Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography.
We present a simple and fast method for large-scale purification of RNA oligonucleotides suitable for biochemical and structural studies. RNAs are transcribed in vitro with T7 RNA polymerase using linearized plasmid DNA templates. After addition of EDTA, the crude transcription reaction is subjected directly to weak anion-exchange chromatography using DEAE-sepharose to separate the T7 RNA polymerase, unincorporated rNTPs, small abortive transcripts, and the plasmid DNA template from the desired RNA product. The novel method does neither require tedious phenol/chloroform extraction of the T7 RNA polymerase nor denaturation of the RNA, which is desirable especially for larger RNAs. In addition, isotopically labeled rNTPs can be easily recycled from the column flow-through and oligomeric RNA aggregates can be separated from the natively folded monomeric RNA product.
Easton
Laura E
LE
Structural Studies Division, Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
Shibata
Yoko
Y
Lukavsky
Peter J
PJ
D002853:Chromatography, Liquid / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism
D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases