Interrogating TREX1 with the Transcreener dAMP Exonuclease Assay

Exonuclease Assay Methods

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Exonuclease Assay Services

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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

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

Learn More About the dAMP Assay in this Poster

Transcreener dAMP Exonuclease Assay Tech Manual

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.

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

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

Linear Response

TREX1 Assay Enzyme Titration Product Formed

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Far Red FP Readouts Validated on Major Multimode Readers

Supplier	Instrument	FP Assays
	TriStar²S LB 942	In Review
	Tristar 5	In Review
	Mithras² LB 943	In Review

	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	Not Capable
	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	Not Capable
	SpectraMax^® Paradigm	Validated
	SpectraMax^® iD3/iD5	Validated
	SpectraMax^® i3x	Validated

	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
dAMP Alexa Tracer, 400 nM	AlexaFluor^® 633 labeled tracer in 2 mM HEPES (pH 7.5), 0.01% Brij-35. When displaced, changes in fluorescence can be read in an FP format.
dAMP Antibody	Antibody specific to the nucleotide dAMP. 3.5 mg/mL mouse polycolonal antibody in PBS, pH 7.2 with 5% Glycerol.
Stop & Detect Buffer B, 10X	The Stop & Detect Buffer B components will stop enzyme reactions that require Mg2+. To ensure that the enzyme reaction is stopped completely, confirm that the EDTA concentration is at least equimolar to the magnesium ion concentration in the reaction. The final concentration of Stop & Detect Buffer B at the time of FP measurement is 0.5X.
dAMP	The dAMP in this kit can be used to generate a standard curve which can be used to convert mP values to dAMP product formed.

What You Will Need


Component	Notes
Exonuclease Enzyme(s)	Transcreener Assays are designed for use with purified enzyme preparations. Contaminating enzymes can produce background signal and reduce the assay window.
Enzyme Buffer Components	User-supplied enzyme buffer components include enzyme, buffer, acceptor substrate, MgCl₂ or MnCl₂, EGTA, Brij-35, and test compounds.
Assay Plates	Assay Plates Can Be Purchased Separately From BellBrook Labs. FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514
Plate Reader	A microplate reader configured to measure FP is required for the assay.
Liquid Handling Devices	Use liquid handling devices that can accurately dispense a minimum volume of 0.5 µL into 384-well plates.
Ultrapure Water	Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates, RNA aptamers, and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem.

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Transcreener dAMP Exonuclease Assay Kits

* 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
3028-1K	Transcreener dAMP Exonuclease FP Assay (1,000 Assay, 384 Well)	$725.00	Add to Cart
3028-10K	Transcreener dAMP Exonuclease FP Assay (10,000 Assay, 384 Well)	$4,199.00	Add to Cart
3028-100K	Transcreener dAMP Exonuclease FP Assay (10 X 10,000 Assay, 384 Well)	$20,995.00	Add to Cart

Assay Plates for FP

Cat. No.	Product Description	Price	Add to Cart
2233	Corning 96-Well Black Assay Plates (#3686), 3-Pack (200+ Assays)	$55.00	Add to Cart
2229	Corning 384-Well Black Assay Plates (#4514), 3-Pack (1,000+ Assays)	$60.00	Add to Cart
2230	Corning 384-Well Black Assay Plates (#4514), 30-Pack (10,000+ Assays)	$479.00	Add to Cart

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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.

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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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.

Transcreener dAMP Exonuclease Assay Kit

Interrogating TREX1 with the Transcreener dAMP Exonuclease Assay

Exonuclease Assay Methods

Ordering Information

Exonuclease Assay Services

Related Resources

Transcreener dAMP Exonuclease Assay

How does the Transcreener dAMP Exonuclease Assay Work?

Direct Detection dAMP with a Fluorescent FP Readout

Applications

Features

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

Robust Assay Yields Quality Data

Sensitive Tunable Assay

Obtain Dose-Response Data Fast

Detection of dAMP to Measure TREX1 Activity

TREX1 Enzyme Titration

Linear Response

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Far Red FP Readouts Validated on Major Multimode Readers

What's Included

What You Will Need

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Transcreener dAMP Exonuclease Assay Kits

Assay Plates for FP

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Exonuclease Assay Services

Lead Discovery Services Include:

Contact Us to Learn More

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Exonucleases As Drug Targets

Related Resources

TREX1 Inhibitor Assays May Aid the Discovery Novel HIV Treatments

Fighting Infection’s Ghost

PRMT5 is Innately Interesting

Targeting ENPP1 for Cancer Immunotherapy: Development of an HTS Method Using the Transcreener® AMP2/GMP2 Assay

Posters and Presentations

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