title: Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).
abstract: New regulatory roles continue to emerge for both natural and engineered noncoding RNAs, many of which have specific secondary and tertiary structures essential to their function. Thus there is a growing need to develop technologies that enable rapid characterization of structural features within complex RNA populations. We have developed a high-throughput technique, SHAPE-Seq, that can simultaneously measure quantitative, single nucleotide-resolution secondary and tertiary structural information for hundreds of RNA molecules of arbitrary sequence. SHAPE-Seq combines selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry with multiplexed paired-end deep sequencing of primer extension products. This generates millions of sequencing reads, which are then analyzed using a fully automated data analysis pipeline, based on a rigorous maximum likelihood model of the SHAPE-Seq experiment. We demonstrate the ability of SHAPE-Seq to accurately infer secondary and tertiary structural information, detect subtle conformational changes due to single nucleotide point mutations, and simultaneously measure the structures of a complex pool of different RNA molecules. SHAPE-Seq thus represents a powerful step toward making the study of RNA secondary and tertiary structures high throughput and accessible to a wide array of scientific pursuits, from fundamental biological investigations to engineering RNA for synthetic biological systems.
authors.affiliation: Department of Bioengineering, University of California, Berkeley, CA 94720, USA. jblucks@cornell.edu
authors.forename: Julius B
authors.initials: JB
authors.lastname: Lucks
authors.forename: Stefanie A
authors.initials: SA
authors.lastname: Mortimer
authors.forename: Cole
authors.initials: C
authors.lastname: Trapnell
authors.forename: Shujun
authors.initials: S
authors.lastname: Luo
authors.forename: Sharon
authors.initials: S
authors.lastname: Aviran
authors.forename: Gary P
authors.initials: GP
authors.lastname: Schroth
authors.forename: Lior
authors.initials: L
authors.lastname: Pachter
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D019295:Computational Biology; D058893:DNA Barcoding, Taxonomic; D017931:DNA Primers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000706:statistics & numerical data; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D012313:RNA / Q000737:chemistry* / Q000235:genetics*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics; D043262:Ribonuclease P / Q000737:chemistry / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods* / Q000706:statistics & numerical data
chemical_list: D017931:DNA Primers; D015335:Molecular Probes; D016337:RNA, Catalytic; D012313:RNA; D043262:Ribonuclease P