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

Recent discoveries of noncanonical RNA caps, such asnicotinamideadenine dinucleotide (NAD(+)) and 3 & PRIME;-dephospho-coenzymeA (dpCoA), have expanded our knowledge of RNA caps. Although dpCoAhas been known to cap RNAs in various species, the identities of itscapped RNAs (dpCoA-RNAs) remained unknown. To fill this gap, we developeda method called dpCoA tagSeq, which utilized a thiol-reactive maleimidegroup to label dpCoA cap with a tag RNA serving as the 5 & PRIME; barcode.The barcoded RNAs were isolated using a complementary DNA strand ofthe tag RNA prior to direct sequencing by nanopore technology. Ourvalidation experiments with model RNAs showed that dpCoA-RNA was efficientlytagged and captured using this protocol. To confirm that the taggedRNAs are capped by dpCoA and no other thiol-containing molecules,we used a pyrophosphatase NudC to degrade the dpCoA cap to adenosinemonophosphate (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 andcharacterizing the capped RNAs. This strategy provides a viable approachto identifying dpCoA-RNAs that allows for further functional investigationsof the cap.