The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions

Abstract

Although RNA-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed RNA generally relies on gel-based methods that are time-consuming, tedious and denature the RNA. Here, we present a reliable procedure for affinity batch purification of RNA, which exploits the high-affinity interaction between the boxB RNA and the N-peptide from bacteriophage λ. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the λBoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/λN-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/λN-fusion proteins and ARiBo tags were tested to optimize RNA yield and purity. The optimized procedure enables one to quickly obtain (3 h) highly pure RNA (>99%) under native conditions and with yields comparable to standard denaturing gel-based protocols. It is widely applicable to a variety of RNAs, including riboswitches, ribozymes and microRNAs. In addition, it can be easily adapted to a wide range of applications that require RNA purification and/or immobilization, including isolation of RNA-associated complexes from living cells and high-throughput applications.

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

“…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.…”

“…The ARiBo tag sequence used here (ARiBo4) (Supplemental Fig. S1) is slightly different from our original ARiBo1 tag (Di Tomasso et al 2011) in that the glmS ribozyme sequence was modified to help reduce misfolding and improve GlcN6P-induced cleavage of ARiBo-fusion RNAs. Using this ARiBo tag, the SLI RNA of the correct size was successfully detected in the elution fractions.…”

“…To investigate the compatibility of the Cse3/CRISPR system with affinity purification of RNA, we modified our ARiBobased affinity purification procedure (Di Tomasso et al 2011) by incorporating a Cse3 cleavage step after the transcription and before affinity immobilization (Fig. 5A).…”

“…Thus, endoribonucleases that do not rely on structural reorganization of the target RNA present a distinct advantage for trimming the ends of RNA transcripts. For example, the VS, HDV, and glmS ribozymes have little sequence or structure requirements 5 ′ of the cleavage site and can be incorporated at the 3 ′ end of an RNA to ensure its 3 ′ -sequence homogeneity (Ferré-D'Amaré and Doudna 1996; Kieft and Batey 2004;Batey and Kieft 2007;Di Tomasso et al 2011). We are unaware of an equivalent ribozyme (or DNAzyme) with minimal sequence and structure requirements 3 ′ of the cleavage site that has been used to achieve 5 ′ -sequence homogeneity.…”

“…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.…”