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Explore RIG-I in the Innate Immune Response

Enzolution™ RIG-I ATPase Assay System

The Enzolution RIG-I ATPase Assay System includes RIG-I (Retinoic acid-inducible gene I) enzyme, substrate, assay plates, and buffers required for a RIG-I enzymatic reaction. The Transcreener ADP2 Assay (sold separately), a far-red competitive assay designed for HTS and inhibitor dose-response measurements, measures ADP produced to determine activity. Used together, the Enzolution RIG-I Assay System and Transcreener ADP2 Assay provide all reagents required to screen and profile RIG-I inhibitors. 

The Enzolution Assay System is a valuable tool to advance drug discovery efforts in innate immune system pathways. RIG-I is a critical sensor of RNA viral infections, recognizing double-stranded RNA, 4-triphosphylated RNAs, and other pathogen-associated molecular patterns (PAMPs) that indicate the presence of a virus. It then induces a Type I IFN response via the NFkB and IRF3 pathways. 

The Transcreener ADP2 Assay is available for separate purchase through BellBrook Labs. For ADP assay ordering information, please view Transcreener ADP2 Assay. The Transcreener ADP2 Assay is available with an FP, FI, or TR-FRET configuration. When purchasing an Enzolution Assay System, it is important to choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP2 Assay Kit.

The All-in-One Assay Format to Determine RIG-I Activity

Enzolution Assay System includes enzyme, substrate, & buffers for the RIG-I reaction. Transcreener's ADP2 Assay determines resulting ADP activity.

How Does The Procedure Work?

The RIG-I reaction is initiated by the addition of a dsRNA/ATP substrate mixture. After the enzyme reaction is complete, ADP detection reagents (Transcreener ADP2 Antibody & Tracer) and EDTA are added. Read the plate with a compatible microplate reader. 

RIG-I Assay Procedure

Excellent Z' Data Amenable to HTS

The Enzolution RIG-I Assay System used with the Transcreener ADP assay produces robust results amenable to HTS. Z' measurement using optimized RIG-I assay conditions (n=16). Z' = 0.85.

Z' Data for RIG-I ATPase

Measure ADP to Determine RIG-I Activity Under Initial Velocity Assay Conditions

Raw polarization signal (mP) is converted to ADP formed using a standard curve. Conversion of raw data shows assay linearity. Here, we use 100 µM ATP with 16.2 nM dsRNA substrate. The enzyme buffer included 50 mM Tris, 2.5 mM MgCl2, 50 mM NaCl, 0.01% Brij-35, and 5 mM DTT. The enzyme reaction took place for 60 minutes at 30°C. The ADP Detection Mix included 1X Stop & Detect Buffer B, 4 nM ADP2 Alexa Fluor 633 Tracer, and 82 µg/mL ADP antibody. The Detection Mix was added and incubated for 60 minutes at room temperature.

Enzyme Titration - FP Readout

RIG-I Assay Titration with FP Readout

Linear Response

RIG-I Linear Response Curve

Screen and Profile RIG-I Inhibitors for Small Molecule Drug Discovery

Transcreener Assays are designed for screening compound libraries and obtaining dose responses in a high throughput format. Follow-up SAR can be performed using the assay to determine inhibitor potency with ease. Assay conditions included 100 μM ATP, 16.2 nM 19-bp-dsRNA oligomer, 50 mM Tris, 2.5 mM MgCl2, 50 mM NaCl, 0.01% Brij-35, 5 mM DTT, 1X Stop & Detect Buffer B, 4 nM ADP2 AlexaFluor 633 Tracer, and 82 μg/mL ADP2 Antibody.

Dose Response Curve

RIG-I DRC with Suramin and BVT 948

IC50 (BVT 948) = 3.56 μM

IC50 (Suramin) = 6.20 μM

Three Fluorescent Detection Mode Options

Choose the readout configuration that is the best fit for your lab based on preference and plate reader compatibility. 

Fluorescent Intensity

RIG-I Assay Titration with FI Detection

FI Detection Mix (10 μL): 1X Stop & Detect Buffer B, 93.7 μg/mL ADP2 Antibody-IRDye QC-1, 8 nM ADP2 AlexaFluor 594 Tracer.

TR-FRET

RIG-I Assay Titration with TR-FRET Detection

TR-FRET Detection Mix (10 μL): 1X Stop & Detect Buffer C, 8 nM ADP2 Antibody-Terbium Conjugate, and 197.8 nM ADP HiLyte647 Tracer.

Overview of the Transcreener ADP Assay (Sold Separately)

ADP Assay Schematic

The Transcreener® ADP2 Assay is a far-red, competitive assay that measures ADP production to determine enzymatic activity. The technology uses a simple but highly effective method that consists of an antibody selective to ADP over ATP and a far-red fluorescent tracer. ADP produced in the reaction competes with the tracer, changing the fluorescent properties and providing a fluorescent readout.

The Transcreener® assay is designed specifically for high-throughput screening (HTS), with a single addition, mix-and-read format. It offers reagent stability and compatibility with commonly used multimode plate readers. The assay is available as an FP, FI, or TR-FRET configuration.

Recombinant Full-Length RIG-I Enzyme

Active, human Retinoic acid-inducible gene I enzyme (AA 1-925), full-length, C-terminal 6xHis-tagged protein expressed and purified from insect cells. The enzyme has been thoroughly validated with the Transcreener ADP Assay Kit. For specific activity, please refer to the Certificate of Analysis for each individual enzyme lot. Purity is >90% in a composition of 50 mM Tris, 500 mM NaCl, and 10% Glycerol (pH 8.0). The RIG-I protein is currently used with the Transcreener assay at a concentration of 1.31 nM in a 10 μL reaction.

RIG-I Enzyme Graphic

RIG-I ATPase Profilling Services

Interested in moving your program forward with BellBrook's RIG-I assay services? BellBrook scientists will use their extensive biochemistry and enzymology expertise to work with you and accelerate your RIG-I discovery program.

Scientist Studying DDX41

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

Contact Us to Learn More

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What's Included

What You Will Need

Enzolution RIG-I ATPase Assay System

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

Please choose the assay system readout corresponding to the FP, FI, or TR-FRET configuration of your Transcreener ADP Assay Kit.

Transcreener ADP Assay Kits

* 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-I/MDA5
  • DDX3
  • CD39
  • CD73
DNA Damage Response and Innate Immunity Preview
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