SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts

Hao Hu, Nora Flynn, Hailei Zhang, Chenjiang You, Runlai Hang, Xufeng Wang, Huan Zhong, Zhulong Chan, Yiji XIA*, Xuemei Chen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5′ cap in Escherichia coli, yeast, mammals, and Arabidopsis. Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide–alkyne cycloaddition reaction (SPAAC). We examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis. Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.

Original languageEnglish
Article numbere2025595118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number13
DOIs
Publication statusPublished - 30 Mar 2021

Scopus Subject Areas

  • General

User-Defined Keywords

  • MG-RNA
  • NAD
  • NAD captureSeq
  • NAD-RNA
  • SPAAC-NAD-seq

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