Supplementary Figure 5: Biochemical assays and optimization of demethylation activity of purified AlkB enzyme against m1A.
From: Evolution of a reverse transcriptase to map N1-methyladenosine in human messenger RNA
a, Shown are LC-MS/MS data of m1A level before and after AlkB treatment for synthetic m1A15 RNA (left; mean ± s.d. from n = 2 technical replicates) and polyA-enriched RNA from HEK293T cells (right; mean ± s.d. from n = 12 replicates — that is, 3 cell culture replicates and 4 LC–MS/MS injection replicates). b, Schematics for the RT-PCR-IVT assay for detecting AlkB activity using m1A18 RNA. Fluorescence data of positive (m1A18 treated by AlkB) and negative control (no AlkB added or AlkB added together with 5mM EDTA) experiments are shown on the right. Error bars represent s.e.m. from n = 2 independent assays for –AlkB and from n = 6 independent assays for +AlkB and +AlkB+EDTA. c, Optimization of the AlkB reaction condition by the RT-PCR-IVT assay for a 2-h reaction at 37 °C. Fluorescence intensities at 60 min are plotted versus various reaction components with the reaction conditions as 50 mM MES (pH 5.0), 50 μM (NH4)2Fe(SO4)2, 300 μM 2-ketoglutarate, 2 mM l-ascorbic acid, 2 mM MgCl2 and RNase inhibitor unless specified on the plot. Error bars represent s.d. from n = 2 or 3 independent assays. d, Optimization of iron concentration and reaction temperatures by the RT-PCR-IVT assay under the condition 50 mM MES (pH 5.0), 300 μM 2-ketoglutarate, 2 mM l-ascorbic acid and RNase inhibitor and noted concentrations of (NH4)2Fe(SO4)2 on the plot. Shown are mean ± s.d. from n = 2 independent assays. e, Optimization of iron concentration and reaction temperatures by LC–MS/MS assay with total RNA sample from HEK293T cells with small RNA removal. Shown are mean ± s.d. from n = 6 replicates — that is, 2 independent AlkB assays and 3 LC–MS/MS injection replicates.
