AptaFluor SAH Assay directly measures SAH produced by NSP14 (Nonstructural Protein 14) in an HTS format. The assay relies on a naturally occurring SAH-sensing RNA aptamer coupled with TR-FRET detection. SAH produced drives assembly of the split aptamer allowing for FRET signal between a Terbium chelate donor and DyLight 650 acceptor.
Current non-radioactive SAH detection methods are not sufficiently sensitive to allow detection of many methyltransferases using physiological concentrations of SAM. The AptaFluor Assay overcomes technical gaps of current detection methods that detect specific methylation events or are not sensitive enough for enzymes with low turnovers and/or high SAM affinities.
The kit comes complete with the detection reagents required to measure activity. NSP14 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. AptaFluor assays are not validated with cell lysates, blood, serum, or other biological samples.
This application note describes methods for using the AptaFluor SAH Methyltransferase Assay to measure NSP14 enzymatic activity in a high-throughput (384-well) format, e.g., for inhibitor screening and/or dose-response measurements.
The AptaFluor Assay is the most sensitive HTS methyltransferase assay, allowing a LLD of 0.6 nM SAH. This drastically reduces enzyme usage and allows the assay to be run at or below KM for SAM.
Run your enzyme reaction, add quenching reagent, add AptaFluor reagents, and read your plates. The assay is compatible with 96, 384, and 1536-well formats.
Excellent Z’ under a variety of assay 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 = 0.80.
The assay demonstrates linearity when raw data is converted to SAH using a standard curve. Here, we used 50 μM RNA Substrate and 100 nM SAM. The assay buffer included 20 mM Tris-HCl (pH 8.0), 3 mM MgCl2, 0.01% BSA, 1 mM EDTA and 1 mM DTT. The Enzyme Stop Mix included 1X Enyzme Stop Reagent and 1X SAH Detection Buffer. The SAH Detection Mix included 20 nM P1-Terbium Mix, 40 nM P2-Dylight 650 nM, and 1X SAH Detection Buffer. The enzyme reaction took place for 90 minutes at 37°C. The Detection Mix was added and incubated for 180 minutes at room temperature and read with a Pherastar Plus/FS plate reader.
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| Component | Notes |
|---|---|
| SAM | SAM supplied in the kit should be used for MT enzyme reactions and to create a SAM/SAH standard curve. The NSP14 enzyme reaction contained 100 nM SAM. |
| SAH | SAH is used to create a SAM/SAH standard curve. |
| Enzyme Stop Reagent, 10X | Enzyme Stop Reagent is diluted to 1X in SAH Detection Buffer and used to quench the activity of MT enzyme components. |
| SAH Detection Buffer, 10X | This buffer is used to prepare the SAH Detection Mix and the Enzyme Stop Mix. |
| P1-Biotin | P1-Biotin is a 55-base RNA oligomer with a biotin molecule at the 5' end. It is incubated with TB-Streptavidin to produce P1-Terbium, the donor piece of the SAH aptamer. |
| P2-Dylight 650 | P2-Dylight 650 is an 18-base RNA oligomer with a Dylight 650 molecule at the 3' end. It is the acceptor piece of the SAH aptamer. |
| Tb-Streptavidin | Tb-Streptavidin is used to produce the Tb-labeled (donor) piece of the SAH aptamer. |
| Conjugation Buffer | This buffer is used for the P1-Biotin/Tb-Streptavidin conjugation reaction. |
| Component | Notes |
|---|---|
| NSP14 Enzyme | We have successfully used recombinant human NSP14 Enzyme Complex from Abcam Cat. #ab277616. |
| RNA Cap Substrate | G(5')ppp(5')A RNA Cap Structure Analog from New England BioLabs Inc. Cat #S1406L. |
| Enzyme Buffer | 20 mM Tris-HCl (pH 8.0), 3 mM MgCl2, 0.01% BSA, 1 mM EDTA, and 1 mM DTT. Enzyme buffer components may be optimized as required. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs 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 read TR-FRET is required for the assay. |
| 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 and RNA aptamers. Careful handling and use of ultrapure water eliminates this potential problem. |
The AptaFluor SAH Methyltransferase Assay is a high-throughput enzymatic assay for SAM-dependent methyltransferases. The assay relies on a naturally occurring SAH-sensing RNA aptamer, or riboswitch, that binds SAH with nanomolar affinity and exquisite selectivity. Upon binding to as low as 1 nM SAH, these split-aptamer sensors generate positive TR-FRET signals. As a result, the assay overcomes technical gaps of current detection methods that detect specific methylation events or are not sensitive enough for enzymes with low turnovers and/or high SAM affinities.
Here, we demonstrate how the AptaFluor Assay provides a reliable and robust tool for detecting activity of four methyltransferases, the sensitivity, selectivity, & stability of the assay, robust detection of SAH (Z’ > 0.7) at nM concentration of enzyme, and profiling dose response curves for known inhibitors. Selected enzymes are representative of typical low Km (100 nM to 5 µM) and have attracted interest as therapeutic targets.
NSP14 is a bifunctional protein containing two functional domains – an ExoN domain and N7-MTase domain. NSP14’s ExoN domain allows SARS-CoV virus to proofread by catalyzing nucleoside monophosphate excision from nucleic acids, which is critical for high-fidelity replication. NSP14’s N7-MTase domain is involved in viral mRNA capping, which hides the virus from the immune system.
The NSP14 assay provides an excellent tool to screen compound libraries for NSP14 modulators.
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