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Application Note: Detection of WRN Exonuclease Activity with the Transcreener dAMP Assay

WRN Exonuclease Assay - A Transcreener dAMP Assay Application

The WRN Exonuclease Assay uses the Transcreener® dAMP Assay to directly measure dAMP produced during the degradation of DNA. It is a far-red, competitive assay designed for high-throughput screening (HTS), with a single-addition, mix-and-read format. The assay is an excellent tool for researchers screening compound libraries to discover WRN modulators. 

The kit comes complete with the detection reagents required to measure activity. WRN exonuclease 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.

An Overview of the WRN Exonuclease Assay Method

The Transcreener WRN Exonuclease Assay determines enzyme activity by directly measuring dAMP formed by the enzyme using the Transcreener dAMP Exonuclease Assay. This biochemical HTS assay provides a universal method to assess the activity of virtually any exonuclease that produces dAMP.

Transcreener dAMP technology uses polyclonal mouse antibodies that selectively bind to dAMP and a far-red fluorescent tracer. dAMP produced displaces the tracer, changing the fluorescent properties and providing a fluorescent readout.

The WRN exonuclease assay is an easy-to-use, mix-&-read, HTS-ready assay that yields robust results. All you need to do is perform the enzyme reaction, add Transcreener reagents, and read your plates. It is available with an FP fluorescent readout.

Directly Detect dAMP to Measure WRN Exonuclease Activity with an FP Readout

Transcreener WRN Exonuclease Assay Schematic

Applications

  • Measure Enzymatic Activity of WRN Exonuclease
  • Screen Compound Libraries for WRN Exonuclease Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling

Features

  • Direct detection of unlabeled dAMP
  • Easy to use, homogenous, one-step format
  • Robust Assay Z’ > 0.7 under initial velocity conditions
  • Far-red fluorescent readouts minimize compound interference
  • A safe, non-radioactive method
  • Available in a robust FP fluorescent readout

Easy-to-Use, Mix-and-Read, HTS-Ready Assay

Run your enzyme reaction, add Transcreener reagents, and read your plates. The WRN exonuclease assay is compatible with 96, 384, and 1536-well formats. 

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurements using optimized 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' shown here = 0.89.

Robust Assay Z' Data WRN Exonuclease

Detection of dAMP Under WRN Exonuclease Initial Velocity Conditions

The assay demonstrates linearity when raw data is converted to dAMP using a standard curve. We used 1 μM Annealed DNA substrate. The enzyme buffer included 50 mM HEPES (pH 7.5), 5 mM MnCl2, 5 mM MgCl2, and 1 mM DTT. The Detection Mix included 28 μg/mL dAMP antibody, 4 nM dAMP Alexa Fluor 633 tracer, and 1X Stop & Detect Buffer B in 10 μL. The WRN Exonuclease enzyme reaction took place for 60 minutes at room temperature. The Detection Mix was added and incubated for 90 minutes and read with a CLARIOstar plate reader.

WRN Exonuclease Enzyme Titration

Enzyme Titration with WRN Exonuclease Assay

Linear Response

WRN Exonuclease Assay Converted 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 inhibitor potency with ease. Assay conditions include 1 nM WRN exonuclease, 50 mM HEPES (pH 7.5), 5 mM MnCl2, 5 mM MgCl2, 1 mM DTT, and 1 μM Annealed DNA. The Detection Mix consists of 1X Stop & Detect Buffer B, 28 μg/mL dAMP Ab, and 4 nM dAMP tracer.

Dose-Response - Suramin

WRN Exonuclease Assay Dose Response Curve with Suramin

IC50 = 0.11 µM

WRN Exonuclease Assay Services

BellBrook Labs offers WRN exonuclease assay services using our trusted Transcreener dAMP Assay. BellBrook scientists will use their extensive biochemical and enzymology expertise to work with you and accelerate your exonuclease program. We tailor our efforts to meet your specific needs, saving you both time and money. Accelerate your WRN exonuclease program with our drug discovery services. 

Scientist Studying DDX41

Drug 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.
  • Triaging Non-stoichiometric Inhibitors - Run assays under different conditions, such as varying detergent and enzyme concentrations.

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 FP Readouts Validated on Major Multimode Readers

SupplierInstrumentFP Assays
TriStar2S LB 942In Review
Tristar 5In Review
Mithras2 LB 943In Review
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® OmegaNot Capable
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® 3Not Capable
SpectraMax® ParadigmClick Me ArrowValidated
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

Transcreener dAMP Assay Kits - For WRN Exonuclease Activity Assays

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

Related Resources

Interconnections Between the DNA Damage Response and Innate Immunity

There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.

In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:

DDR

  • POLQ
  • WRN
  • PARP1
  • PARG
  • DNA PK

Innate Immunity

  • cGAS
  • TREX1
  • TBK1
  • IKKβ
  • IRAK4
  • ENPP1
  • CD38
  • OAS-1
  • RIG-1/MDA5
  • DDX3
  • CD39
  • CD73
DNA Damage Response and Innate Immunity Preview

WRN As a Therapeutic Target

WRN is one of the 5 evolutionarily conserved RecQ helicases found in humans. All human RecQ helicases hydrolyze ATP, unwind double stranded DNA, and are proficient at repairing DNA breaks. WRN alone possesses an exonuclease function. The helicase and exonuclease domains can act alone or in concert to address a variety of DNA substrates, such as double stranded DNA breaks, DNA duplexes, replication forks, Holliday junctions, and G-quadruplexes.

Deficient or impaired production of WRN produces accelerated senescence and a greatly increased risk of cancer. The WRN exonuclease assay accelerates and simplifies the process to discover WRN modulators. 

Read more about WRN in this article from BellBrook labs: Can WRN Helicase Inhibitors Treat MSI-H Cancers?

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