Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq

Yiliang Ding, Chun Kit Kwok, Yin Tang, Philip C. Bevilacqua, Sarah M. Assmann

Research output: Contribution to journalArticle

42 Scopus citations

Abstract

Structure-seq is a high-throughput and quantitative method that provides genome-wide information on RNA structure at single-nucleotide resolution. Structure-seq can be performed both in vivo and in vitro to study RNA structure-function relationships, RNA regulation of gene expression and RNA processing. Structure-seq can be carried out by an experienced molecular biologist with a basic understanding of bioinformatics. Structure-seq begins with chemical RNA structure probing under single-hit kinetics conditions. Certain chemical modifications, e.g., methylation of the Watson-Crick face of unpaired adenine and cytosine residues by dimethyl sulfate, result in a stop in reverse transcription. Modified RNA is then subjected to reverse transcription using random hexamer primers, which minimizes 3′ end bias; reverse transcription proceeds until it is blocked by a chemically modified residue. Resultant cDNAs are amplified by adapter-based PCR and subjected to high-throughput sequencing, subsequently allowing retrieval of the structural information on a genome-wide scale. In contrast to classical methods that provide information only on individual transcripts, a single structure-seq experiment provides information on tens of thousands of RNA structures in ∼1 month. Although the procedure described here is for Arabidopsis thaliana seedlings in vivo, structure-seq is widely applicable, thereby opening new avenues to explore RNA structure-function relationships in living organisms.

Original languageEnglish (US)
Pages (from-to)1050-1066
Number of pages17
JournalNature Protocols
Volume10
Issue number7
DOIs
StatePublished - Jul 27 2015

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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