET

The Role of METTL3/METTL14 As a Therapeutic Target

Related Resources

Detection of METTL3/METTL14 Activity with the AptaFluor SAH Methyltransferase Assay

Interpolating Data from a Standard Curve to Generate a Dose-Response Using GraphPad Prism

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

IC₅₀ = 0.01587 μM

IC₅₀ = 0.01715 μM