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

[Image: see text] 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)...

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Autores principales: Shao, Xiaojian, Zhang, Hailei, Zhu, Zhou, Ji, Fenfen, He, Zhao, Yang, Zhu, Xia, Yiji, Cai, Zongwei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2023
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10372868/
https://www.ncbi.nlm.nih.gov/pubmed/37439785
http://dx.doi.org/10.1021/acs.analchem.3c02063
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author Shao, Xiaojian
Zhang, Hailei
Zhu, Zhou
Ji, Fenfen
He, Zhao
Yang, Zhu
Xia, Yiji
Cai, Zongwei
author_facet Shao, Xiaojian
Zhang, Hailei
Zhu, Zhou
Ji, Fenfen
He, Zhao
Yang, Zhu
Xia, Yiji
Cai, Zongwei
author_sort Shao, Xiaojian
collection PubMed
description [Image: see text] 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.
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spelling pubmed-103728682023-07-28 DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction Shao, Xiaojian Zhang, Hailei Zhu, Zhou Ji, Fenfen He, Zhao Yang, Zhu Xia, Yiji Cai, Zongwei Anal Chem [Image: see text] 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. American Chemical Society 2023-07-13 /pmc/articles/PMC10372868/ /pubmed/37439785 http://dx.doi.org/10.1021/acs.analchem.3c02063 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by/4.0/Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Shao, Xiaojian
Zhang, Hailei
Zhu, Zhou
Ji, Fenfen
He, Zhao
Yang, Zhu
Xia, Yiji
Cai, Zongwei
DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction
title DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction
title_full DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction
title_fullStr DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction
title_full_unstemmed DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction
title_short DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction
title_sort dpcoa tagseq: barcoding dpcoa-capped rna for direct nanopore sequencing via maleimide-thiol reaction
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10372868/
https://www.ncbi.nlm.nih.gov/pubmed/37439785
http://dx.doi.org/10.1021/acs.analchem.3c02063
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