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

NSP14 Assay - An AptaFluor SAH Assay Application

AptaFluor SAH Assay directly measures SAH produced by NSP14 (Nonstructural Protein 14) in an HTS format. The assay relies on a naturally occurring SAH-sensing RNA aptamer coupled with TR-FRET detection. SAH produced drives assembly of the split aptamer allowing for FRET signal between a Terbium chelate donor and DyLight 650 acceptor. 

Current non-radioactive SAH detection methods are not sufficiently sensitive to allow detection of many methyltransferases using physiological concentrations of SAM. The AptaFluor Assay overcomes technical gaps of current detection methods that detect specific methylation events or are not sensitive enough for enzymes with low turnovers and/or high SAM affinities.

The kit comes complete with the detection reagents required to measure activity. NSP14 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 - Nonstructural Protein 14 Activity with the AptaFluor SAH Methyltransferase Assay

This application note describes methods for using the AptaFluor SAH Methyltransferase Assay to measure NSP14 enzymatic activity in a high-throughput (384-well) format, e.g., for inhibitor screening and/or dose-response measurements.

Application Note Preview Image

Direct Detection of SAH to Meaure NSP14 Activity

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

NSP14 Assay Schematic

An Ultra-Sensitive Methyltransferase Assay

The AptaFluor Assay is the most sensitive HTS methyltransferase assay, allowing 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 Format

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

AptaFluor Methyltransferase Assay Protocol Outline

Applications

  • Measure enzymatic activity of NSP14
  • Screen Compound Libraries for NSP14 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 Perfect for HTS

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 = 0.80.

Z' Data for NSP14

NSP14 Enzyme Titration & Linear Response Under Initial Velocity Conditions

The assay demonstrates linearity when raw data is converted to SAH using a standard curve. Here, we used 50 μM RNA Substrate and 100 nM SAM. The assay buffer included 20 mM Tris-HCl (pH 8.0), 3 mM MgCl2, 0.01% BSA, 1 mM EDTA and 1 mM DTT. 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 37°C. The Detection Mix was added and incubated for 180 minutes at room temperature and read with a Pherastar Plus/FS plate reader.

NSP14 Enzyme Titration

Enzyme Titration with NSP14 Assay

Linear Response

NSP14 Linear Response Data

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.

Sinefungin Dose Response Measurements with NSP14 Assay

IC50 = 0.028 μM

NSP14 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 NSP14 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.
  • Evaluate Compound-Target Binding – Run thermal shift assays to confirm compound-target engagement via shifts in melting temperature.

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 NSP14 Activity Assays

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

Related Resources

Poster: A Homogenous, Universal Assay for Histone, RNA, & DNA Methyltransferases. Case Study for NSP14, MLL4, PRMT5, and METTL3/14

The AptaFluor SAH Methyltransferase Assay is a high-throughput enzymatic assay for SAM-dependent methyltransferases. The assay relies on a naturally occurring SAH-sensing RNA aptamer, or riboswitch, that binds SAH with nanomolar affinity and exquisite selectivity. Upon binding to as low as 1 nM SAH, these split-aptamer sensors generate positive TR-FRET signals. As a result, the assay overcomes technical gaps of current detection methods that detect specific methylation events or are not sensitive enough for enzymes with low turnovers and/or high SAM affinities.

Here, we demonstrate how the AptaFluor Assay provides a reliable and robust tool for detecting activity of four methyltransferases, the sensitivity, selectivity, & stability of the assay, robust detection of SAH (Z' > 0.7) at nM concentration of enzyme, and profiling dose response curves for known inhibitors. Selected enzymes are representative of typical low Km (100 nM to 5 µM) and have attracted interest as therapeutic targets.

AptaFluor Poster Blurred Preview

What is the Role of NSP14?

NSP14 is a bifunctional protein containing two functional domains – an ExoN domain and N7-MTase domain. NSP14's ExoN domain allows SARS-CoV virus to proofread by catalyzing nucleoside monophosphate excision from nucleic acids, which is critical for high-fidelity replication. NSP14's N7-MTase domain is involved in viral mRNA capping, which hides the virus from the immune system.

The NSP14 assay provides an excellent tool to screen compound libraries for NSP14 modulators.

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