High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.
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.
Shields
T P
TP
Department of Chemistry and Biochemistry, University of Colorado at Boulder 80309-0215, USA.
Mollova
E
E
Ste Marie
L
L
Hansen
M R
MR
Pardi
A
A
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
D016337:RNA, Catalytic; D015636:Magnesium Chloride; D012313:RNA