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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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Detalles Bibliográficos
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
Descripción
Sumario:[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.