Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5′ cap in Escherichia coli, yeast, mammals, and Arabidopsis. Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information.
A team led by researchers from the University of California, Riverside have developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide–alkyne cycloaddition reaction (SPAAC). They examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. The researchers demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis. Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.
m7G-RNA depletion promotes the identification of low-abundant, authentic NAD-RNAs
(A) Schematic workflow of CuAAC- or SPAAC-NAD-seq. mRNAs were isolated and treated by the anti-m7G antibody for m7G-RNA depletion. CuAAC- or SPAAC-NAD reactions were subsequently performed to isolate NAD-RNAs. NAD-RNA libraries were constructed for high-throughput sequencing. NAD-RNAs were defined as ones with a twofold change in FPKM and adjusted P value < 0.05 in NAD-seq compared to mRNA-seq. (B) Scatter plots showing NAD-RNAs identified by CuAAC- and SPAAC-NAD-seq in m7G-depletion and mock-depletion samples. The log2 FPKM ratio between NAD-seq samples and mRNA-seq samples is plotted against the log2 FPKM of mRNA-seq. The green and orange dots represent the NAD-RNAs identified by CuAAC-NAD-seq or SPAAC-NAD-seq, respectively.