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. 2021 Mar 29;118(14):e2026183118. doi: 10.1073/pnas.2026183118

Fig. 3.

Fig. 3.

Specificity of the SPAAC-based tagging process. (A) Synthetic NAD-RNAs were tagged through SPAAC tagging, but 5′-monophosphate RNA (pA-RNA), 5′-triphosphate RNA (pppA-RNA), and FAD-capped RNA were not tagged with the synthetic RNA tag. (B) The 5′-hydroxyl RNA (5′ OH-RNA) and 5′-diphosphate RNA (ppA-RNA) were not tagged with the synthetic RNA tag. (C) Ap4A-capped RNA (Ap4A-RNA) was not tagged with the synthetic RNA tag. (D) A very small portion of synthetic dpCoA-capped RNAs was tagged through the SPAAC tagging process, irrespective of the presence of ADPRC. (E) The dpCoA-RNAs were first incubated with IAA and then subjected to the same tagging process as in D. No ligation product was detected. (F) A small portion of m7GpppA-RNAs was tagged through SPAAC tagging. (G) m7GpppA-RNAs were reacted with 4-pentyn-1-ol in the presence or absence of ADPRC and labeled with biotin through CuAAC tagging. m7GpppA-RNAs were found tagged with biotin in the presence of ADPRC, which were detected by Streptavidin-HRP. (H) Mass spectrometry analysis of alkyne- and azide-modified m7GpppA-RNAs after ADPRC reaction.