The Transcreener NSP13 Assay determines NSP13 enzyme activity by directly measuring the ADP formed by the enzyme using the Transcreener ADP Assay. By detecting ADP output, the assay provides a universal method to assess the activity of any ADP-producing enzyme in real-time.
Transcreener NSP13 Assay 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 fluorescent readout.
The NSP13 assay is available with FP, FI, and TR-FRET. It is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS.
Run your enzyme reaction, add Transcreener reagents, and read your plates. The NSP13 assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized NSP13 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 = 0.75.
The assay demonstrates linearity when raw data is converted to ADP using a standard curve. Here we use 500 µM ATP with 20 µM dsRNA substrate. Linearity is shown here under initial velocity conditions when raw data is converted to ADP formed. The buffer includes 20 mM Tris pH 7.5, 1 mM MgCl2, 2.5 mM NaCl, 0.005% Brij, 0.005% BSA, 225 μg/mL ADP2 antibody, and 2 nM ADP Alexa 633 tracer. The NSP13 enzyme reaction took place for 60 minutes at 37ºC in kinetic mode.
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| Component | Notes |
|---|---|
| ADP² Antibody | Antibody specific to the nucleotide ADP. Suggested ADP² antibody concentration (in the Detection Mixture) for FP is 225 μg/mL for 500 μM ATP. Yours may vary depending on, enzyme, and buffer composition. |
| ADP Tracer | When displaced, changes in fluorescence can be read in an FP, FI, or TR-FRET depending on the assay format. |
| ATP and ADP | ATP and ADP are used to create the ATP/ADP standard curve. ATP is used as a phosphate donor to start the reaction. We suggest using a starting ATP concentration of 500 μM for NSP13. |
| Component | Notes |
|---|---|
| NSP13 Enzyme | We have successfully used recombinant human NSP13 from Cayman Chemical Cat.# 30589. An NSP13 concentration of 125 pM was used for screening and SAR. |
| Enzyme Buffer | 20 mM Tris (pH 7.5), 1 mM MgCl2,, 2.5 mM NaCl, 0.005% Brij-35, and 0.005% BSA. Enzyme buffer components may be optimized as required. |
| dsRNA Substrate | Here we used 28 bp 5'-3' dsRNA at 20 μM. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs FP - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 FI - An entirely black plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4514 TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to measure the output of the ADP tracer is required. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products, reducing assay performance. Careful handling and use of ultrapure water eliminates this potential problem. |
COVID-19 led to intense research to find new therapeutics for the disease. As a result, a variety of different targets are currently being explored to help fight the pandemic. As an antiviral target, NSP13 is critical for SARS-CoV-2 replication, thus making it an area of extreme interest.
As one of the 16 known coronavirus nonstructural proteins, NSP13 is the most similar to other forms of the virus, such as MERS-CoV and SARS-CoV, making it an exciting target not only for the current pandemic but for future outbreaks as well (White et al. 2020).
The Transcreener ADP2 NSP13 Assay is an excellent tool for researchers examining the therapeutic effects of NSP13.
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