title: Native Purification and Analysis of Long RNAs.
abstract: The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation-renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2'-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing.
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.
authors.forename: Isabel
authors.initials: I
authors.lastname: Chillón
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.
authors.forename: Marco
authors.initials: M
authors.lastname: Marcia
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.
authors.forename: Michal
authors.initials: M
authors.lastname: Legiewicz
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.
authors.forename: Fei
authors.initials: F
authors.lastname: Liu
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Somarowthu
authors.affiliation: Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA; Howard Hughes Medical Institute, Chevy Chase, Maryland, USA; Department of Chemistry, Yale University, New Haven, Connecticut, USA. Electronic address: anna.pyle@yale.edu.
authors.forename: Anna Marie
authors.initials: AM
authors.lastname: Pyle
mesh_terms: D000215:Acylation; D002413:Cations, Divalent; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005822:Genetic Vectors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry; D066298:In Vitro Techniques / Q000295:instrumentation / Q000379:methods*; D008274:Magnesium / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry* / Q000378:metabolism; D059370:RNA Folding; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D017423:Sequence Analysis, RNA; D013463:Sulfuric Acid Esters / Q000737:chemistry; D014158:Transcription, Genetic; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D002413:Cations, Divalent; D015335:Molecular Probes; D062085:RNA, Long Noncoding; D013463:Sulfuric Acid Esters; D017665:Hydroxyl Radical; D008274:Magnesium; C007482:dimethyl sulfate
keywords: Analytical ultracentrifugation; Chemical probing; DMS; Long noncoding RNA; Native purification; RNA folding; SHAPE; Secondary structure; Size-exclusion chromatography