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

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.

Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

“…The increased reaction volume may decrease effective local reactant concentrations, reducing effective collision between active reactants. Therefore, higher initial reactant concentrations of NTPs and Mg 2+ both positively affect in vitro transcription yield, consistent with previous reports [18,19,21,43].…”

“…Alternative approaches such as ion exchange [18,19] require specific conditions for different RNA molecules. Previous SEC-based studies [20,21,52] require either toxic phenol/chloroform extraction or addition of proteinase K to remove RNAP.…”

Efficient Large-Scale Preparation and Purification of short Single-Stranded RNA Oligonucleotides

“…Once the optimum Mg 2+ concentration is determined, transcription reactions can be scaled up. Since sufficient amounts of all enzymes participating in the transcription reaction are essential for the preparation of large quantities of RNA, which could become expensive if commercial enzymes are used, both T7 RNA polymerase and inorganic pyrophosphatase, which prevents Mg 2+ depletion during transcription, are prepared in house (11, 20). This also helps reduce the cost large-scale production of small ssRNA oligonucleotides.…”

“…RNA in vitro transcription with T7 RNA polymerase allows the production of milligram quantities of RNA oligonucleotides (7, 8), and some methods for in vitro RNA oligonucleotide preparation and purification are currently available (915). However, the existing approaches either require the design of an RNA for trans-cleavage (14) or include multiple purification steps that lead to product losses (11). Other drawbacks of existing protocols include the requirements for starting sequence, low yield (13), use of RNase H in the transcription reaction (15), and lack of robust ssRNA oligonucleotide purification procedures (10).…”

Nonorthogonal tRNAcysAmber for protein and nascent chain labeling

“…2), which are sufficient for the production of RNCs under common experimental conditions. Therefore, although the purification of overexpressed-tRNA may be more labor intensive than the purification of tRNA transcribed in vitro (Table 4; Lukavsky and Puglisi 2004; Easton et al 2010;Koubek et al 2013), the former is advantageous in its stability.…”