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

Nat Protoc. 2015 Jul;10(7):1050-66. doi: 10.1038/nprot.2015.064. Epub 2015 Jun 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Arabidopsis / chemistry
  • Arabidopsis / genetics
  • Gene Expression Profiling / methods*
  • Genomic Library
  • High-Throughput Nucleotide Sequencing / methods
  • Nucleic Acid Conformation
  • RNA / chemistry*
  • RNA / genetics
  • RNA, Plant / chemistry
  • RNA, Plant / genetics
  • Sequence Analysis, RNA / methods


  • RNA, Plant
  • RNA