Structural Studies Division, Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom.
RNA. 2010 Mar;16(3):647-53. doi: 10.1261/rna.1862210. Epub 2010 Jan 25.
We present a simple and fast method for large-scale purification of RNA oligonucleotides suitable for biochemical and structural studies. RNAs are transcribed in vitro with T7 RNA polymerase using linearized plasmid DNA templates. After addition of EDTA, the crude transcription reaction is subjected directly to weak anion-exchange chromatography using DEAE-sepharose to separate the T7 RNA polymerase, unincorporated rNTPs, small abortive transcripts, and the plasmid DNA template from the desired RNA product. The novel method does neither require tedious phenol/chloroform extraction of the T7 RNA polymerase nor denaturation of the RNA, which is desirable especially for larger RNAs. In addition, isotopically labeled rNTPs can be easily recycled from the column flow-through and oligomeric RNA aggregates can be separated from the natively folded monomeric RNA product.
我们提出了一种简单快速的方法，用于大规模纯化适用于生化和结构研究的 RNA 寡核苷酸。使用线性化质粒 DNA 模板，T7 RNA 聚合酶在体外转录 RNA。加入 EDTA 后，粗转录反应直接进行弱阴离子交换层析，使用 DEAE-琼脂糖从所需的 RNA 产物中分离 T7 RNA 聚合酶、未结合的 rNTP、小的无意义转录本和质粒 DNA 模板。该新方法既不需要繁琐的 T7 RNA 聚合酶酚/氯仿抽提，也不需要 RNA 变性，这对于较大的 RNA 尤为重要。此外，同位素标记的 rNTP 可以从柱流中轻松回收，寡聚 RNA 聚集体可以与天然折叠的单体 RNA 产物分离。
