title: Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq.
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.
authors.affiliation: 1] Department of Biology, Pennsylvania State University, University Park, Pennsylvania, USA. [2] Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA. [3] Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Yiliang
authors.initials: Y
authors.lastname: Ding
authors.affiliation: 1] Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA. [2] Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Chun Kit
authors.initials: CK
authors.lastname: Kwok
authors.affiliation: 1] Department of Biology, Pennsylvania State University, University Park, Pennsylvania, USA. [2] Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA. [3] Bioinformatics and Genomics Graduate Program, Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Yin
authors.initials: Y
authors.lastname: Tang
authors.affiliation: 1] Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA. [2] Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA. [3] Plant Biology Graduate Program, Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Bevilacqua
authors.affiliation: 1] Department of Biology, Pennsylvania State University, University Park, Pennsylvania, USA. [2] Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA. [3] Bioinformatics and Genomics Graduate Program, Pennsylvania State University, University Park, Pennsylvania, USA. [4] Plant Biology Graduate Program, Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Assmann
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods*; D015698:Genomic Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D018749:RNA, Plant / Q000737:chemistry / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D018749:RNA, Plant; D012313:RNA