Affinity Purification of RNA Using an ARiBo Tag

Abstract

The increased awareness of the importance of RNA in biology, illustrated by the recent attention given to RNA interference research and applications, has spurred structural and functional investigations of RNA. For these studies, the traditional purification method for in vitro transcribed RNA is denaturing polyacrylamide gel electrophoresis. However, gel-based procedures denature the RNA and can be very tedious and time-consuming. Thus, several alternative schemes have been developed for fast non-denaturing purification of RNA transcribed in vitro. In a recent report, a quick affinity purification procedure was developed for RNAs transcribed with a 3'-ARiBo tag and shown to provide RNA with exceptionally high purity and yield. The ARiBo tag contains the λboxB RNA and the glmS ribozyme, allowing immobilization on GSH-Sepharose resin via a λN-GST fusion protein and elution by activation of the glmS ribozyme with glucosamine-6-phosphate. This Chapter outlines the experimental details for affinity batch purification of RNAs using ARiBo tags. Although the procedure was originally developed for purification of a stable purine riboswitch mutant, it is demonstrated here for purification of the terminal loop of the let-7g precursor miRNA, an important target of the pluripotency factor Lin28.

Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags

“…For VS ribozyme cleavage, the Avapl ribozyme was prepared by in vitro T7 RNAP transcription from the pAvapl-ARiBo1 plasmid derived from pAvapl (Bouchard et al 2008;Di Tomasso et al 2012a), linearized with EcoRI, and purified by affinity, as previously described (Di Tomasso et al 2011, 2012a. The VS ribozyme cleavage step was performed for 30 min at 37°C in 400 µL of VS cleavage buffer (50 mM HEPES at pH 7.5, 25 mM MgCl 2 , 50 mM KCl, 0.5 U RNasin [Promega], and 9.75 nM Avapl ribozyme) followed by an incubation of 5 min at room temperature prior to centrifugation.…”

“…Given that gel purification is a tedious and long procedure, we were interested in speeding up the process using batch affinity purification with an ARiBo tag (Di Tomasso et al 2011, 2012a. The first SLI RNA tested was SLI(2) (Fig.…”

“…In contrast, the Cse3/CRISPR system is well suited for batch affinity purification using the ARiBo tag, and the combination of the CRISPR and ARiBo tags allows for rapid production of RNA with homogeneity at both the 5 ′ and the 3 ′ ends. The practicality of the ARiBo-based affinity purification has already been demonstrated for several RNAs, including the small SLI RNA hairpin (29 nt) (this study), the let-7g pre-miRNA (46 nt) (Di Tomasso et al 2012a), a purine riboswitch variant (69 nt) (Di Tomasso et al 2011), and the Neurospora VS ribozyme (138 nt) (this study). Here, we described two approaches to ensure that the affinity-purified RNA has a homogeneous 5 ′ end, either choosing an appropriate GNN/ANN starting sequence or incorporating the Cse3/CRISPR system in the purification scheme.…”

“…Recently, we reported a quick procedure for affinity batch purification of in vitro-transcribed RNA using a 3 ′ -ARiBo tag (Di Tomasso et al 2011, 2012a) composed of an activatable ribozyme, the glmS ribozyme, and the λBoxB RNA. The λboxB RNA allows immobilization on glutathione-Sepharose (GSH-Sepharose) resin via a λN-GST fusion protein, whereas the glmS ribozyme is activated with glucosamine-6-phosphate (GlcN6P) for RNA elution.…”

ARiBo pull-down for riboproteomic studies based on label-free quantitative mass spectrometry

“…Over the past few years, our laboratory has optimized the ARiBo procedure for affinity purification of in vitro synthesized RNAs with a 3 ′ -ARiBo tag, which contains an Activatable glmS Ribozyme and the BoxB RNA from bacteriophage λ (Di Tomasso et al 2011Tomasso et al , 2012a. The λboxB RNA part allows the specific immobilization of an ARiBo-tagged RNA on Glutathione-Sepharose (GSH-Sepharose) resin via its high affinity to the λN-glutathione-S-transferase (GST) fusion protein, whereas the glmS ribozyme part can be activated by glucosamine-6-phosphate (GlcN6P) to liberate the RNA of interest and concomitantly produce a homogeneous 3 ′ -end.…”