The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions.
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.
Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, H3C 3J7 Canada.
Geneviève
G
Di Tomasso
Philipe
P
Lampron
Pierre
P
Dagenais
James G
JG
Omichinski
Pascale
P
Legault
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D005821:Genetic Techniques; D007202:Indicators and Reagents; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification*; D016337:RNA, Catalytic; D012367:RNA, Viral / Q000737:chemistry
D007202:Indicators and Reagents; D016337:RNA, Catalytic; D012367:RNA, Viral; D012313:RNA