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DOI	10.1073/pnas.2025595118
	SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts
	Hu H.; Flynn N.; Zhang H.; You C.; Hang R.; Wang X.; Zhong H.; Chan Z.; Xia Y.; Chen X.
发表日期	2021
ISSN	00278424
卷号	118 期号:13
英文摘要	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. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	M7G-RNA; NAD; NAD captureSeq; NAD-RNA; SPAAC-NAD-seq
语种	英语
scopus关键词	bacterial RNA; nicotinamide adenine dinucleotide; Arabidopsis; Article; controlled study; cycloaddition; Escherichia coli; high throughput sequencing; measurement accuracy; nonhuman; priority journal; RNA transcription; sensitivity and specificity; strain promoted azide alkyne cycloaddition reaction
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/180068
作者单位	Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521, United States; Key Laboratory for Biology of Horticultural Plants, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, China; Department of Biology, Hong Kong Baptist University, Hong Kong; State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, 200438, China; Collaborative Innovation Center of Genetics and Development, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, 200438, China; Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518060, China; State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong
推荐引用方式 GB/T 7714	Hu H.,Flynn N.,Zhang H.,et al. SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts[J],2021,118(13).
APA	Hu H..,Flynn N..,Zhang H..,You C..,Hang R..,...&Chen X..(2021).SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts.Proceedings of the National Academy of Sciences of the United States of America,118(13).
MLA	Hu H.,et al."SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts".Proceedings of the National Academy of Sciences of the United States of America 118.13(2021).