Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription

Jiri Koubek; Ku Feng Lin; Yet Ran Chen; Richard Ping Cheng; Joseph Jen Tse Huang

doi:10.1261/rna.038117.113

Strong anion-exchange fast performance liquid chromatography as a versatile tool for preparation and purification of RNA produced by in vitro transcription

¹Institute of Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan
²Department of Chemistry, National Taiwan University, Taipei 10617, Taiwan
³Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Academia Sinica, Nankang, Taipei 11529, Taiwan
⁴Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan

↵5 Present address: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA

Abstract

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.

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↵6 Corresponding authors

E-mail jthuang{at}gate.sinica.edu.tw

E-mail rpcheng{at}ntu.edu.tw

Received January 3, 2013.
Accepted June 25, 2013.

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