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 thiolcontaining 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.

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

“…Studies have demonstrated that RNAs can be 5′-capped with metabolites, including dephospho-CoenzymeA (dpCoA), but identification of the capped RNAs is difficult. Recently, Shao et al developed a new method for detection of 5′dpCoA-capped RNA based on the reaction with maleimide linked to a oligonucleotide sequence tag and subsequent nanopore sequencing (dpCoA tagseq). The method was applied to RNA isolated from mouse liver, thereby leading to the identification 44 genes producing 5′dpCoA-capped RNA.…”

“…This is an interesting observation with many potential implications, but in most cases, the frequency of dpCoA capping observed for specific RNAs is likely to be low, making the identification of specific dpCoA-capped RNAs challenging. In a recent study, Shao et al developed the dpCoA-tagseq method and applied it to the detection of dpCoA-tagged RNA among RNA isolated from mouse livers . The dpCoA-tagseq method is based on specific addition of a maleimide containing 25 nt oligonucleotide sequence tag to the 5′ end of dpCoA-capped RNAs.…”

“…In addition, for the nanopore reads containing the tag sequence, the control RNA sequence was located next to the 5′ tag sequence only separated by a short region (10–20 bases), indicating successful tagging of the dpCoA cap and sequencing of the tag. Next, dpCoA-tagseq was applied to RNA isolated from mouse liver, leading to the identification of 44 genes that transcribe dpCoA-RNAs in mouse liver …”

“…Further analysis of data from Shao et al. (a) UCSC genome browser screenshot of Shao et al reads mapping to chr3: 5860338-5860904 region, highlighting the overlap with a LSU rRNA repeat. (b) UCSC genome browser screenshot of Shao et al reads mapping to chr12:69159295-69159591 region, high-lighting the overlap with 7SL ncRNA.…”

Response to the Comment on “DpCoA tagSeq: Barcoding dpCoA-Capped RNA for Direct Nanopore Sequencing via Maleimide-Thiol Reaction”

“…In our recent paper, we reported the dpCoA tagSeq method for the identification of dpCoA-capped RNA (dpCoA-RNA). In this method, synthesized RNA containing thiol-reactive maleimide is used to label dpCoA-RNAs.…”