A general method for rapid and cost-efficient large-scale production of 5' capped RNA.
A general method for rapid and cost-efficient large-scale production of 5' capped RNA.
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DOI：
10.1261/rna.056614.116
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发表时间：
2016-09
期刊：
影响因子：
--
通讯作者：
Sprangers R
中科院分区：
文献类型：
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作者：
Fuchs AL;Neu A;Sprangers R
真核生物mRNA的5′帽结构对于前体mRNA的加工、mRNA的输出、翻译起始和mRNA的稳定性是不可或缺的。尽管如此重要，涉及加帽RNA的结构和生物物理研究是具有挑战性的，并且由于缺乏制备足够量mRNA的通用方法而罕见。在这里，我们表明，牛痘加帽酶可用于生产加帽RNA的量，所需的大规模结构研究。因此，我们设计了一个有效的表达和纯化方案的牛痘加帽酶。使用这种方法，可以以成本有效的方式增加反应规模，其中加帽RNA的产率仅取决于可用的未加帽RNA靶标的量。使用大量的RNA底物，我们表明，加帽反应的效率在很大程度上是独立的序列，长度和二级结构的RNA，这使得我们的方法普遍适用。我们证明，加帽的RNA可以直接用于定量生物物理研究，包括荧光各向异性和高分辨率NMR光谱。与13 C-甲基标记的S-腺苷甲硫氨酸组合，可以标记RNA中的甲基用于甲基TROSY NMR光谱。最后，我们证明了我们的方法可以大量产生cap-0和cap-1 RNA。总之，我们在这里介绍了一个通用的和简单的方法，打开了新的手段，蛋白质和酶的结构和功能的研究复杂的加帽RNA。
The eukaryotic mRNA 5′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and high-resolution NMR spectroscopy. In combination with 13C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.