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Related Concept Videos

RNA-seq03:21

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Related Experiment Video

Updated: Nov 11, 2025

3' End Sequencing Library Preparation with A-seq2
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SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts.

Hao Hu1,2, Nora Flynn1, Hailei Zhang3

  • 1Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521.

Proceedings of the National Academy of Sciences of the United States of America
|March 23, 2021
PubMed
Summary

Nicotinamide adenine diphosphate (NAD+) caps RNA in various organisms. A new method, SPAAC-NAD-seq, specifically identifies these NAD-RNAs, improving accuracy and retaining full-length sequence information.

Keywords:
NADNAD captureSeqNAD-RNASPAAC-NAD-seqm7G-RNA

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Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Nicotinamide adenine diphosphate (NAD+) functions as a novel RNA 5' cap in prokaryotes and eukaryotes.
  • Existing methods like NAD captureSeq have limitations in specificity and RNA integrity.

Purpose of the Study:

  • To develop a more specific and efficient method for identifying NAD+-capped RNAs (NAD-RNAs).
  • To overcome the limitations of previous techniques regarding specificity and RNA fragmentation.

Main Methods:

  • Development of a copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) based method for NAD-RNA capture.
  • Comparative analysis of CuAAC and SPAAC specificity for NAD-RNAs versus m7G-capped RNAs (m7G-RNAs).
  • Combination of SPAAC with immunodepletion of m7G-RNAs for enhanced NAD-RNA identification.

Main Results:

  • Both CuAAC and SPAAC reactions show preference for NAD-RNAs but also react with m7G-RNAs.
  • SPAAC-NAD sequencing, coupled with m7G-RNA depletion, accurately and sensitively identifies NAD-RNAs.
  • New NAD-RNA profiles were discovered in Arabidopsis using the improved method.
  • SPAAC-NAD-seq preserves full-length RNA sequence information.

Conclusions:

  • SPAAC-NAD-seq offers a specific and efficient approach for discovering NAD-RNAs.
  • This method is suitable for both prokaryotic and eukaryotic systems, especially when combined with m7G-RNA depletion.