HPLC purification of in vitro transcribed long RNA.
HPLC purification of in vitro transcribed long RNA.
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DOI：
10.1007/978-1-62703-260-5_3
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发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Kariko, Katalin
中科院分区：
文献类型：
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作者：
Weissman, Drew;Pardi, Norbert;Kariko, Katalin
利用噬菌体RNA聚合酶体外转录DNA是目前生产长序列特异性RNA的最有效方法。虽然该反应可以产生大量的RNA，但由于聚合酶的各种不需要的活性，它含有杂质。在这里，我们描述了一种容易进行的HPLC纯化，从体外转录的RNA中去除多种污染物，并且是可扩展的。纯化的RNA以更高的水平翻译，特别是在原代细胞和体内。含有抑制RNA介导的先天免疫传感器活化的修饰核苷的RNA的HPLC纯化产生具有上级翻译能力的非免疫原性RNA。
In vitro transcription of DNA with phage RNA polymerases is currently the most efficient method to produce long sequence-specific RNA. While the reaction can yield large quantities of RNA, it contains impurities due to various unwanted activities of the polymerases. Here, we described an easily performed HPLC purification that removes multiple contaminants from in vitro transcribed RNA and is scalable. The purified RNA is translated at much greater levels, especially in primary cells and in vivo. HPLC purification of RNA containing modified nucleosides that suppress RNA-mediated activation of innate immune sensors leads to a non-immunogenic RNA with superior translational capacity.