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 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. 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 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.

Keywords: NAD; NAD captureSeq; NAD-RNA; SPAAC-NAD-seq; m7G-RNA.