The methyltransferase activity was measured using a filter-binding assay, performed according to the method described previously (Paesen et al., 2015 (link)). A 50 nM solution of RSV L–P complex was incubated with 1.8 μM purified synthetic RNA, 0.17 μM S-adenosyl methionine (SAM) and 0.8 μM 3H-SAM (Perkin Elmer) in 50 mM Tris-HCl pH 8.0. After 3 h incubation at 30°C, reactions were quenched by a 20-fold dilution in cold water. Samples were transferred to DEAE filtermats (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA-retaining mats were washed twice with 10 mM ammonium formate pH 8.0, twice with water and once with ethanol. They were then soaked with scintillation fluid (Perkin Elmer), and 3H-methyl transfer to the RNA substrates was determined using a Wallac MicroBeta TriLux Liquid Scintillation Counter (Perkin Elmer).
Wallac microbeta trilux liquid scintillation counter
Manufactured by PerkinElmer
The Wallac MicroBeta TriLux is a liquid scintillation counter designed for high-throughput radioactivity detection. It is capable of quantifying low-level radioactive signals in biological, chemical, and environmental samples.
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4 protocols using wallac microbeta trilux liquid scintillation counter
1
Methyltransferase activity assay of RSV L-P complex
2
Quantitative RNA Methyltransferase Assay
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The MTase activity was measured using a filter-binding assay, performed by combining 25 nM of RSV MTase-CTD with 0.7 μM of synthetic RNAs, 2 μM SAM, and 0.33 μM 3H-SAM (Perkin Elmer) in 40 mM Tris-HCl pH 7.5. After 3-h incubation at 30°C, reactions were quenched by 10-fold dilution in ice-cold 100 μM SAH, and samples were transferred to DEAE filtermats (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA-retaining mats were washed twice with 10 mM ammonium formate (pH 8.0), twice with water, and once with ethanol. They were then soaked with liquid scintillation fluid for measuring 3H-methyl transfer to the RNA substrates using a Wallac MicroBeta TriLux Liquid Scintillation Counter (Perkin Elmer).
3
SARS-CoV-2 nsp14 MTase Enzymatic Assay
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The SARS-CoV-2 nsp14 MTase assay was carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl2, 1.9 μM SAM, and 0.1 μM 3H-SAM (Perkin Elmer)] in the presence of 0.7 μM GpppAC4 synthetic RNA23 (link) and the MTase (10 nM). Briefly, the enzyme was first mixed with the compound suspended in 50% DMSO (2.5% final DMSO) before the addition of RNA substrate and SAM and then incubated at 30 °C. Reactions mixtures were stopped after 30 min by their 10-fold dilution in ice-cold water. Samples were transferred to diethylaminoethyl (DEAE) filtermat (Perkin Elmer) using a Filtermat Harvester (Packard Instruments). The RNA-retaining mats were washed twice with 10 mM ammonium formate pH 8.0, twice with water and once with ethanol. They were soaked with scintillation fluid (Perkin Elmer), and 3H-methyl transfer to the RNA substrates was determined using a Wallac MicroBeta TriLux liquid scintillation counter (Perkin Elmer). For IC50 measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation: Y = 100/[1 + ((X/IC50)^Hillslope)]. IC50 is defined as the inhibitory compound concentration that causes 50% reduction in enzyme activity.
4
Methyltransferase Activity Assay Using [Methyl-3H]-SAM
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MTase activity assays were performed by combining 0.5 μM of enzymes with 1 μM of GpppAC4 or mGpppAC4 RNAs, 2 μM SAM and 0.33 μM [methyl-3H]-SAM (Perkin Elmer, Waltham, MA, USA) in 40 mM Tris–HCl (pH 8.5), 1 mM DTT. Reactions at 30°C were stopped by a 10-fold dilution in 100 μM ice-cold SAH and the samples transferred to DEAE (Perkin Elmer) using a Filtermats Harvester (Packard Instruments, Downers Grove, IL, USA). The RNA-retaining mats were washed twice with 10 mM ammonium formate (pH 8.0), twice with water, and once with ethanol. They were then soaked with liquid scintillation fluid, allowing the measurement of [methyl-3H] transfer to the RNA substrates using a Wallac MicroBeta TriLux Liquid Scintillation Counter (Perkin Elmer).
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