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Interrogating TREX1 with the Transcreener dAMP Exonuclease Assay

Transcreener dAMP Exonuclease Assay

The Transcreener dAMP Exonuclease Assay directly measures dAMP produced by exonuclease enzymes as they cleave polynucleotides. These dAMP measurements allow researchers to effectively determine the activity of the enzyme. The assay provides a powerful tool to screen compound libraries for exonuclease modulators to help find new therapies for disease. One example is TREX1, a crucial component of the innate immune response where it acts as the main exonuclease responsible for degrading cytosolic DNA.

The kit comes complete with detection reagents required to measure activity. Exonuclease 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.

How does the Transcreener dAMP Exonuclease Assay Work?

The Transcreener dAMP Exonulcease Assay determines exonuclease enzyme activity by directly measuring dAMP (deoxyadenosine monophosphate, deoxyadenylic acid, deoxyadenylate, or 2'-Deoxyadenosine-5'-monophosphate) that can be produced during biological functions like the degradation of DNA. By directly detecting dAMP in a homogenous format, it is possible to perform screening and enzymatic studies with virtually any enzyme that produces dAMP. 

Transcreener dAMP technology uses polyclonal mouse antibodies that selectively bind to dAMP and a far-red fluorescent tracer. As dAMP produced in the reaction competes with the tracer changing the fluorescent properties and providing fluorescent readout.

The Transcreener dAMP Exonuclease Assay is available with an FP fluorescent readout. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagents, and measure. The simplicity of the system yields robust results that also make it extremely amiable to HTS.

Direct Detection dAMP with a Fluorescent FP Readout

Transcreener dAMP Exonuclease Assay

dAMP tracer bound to anti-dAMP antibody is competed off with dAMP produced from an exonuclease resulting in measurable FP signal.

Applications

  • Measure Enzymatic Activity of Exonucleases
  • Screen Compound Libraries for Exonuclease Modulators
  • Quantify Inhibitor Potency
  • Inhibitor Selectivity Profiling
  • Measure Drug-Target Residence Time

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 dAMP assay is compatible with 96, 384, and 1536-well formats.

Transcreener Mix and Read Assay

Robust Assay Yields Quality Data

Z’ measurement using optimized TREX1 assay conditions (n=16). A Z’ of 0.88 demonstrates a robust assay method amenable to HTS.

Sensitive Tunable Assay

dAMP Standard Curve Different Antibody Concentrations

Competition curves indicate displacement of tracer by dAMP and show the dependence of antibody concentration on the dynamic range. Detection limits to 1 nM can be achieved by using 5 µg/mL Ab in the final 20 µL reaction volume.

Obtain Dose-Response Data Fast

Trex1 dose-response curves v3

The Transcreener dAMP Exonuclease Assay allows selectivity profiling for TREX1. Suramin IC50 = 3.0 µM.

Detection of dAMP to Measure TREX1 Activity

The enzyme buffer includes 10 mM Tris pH 7.5, 7.5 mM MgCl2, 0.005% BSA, and 0.01% Brij-35. Here we use 35 nM ISDna. The Detection mix included 20 μg/mL dAMP antibody, 2 nM dAMP Alexa 633 tracer, and 1X Stop & Detect Buffer B in 10 μL. The TREX1 enzyme reaction took place for 60 minutes at 30°C. The Detection Mix was added and incubated for 90 minutes at room temperature and read with a CLARIOstar plate reader. Linearity is shown here when raw data is converted to dAMP formed using a standard curve.

TREX1 Enzyme Titration

TREX1 Assay Enzyme Titration

Linear Response

TREX1 Assay Enzyme Titration Product Formed

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 Exonuclease Assay Kits

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

Exonuclease 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 Exonuclease 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

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Exonucleases As Drug Targets

One current model exonuclease target is Three Prime Repair Exonuclease 1 (TREX1) a crucial component of the innate immune response acting as the main exonuclease responsible for degrading cytosolic DNA. Reduction of cytosolic DNA leads to inactivation of the cGAS/STING Pathway, which is essential for intrinsic antitumor immunity. As a result, TREX1 plays a pro-tumorigenic role in cancer, and its inactivation is a promising strategy for future monotherapy and combination treatments.

The Discovery of TREX1 antagonists to reign in overactivity requires selective technologies that can help assess the ability to interact with the target. Robust, HTS-ready assays that are accurate yet flexible provide a powerful tool to accelerate discovery. 

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