Fig. 4: Guanine-N7 methyltransferase activity of TgCmt.

A–C The expression and localization of TgCmt. Coomassie blue-stained SDS-PAGE gel of recombinant His_6- TgCmt_716-1283 protein (A), Western blot analysis of TgCmt expression levels in tachyzoites and bradyzoites (B). Immunofluorescence analysis in RH/ME49 parasites with α-TgCmt antibody (C). Loading controls: TgAldolase (TgALD) - parasite proteins, TgSAG1 and TgBAG1, tachyzoite- and bradyzoite stage-specific markers, respectively. Tz tachyzoite, and Bz bradyzoite. Scale bar 5 µm. D Experimental workflow showing steps involved to generate 32-mer (N₃₂) RNA variants, such as triphosphate RNA (pppN₃₂RNA), diphosphate RNA (ppN₃₂RNA), Guanylated RNA (GpppN₃₂RNA), 7-methylguanosine RNA (m⁷GpppN₃₂RNA). E Immuno-dot blot analysis of RNA with α-m⁷G antibody. RNA variants were generated by incubating IVT RNA with or without capping enzymes as specified. Vaccinia capping enzyme (VCE) was used as a positive control. Methylene blue stain serves as an RNA loading control. Graphical representation is Mean ± S.D. (n = 3 replicates) analyzed by one-way ANOVA. F SAM-dependent RNA methylation. A capping reaction was performed with or without Sinefungin at the specified concentration. A–F: n = 3 replicates. E: mean ± SD, one way ANOVA; Dunnett’s comparison test; ns not significant; p values: *<0.05; *** <0.001; ****<0.0001. Source data are provided as a source data file.