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. 2021 Mar 22;118(13):e2025595118. doi: 10.1073/pnas.2025595118

Fig. 3.

Fig. 3.

CUAAC- and SPAAC-NAD reactions after m7G-RNA depletion. (A) A gel blot assay to evaluate the efficacy of m7G-RNA depletion. A mixture of in vitro-transcribed NAD-RNA (1 μg) and m7G-RNA (100 μg) (molar ratio, 1:225) was incubated with (m7G-depletion sample) or without the m7G antibody (mock-depletion sample). Protein G beads were then used to remove the m7G-RNA. Afterward, RNAs were subjected to the SPAAC-NAD reaction and biotin signals were detected with the chemiluminescent nucleic acid detection module kit after probing with streptavidin–horseradish peroxidase. The left two lanes (marker RNA 1 and marker RNA 2) are 70- and 31-nt NAD-RNAs after the SPAAC-NAD reaction; they are used as size markers. The input sample did not undergo mock or m7G-RNA depletion. (B) A dot blot assay demonstrating that m7G-RNAs in mRNAs were largely removed. Arabidopsis mRNA samples were treated with (m7G-depletion sample) or without the m7G antibody (mock-depletion sample). Protein G beads were then applied to specifically capture the m7G antibody. The mRNAs collected from the flow-through and beads were spotted onto a membrane and probed with the anti-m7G antibody or stained with methylene blue. (C) LC-MS analysis demonstrating that m7G-RNAs were largely removed from mRNAs but NAD-RNAs were not affected by immunodepletion. mRNA samples after m7G antibody treatment were subjected to nuclease P1 digestion and analyzed by HPLC-MS. Targeted detection of m7GpppA and NAD+ released by nuclease P1 indicates the amounts of m7G-RNAs and NAD-RNA, respectively. The bar plots show relative m7GpppA or NAD+ levels as calculated by the peak area among the samples. The error bars represent SE calculated from three independent experiments (two-tailed Student’s t test: *P < 0.05; ***P < 0.001; NS, not significant). (D) A gel blot assay of mRNAs that were pretreated with the anti-m7G antibody followed by CuAAC or SPAAC-NAD reactions. The anti-m7G antibody treatment and biotin signal detection were performed similarly to A. m7G dep, m7G depletion; mock dep, mock depletion.