PMC:7291971 / 85505-86967
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7291971","sourcedb":"PMC","sourceid":"7291971","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7291971","text":"The transfer of tritiated methyl from [3H] SAM onto RNA substrate was monitored by filter binding assay, performed according to the method described previously [40]. Assays were carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl2, 2 μM SAM and 0.1 μM 3H-SAM (PerkinElmer)] in the presence of 0.7 μM GpppAC4 synthetic RNA and SARS-CoV nsp14 (50 nM), vaccinia virus capping enzyme (D1-D12) (41 U), or human RNA N7 MTase (hRNMT) (50 nM). The enzymes were first mixed with compounds suspended in 100% DMSO (5% final DMSO) before the addition of RNA substrate and SAM and then incubated at 30 °C. Control reactions were performed in the presence of 5% DMSO. Reactions mixtures were stopped after 30 min by their 10-fold dilution in ice-cold water. Samples were transferred to diethylaminoethyl (DEAE) filtermat (PerkinElmer) 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 (PerkinElmer), and 3H-methyl transfer to the RNA substrates was determined using a Wallac MicroBeta TriLux Liquid Scintillation Counter (PerkinElmer). For IC50 measurements, values were normalized and fitted with Prism (GraphPad software) using the following equation: Y = 100/(1+((X/IC50)^Hillslope)). 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