Journal: Nature Communications
Article Title: CompasSeq: epitranscriptome-wide percentage assessment of metabolite-capped RNA at the transcript resolution
doi: 10.1038/s41467-025-61697-y
Figure Lengend Snippet: a In vitro transcription with ATP, m 7 GpppA, NAD, FAD, or dpCoA as the initiating nucleotide. Sequence of the DNA template with the T7 promoter (Top). The “A” in red was the transcription start site and the coding sequence has been underlined. In vitro-transcribed RNAs with different 5′ end caps were resolved in an 8% PAGE gel (Bottom). The arrows indicated the intact FAD-capped species. b Boronate affinity electrophoresis showing in vitro-transcribed 38 nt RNA, including ppp-, m 7 G-, and NAD-capped RNAs. c UREA-PAGE gels showing products of processing of NAD- and m 7 G-RNA 5′-ends by NudC, dRai1, RppH, and yDcpS. d UREA-PAGE gel showing products of processing of FAD- and dpCoA-RNA 5′-ends by NudC. e Analysis of the effect of RNA secondary structure on yDcpS decapping. All m 7 G-capped RNA hairpins and linear 30-mers (1 µg each) were treated by 9 µM yDcpS for 1 h and then resolved in a 12% PAGE gel. f After NudC treatment, NCIN-RNA, such as NAD-RNA (38 nt), could be ligated with the 5′-adapter (27 nt), as shown by the shift of an upper band in the UREA-PAGE gel (left). m 7 G-capped RNA (38 nt) after being processed by yDcpS and CIAP, could not be ligated with the 5′-adapter (middle). RppH-processed m 7 G-RNA, on the other hand, contained a 5′-phosphate group for adapter ligation (right). Source data are provided as a file.
Article Snippet: For other capped RNA forms, the reaction included 4 mM NAD (Macklin, catalog: N814818, for NAD-RNA), 4 mM FAD (Sigma-Aldrich, catalog: F6625, for FAD-RNA), 4 mM dpCoA (Sigma-Aldrich, catalog: D3385, for dpCoA-RNA) or 4 mM m 7 GpppA (NEB, catalog: S1406S, for m 7 GpppA-RNA).
Techniques: In Vitro, Sequencing, Affinity Electrophoresis, Adapter Ligation
