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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.

SUBMITTER: Hu H

PROVIDER: S-EPMC8020637 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:We developed a Copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC) to capture NAD-RNAs without RNA degradation. We examined the specificity of CuAAC and SPAAC reactions towards NAD+ vs. m7G, and found that both prefer NAD+ but also act on m7G. We show that m7G-capped RNA can be immuno-depleted, allowing for the specific identification of NAD-RNA via the SPAAC reaction and sequencing, which we name SPAAC-NAD-seq. Subjecting Arabidopsis RNA to both the original NAD captureSeq and SPAAC-NAD-seq, we found that more NAD+-capped RNA was identified by the latter, particularly those with low abundance. This led to the discovery of new gene ontology terms such as starch biosynthsis, intracellular protein transport and response to cadmium stress associated with genes that produce NAD-RNA. Furthermore, reads were uniformly distributed along gene bodies, which suggested that SPAAC-NAD-seq retained full-length sequence information. SPAAC-NAD-seq enables specific and efficient discovery of NAD-RNA in prokaryotes, and when combined with m7G-RNA depletion, in eukaryotes.

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