SPAAC-NAD-seq, a sensitive and accurate method to profile NAD ⁺ -capped transcripts

Abstract

Nicotinamide adenine diphosphate (NAD⁺) is a novel messenger RNA 5' cap in <i>Escherichia coli</i>, yeast, mammals, and <i>Arabidopsis</i> 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 <i>E. coli</i> 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. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC). We 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. We 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 <i>Arabidopsis</i> 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.