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.

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 assay demonstrates linearity when raw data is converted to dAMP using a standard curve. Here we use 37 nM ISDna and 350 nM TREX1.  Linearity is shown here when raw data is converted to dAMP formed. The enzyme buffer includes 100 mM Tris pH 7.5, 10 mM MgCl2, and 0.01% Brij. 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 room temperature. The Detection Mix was added and incubated for 90 minutes at room temperature and read with a CLARIOstar plate reader.

TREX1 Enzyme Titration

TREX1 Enzyme Titration with Transcreener dAMP Exonuclease Assay

Linear Response

TREX1 Enzyme Titration with Transcreener dAMP Exonuclease Assay Linear Response

Far Red FP Readouts Validated on Major Multimode Readers

Supplier Instrument FP Assays
bioteklogo TriStar²S LB 942 in review
Mithras² LB 943 in review
bioteklogo Cytation™ 5 validated
Cytation™ 3 validated
Cytation™ 1 validated
Synergy™ H1 validated
Synergy™ 2/H4/4 validated
Synergy™ HTX not capable
Synergy™ Neo 2 validated
BMGLABTECH Logo POLARstar® Omega validated
FLUOstar® Omega not capable
PHERAstar® FSX validated
PHERAstar® Plus/FS validated
CLARIOstar® /Plus validated
VANTAstar validated
MDS AT logo Analyst® GT/HT validated
Gemini® XPS/EM not capable
SpectraMax® M2/M2e not capable
SpectraMax® M5/M5e/FlexStation® 3 not capable
SpectraMax® Paradigm validated
perkinElmerLogo EnVision®/EnVision® Xcite validated
tecanLogo Infinite® M1000/M1000Pro/Safire2™ validated
Infinite® M200 not capable
Infinite® F500 validated
Infinite® F200/Ultraevolution contact us
Spark™ 10M validated

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

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.

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. 

TOP