title: High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.
abstract: An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado at Boulder 80309-0215, USA.
authors.forename: T P
authors.initials: TP
authors.lastname: Shields
authors.forename: E
authors.initials: E
authors.lastname: Mollova
authors.forename: L
authors.initials: L
authors.lastname: Ste Marie
authors.forename: M R
authors.initials: MR
authors.lastname: Hansen
authors.forename: A
authors.initials: A
authors.lastname: Pardi
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015636:Magnesium Chloride / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008957:Models, Genetic; D008969:Molecular Sequence Data; D012313:RNA / Q000138:chemical synthesis / Q000302:isolation & purification* / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
chemical_list: D016337:RNA, Catalytic; D015636:Magnesium Chloride; D012313:RNA