Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD+ capping

Hailei Zhang, Huan Zhong, Xufeng Wang, Shoudong Zhang, Xiaojian Shao, Hao Hu, Zhiling Yu, Zongwei Cai*, Xuemei Chen*, Yiji Xia*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

17 Citations (Scopus)


Recent findings regarding nicotinamide adenine dinucleotide (NAD+)-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ noncanonical RNA capping to regulate gene expression. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to label NAD-RNAs. However, copper ions can fragment/degrade RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) for labeling NAD-RNAs, followed by identification of tagged RNA by single-molecule direct RNA sequencing. We used this method to compare NAD-RNA and total transcript profiles of Escherichia coli cells in the exponential and stationary phases. We identified hundreds of NAD-RNA species in E. coli and revealed genome-wide alterations of NAD-RNA profiles in the different growth phases. Although no or few NAD-RNAs were detected from some of the most highly expressed genes, the transcripts of some genes were found to be primarily NAD-RNAs. Our study suggests that NAD-RNAs play roles in linking nutrient cues with gene regulation in E. coli.

Original languageEnglish
Article numbere2026183118
Number of pages11
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number14
Early online date29 Mar 2021
Publication statusPublished - 6 Apr 2021

Scopus Subject Areas

  • Molecular Biology

User-Defined Keywords

  • E. coli
  • Gene regulation
  • NAD tagSeq II
  • NAD+-capped RNAs
  • NAD capping
  • RNA modification
  • gene regulation
  • RNA cap


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