Journal: Nucleic Acids Research
Article Title: NAD + capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp
doi: 10.1093/nar/gkag102
Figure Lengend Snippet: Development and validation of NADbio-northern blotting analysis. ( A ) sibD gene produces NAD-RNAs. The NAD cap content in various RNA samples was determined with LC–MS. The samples analyzed included purified sibD transcripts from the E. coli K12 strain, along with 10 nM NAD + standard. One hundred nanograms of ppp-SibD RNA and NAD-SibD RNA from IVT products were used as negative and positive controls, respectively. ( B ) A schematic illustration of the workflow for the NADbio-northern blotting analysis. NAD and biotin are highlighted in red and blue, respectively. ‘DIG’ highlighted in yellow indicates the digoxin tag in the DNA probes. ( C ) Detection of NAD-RNAs by NADbio-northern blotting with synthetic 5′-ppp-SibD and NAD-SibD. HRP-streptavidin blotting was performed to monitor biotinylation of RNAs mediated by the ADPRC-SPAAC reaction. In addition, a digoxin-tagged sibD DNA probe was used to detect sibD transcripts. ‘ADPRC+’ indicates the biotinylation of NAD-RNAs via the ADPRC-SPAAC reaction with ADPRC, while ‘ADPRC-’ denotes the ADPRC-SPAAC reaction without ADPRC. ( D ) Detection of several NAD-capped cellular RNAs with NADbio-northern blotting analysis. Total RNAs isolated from the wild-type strain were subjected to NADbio-northern blotting analysis. Individual RNAs in the eluate were detected with digoxin-conjugated gene-specific probes. The bands corresponding to individual NAD-RNAs in ADPRC+ lanes were marked with red arrows.
Article Snippet: The hybridized RNAs were enriched and purified with streptavidin beads (NEB), followed by nuclease P1 digestion and liquid chromatography–mass spectrometry (LC–MS) analysis to detect released NAD caps.
Techniques: Biomarker Discovery, Northern Blot, Liquid Chromatography with Mass Spectroscopy, Purification, Isolation
