期刊
NUCLEIC ACIDS RESEARCH
卷 39, 期 3, 页码 -出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkq1084
关键词
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资金
- Canadian Institutes for Health Research (CIHR) [MOP-64341, MOP-86502]
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 lambda. The RNA of interest is synthesized with an ARiBo tag, which consists of an activatable ribozyme (the glmS ribozyme) and the lambda BoxB RNA. This ARiBo-fusion RNA is initially captured on Glutathione-Sepharose resin via a GST/lambda N-fusion protein, and the RNA of interest is subsequently eluted by ribozyme self-cleavage using glucosamine-6-phosphate. Several GST/lambda N-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.
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