Chromas lite

ADAR protein purification and preparation of RNA substrates have been described previously (29 (link),30 (link)). Deamination assays were performed under single turnover conditions with 1 μM enzyme and 10 nM RNA substrate under conditions of 20 mM Tris–HCl pH 7.0, 60 mM KCl, 30 mM NaCl, 1.5 mM ethylenediaminetetraacetic acid, 8% glycerol, 0.003% Nonidet P-40, 0.5 mM DTT, 0.25 mM BME, 160 U/ml RNasin and 1.0 mg/ml yeast tRNA^Phe in a final volume of 10 μl. Each reaction solution was incubated at 30°C for 45 min before adding enzyme and the reaction was allowed to proceed for different times at 30°C prior to being stopped with 10 μl 1% sodium dodecyl sulphate and heating at 95°C for 2 min. Deaminated RNA was purified by phenol-chloroform extraction and ethanol precipitation. RT-PCR was performed on the RNA and the PCR products were purified using an agarose gel. The editing levels were then determined by Sanger sequencing of the RT-PCR products. The sequencing data were quantified using Chromas Lite (Technelysium) and ImageJ and were fit to the equation [P]t = α[1 -e^(-kobs*t)], where [P]t is the fraction of deamination product at time t, α is the fitted reaction end point and k_obs is the fitted rate constant using KaleidaGraph. Each experiment was carried out in triplicate.