DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction

Xiaojian Shao, Hailei Zhang, Zhou Zhu, Fenfen Ji, Zhao He, Zhu Yang*, Yiji Xia*, Zongwei Cai*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

4 Citations (Scopus)

Abstract

Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD+) and 3'-dephospho-coenzyme A (dpCoA), have expanded our knowledge of RNA caps. Although dpCoA has been known to cap RNAs in various species, the identities of its capped RNAs (dpCoA-RNAs) remained unknown. To fill this gap, we developed a method called dpCoA tagSeq, which utilized a thiol-reactive maleimide group to label dpCoA cap with a tag RNA serving as the 5' barcode. The barcoded RNAs were isolated using a complementary DNA strand of the tag RNA prior to direct sequencing by nanopore technology. Our validation experiments with model RNAs showed that dpCoA-RNA was efficiently tagged and captured using this protocol. To confirm that the tagged RNAs are capped by dpCoA and no other thiol-containing molecules, we used a pyrophosphatase NudC to degrade the dpCoA cap to adenosine monophosphate (AMP) moiety before performing the tagSeq protocol. We identified 44 genes that transcribe dpCoA-RNAs in mouse liver, demonstrating the method's effectiveness in identifying and characterizing the capped RNAs. This strategy provides a viable approach to identifying dpCoA-RNAs that allows for further functional investigations of the cap.

Original languageEnglish
Pages (from-to)11124–11131
Number of pages8
JournalAnalytical Chemistry
Volume95
Issue number29
Early online date13 Jul 2023
DOIs
Publication statusPublished - 25 Jul 2023

Scopus Subject Areas

  • Analytical Chemistry

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