Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription.
Here we demonstrate the use of strong anion-exchange fast performance liquid chromatography (FPLC) as a simple, fast, and robust method for RNA production by in vitro transcription. With this technique, we have purified different transcription templates from unreacted reagents in large quantities. The same buffer system could be used to readily remove nuclease contamination from the overexpressed pyrophosphatase, the important reagent for in vitro transcription. In addition, the method can be used to monitor in vitro transcription reactions to enable facile optimization of reaction conditions, and we have compared the separation performance between strong and weak anion-exchange FPLC for various transcribed RNAs, including the Diels-Alder ribozyme, the hammerhead ribozyme tRNA, and 4.5S RNA. The functionality of the purified tRNA(Cys) has been confirmed by the aminoacylation assay. Only the purification by strong anion-exchange FPLC has led to the enrichment of the functional tRNA from run-off transcripts as revealed by both enzymatic and electrophoretic analysis.
Jiri
J
Koubek
Ku Feng
KF
Lin
Yet Ran
YR
Chen
Richard Ping
RP
Cheng
Joseph Jen Tse
JJ
Huang
D000838:Anions / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D066298:In Vitro Techniques; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D012329:RNA, Bacterial / Q000302:isolation & purification; D016337:RNA, Catalytic / Q000302:isolation & purification; D012343:RNA, Transfer / Q000302:isolation & purification; D014158:Transcription, Genetic
C409968:4.5S RNA; D000838:Anions; C518747:Diels-Alder ribozyme; D012329:RNA, Bacterial; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D012313:RNA; D012343:RNA, Transfer; D011755:Pyrophosphatases
RNA purification; fast performance liquid chromatography (FPLC); in vitro transcription; strong anion exchange; tRNA