Dataset Information

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

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.

SUBMITTER: Shao X

PROVIDER: S-EPMC10372868 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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

Shao Xiaojian X Zhang Hailei H Zhu Zhou Z Ji Fenfen F He Zhao Z Yang Zhu Z Xia Yiji Y Cai Zongwei Z

Analytical chemistry 20230713 29

Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD<sup>+</sup>) 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 we ...[more]

PMID: 37439785

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Dataset Information

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

Publications

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

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