Extended Data Fig. 3: PAGE analysis and LC-MS analysis of IVT products generated from plasmid TC.
From: Molecular insight into 5′ RNA capping with NpnNs by bacterial RNA polymerase
PAGE analysis (with APB) shows RNA products obtained from IVT reactions using the TC plasmid and NTPs or their combination with NpnNs radiolabeled with α-[32P]CTP. The IVT reaction was performed in five independent experiments. The controls (CTRL E. coli and CTRL) show the migration of the uncapped RNA (150 nt) produced by E. coli RNAP and Tt RNAP, respectively, and serve as molecular weight markers. Moreover, the formation of the known RNAI encoded in the plasmid (109 nt) and produced by E. coli RNAP was observed. Non-radiolabeled RNA was used for digestion with RNase A (cleaving after pyrimidines) and further analyzed by LC-MS. Extracted ion chromatograms show RNA fragments generated from IVT with or without NpnNs. Uncapped RNA (CTRL) is indicated in gray: solid gray lines represent transcripts initiating at the +1 TSS (defined as uncapped(+1)TSS-RNA), while dashed gray lines represent transcripts initiating at the −1 TSS (defined as uncapped(−1)TSS-RNA). Capped IVT products encoded from the +1 TSS (cap(+1)TSS-RNA) are shown in blue and products encoded from the −1 TSS (cap(−1)TSS-RNA) are shown in red. The sequences and exact masses of the corresponding products are listed in Supplementary Table 4. Created in BioRender. Serianni, V. (2025) https://BioRender.com/pbscak2.
