A general method for rapid and nondenaturing purification of RNAs
A general method for rapid and nondenaturing purification of RNAs
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DOI：
10.1261/rna.7040604
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发表时间：
2004-06-01
期刊：
影响因子：
4.5
通讯作者：
Batey, RT
中科院分区：
文献类型：
--
作者：
Kieft, JS;Batey, RT
RNA结构研究的一个关键瓶颈是制备毫克量的RNA，而目前的技术在十多年来几乎没有变化。为了解决这个问题，我们开发了一种基于亲和标签的RNA寡核苷酸纯化方法。该标签连接到几乎任何所需RNA序列的3 '末端，允许使用已经连接有特异性RNA结合蛋白的亲和柱直接从体外转录反应中快速和特异性地去除感兴趣的RNA。洗涤后，通过向柱中添加咪唑来洗脱感兴趣的RNA，从而激活掺入标签中的突变体HdeltaV核酶。然后可以使用释放蛋白质-RNA标签相互作用而不使蛋白质变性的条件快速再生亲和柱。为了证明这种方法快速产生高质量的RNA，我们已经转录，纯化，并产生衍射质量的晶体的突变形式的嗜热四膜虫P4-P6域在48小时的时间段。
A key bottleneck in RNA structural studies is preparing milligram quantities of RNA, and current techniques have changed little in over a decade. To address this, we have developed an affinity tag-based purification method of RNA oligonucleotides. The tag is attached to the 3'-end of almost any desired RNA sequence, allowing for the rapid and specific removal of the RNA of interest directly from in vitro transcription reactions using an affinity column to which a specific RNA-binding protein has been attached. Following a wash, the RNA of interest is eluted by the addition of imidazole to the column, activating a mutant HdeltaV ribozyme incorporated into the tag. The affinity column can then be rapidly regenerated using conditions that release the protein-RNA tag interaction without denaturing the protein. To demonstrate that this method rapidly generates high-quality RNA, we have transcribed, purified, and generated diffraction-quality crystals of a mutant form of the Tetrahymena thermophila P4-P6 domain in a 48-h time period.