A general method for rapid and nondenaturing purification of RNAs.
A key bottleneck in RNA structural studies is preparing milligram quantities of RNA, and current techniques have changed little in over a decade. To address this, we have developed an affinity tag-based purification method of RNA oligonucleotides. The tag is attached to the 3'-end of almost any desired RNA sequence, allowing for the rapid and specific removal of the RNA of interest directly from in vitro transcription reactions using an affinity column to which a specific RNA-binding protein has been attached. Following a wash, the RNA of interest is eluted by the addition of imidazole to the column, activating a mutant HdeltaV ribozyme incorporated into the tag. The affinity column can then be rapidly regenerated using conditions that release the protein-RNA tag interaction without denaturing the protein. To demonstrate that this method rapidly generates high-quality RNA, we have transcribed, purified, and generated diffraction-quality crystals of a mutant form of the Tetrahymena thermophila P4-P6 domain in a 48-h time period.
Kieft
Jeffrey S
JS
Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. jeffrey.kieft@uchsc.edu
Batey
Robert T
RT
D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D007438:Introns; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D016808:Tetrahymena thermophila / Q000737:chemistry / Q000235:genetics; D020124:Thermotoga maritima / Q000737:chemistry / Q000235:genetics
D000345:Affinity Labels; D016053:RNA, Protozoan; D012313:RNA