PRMT5 Assay – AptaFluor Application

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

PRMT5 Assay - An AptaFluor SAH Assay Application

The AptaFluor Methyltransferase Assay measures PRMT5 (protein arginine methyltransferase 5) activity via direct detection of SAH produced during methylation. The AptaFluor Assay is a high-throughput enzymatic assay for SAM-dependent methyltransferases that uses a naturally occurring SAH-sensing RNA aptamer, or riboswitch, that binds SAH with nanomolar affinity and exquisite selectivity. 

The kit comes complete with the detection reagents required to measure activity. PRMT5 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 Protein Arginine Methyltransferae 5 Activity with the AptaFluor SAH Methyltransferase Assay

This application note describes methods for using the AptaFluor SAH Methyltransferase Assay to measure PRMT5 enzymatic activity in a high-throughput (384-well) format, e.g., for inhibitor screening and/or dose-response measurements.

Application Note Preview Image

How Does This PRMT5 Assay Work?

The PRMT5 Assay determines PRMT5 activity by directly measuring SAH produced during the methylation of SAM. The modified riboswitch that selectively binds with SAH discriminates between SAH and related nucleotides, including SAM (which differs by a methyl group). This produces unambiguous detection of SAH in the presence of excess SAM.

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 PRMT5 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. It is available with a TR-FRET readout.

Direct Detection of SAH to Measure PRMT5 Enzymatic Activity

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

PRMT5 Assay Schematic

The Most Sensitive HTS Methyltransferase Assay

The AptaFluor Assay is an ultra-sensitive assay that allows a LLD of 0.6 nM SAH. This drastically reduces enzyme usage and allows the assay to be run at or below KM for SAM.

Ulta Sensitive Methyltransferase Assay
Data Table Demonstrating AptaFluor Sensitivity

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

Run your enzyme reaction, add quenching reagent, add AptaFluor reagents, and read your plates. The PRMT5 assay is compatible with 96, 384, and 1536-well formats.

AptaFluor Methyltransferase Assay Protocol Outline

Applications

  • Measure enzymatic activity of PRMT5
  • Screen Compound Libraries for PRMT5 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

Robust Assay Yields Quality Data

Excellent Z' under a variety of assay 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' shown here = 0.88.

Z' Data with PRMT5

Detection of SAH Under PRMT5 Initial Velocity Conditions

The assay demonstrates linearity when raw data is converted to SAH using a standard curve. Here, we used 5 μM Histone 2A and 5 μM SAM. The assay buffer included 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, and 0.01% Triton X-100 (pH 8.0). The Enzyme Stop Mix included 1X Enyzme Stop Reagent and 1X SAH Detection Buffer. The SAH Detection Mix included 20 nM P1-Terbium Mix, 40 nM P2-Dylight 650 nM, and 1X SAH Detection Buffer. The enzyme reaction took place for 90 minutes at 30°C. The Detection Mix was added and incubated for 180 minutes at room temperature and read with a Pherastar Plus/FS plate reader.

PRMT5 Enzyme Titration

Enzyme Titration with PRMT5 Assay

Linear Response

PRMT5 Linear Response Data

Screen for Inhibitors & Perform SAR

Transcreener Assays are 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. Assay conditions include 10 nM PRMT5, 5 μM Histone H2A, 5 μM SAM, 20 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.01% Triton X-100 (pH 8.0).

PRMT5 Asay Dose Response Curve with IC50 Values

PRMT5 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 PRMT5 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® i3xIn Review
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 PRMT5 Activity Assays

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

Cat. No.Product DescriptionPriceAdd to Cart
3023-BAptaFluor SAH Methyltransferase Assay (100 Assay, 96 Well)$399.00Add to Cart
3023-1KAptaFluor SAH Methyltransferase Assay (1,000 Assay, 384 Well)$1,299.00Add to Cart
3023-10KAptaFluor SAH Methyltransferase Assay (10,000 Assay, 384 Well)$7,399.00Add to Cart

Assay Plates for TR-FRET

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

The Role of PRMT5 As a Therapeutic Target

PRMT5, a type II arginine methyltransferase, dimethylates arginine residues symmetrically. Its functions extend into T cell development, B cell regulation, hematopoiesis, and mitotic control. Recently, it has also been implicated in aspects of innate immunity via both its traditional role and inhibition of viral replication by a non-arginine methyltransferase modality. 

PRMT5 acts to suppress the cGAS/STING pathway in the cytoplasm via dimethylation of the cytosolic cGAS Arg124 residue, blocking its cytosolic DNA binding abilities. In macrophages, PRMT5 has been shown to associate with nuclear Carbonic Anhydrase 6B (CA VI-B) to upregulate the expression of the pro-inflammatory cytokine IL-12 in an innate response to intracellular bacterial infection. PRMT5 is shown to have a profound antiviral effect on Hepatitis B (HBV) infection.

Read more about PRMT5 as a therapeutic target in this article: PRMT5 is Innately Interesting

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