Synthesis of biotin-AMP conjugate for 5′ biotin labeling of RNA through one-step in vitro transcription
Synthesis of biotin-AMP conjugate for 5′ biotin labeling of RNA through one-step in vitro transcription
复制标题
DOI：
10.1038/nprot.2008.185
复制
发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Guo, Yanlin
中科院分区：
文献类型：
--
作者：
Huang, Faqing;He, Jun;Guo, Yanlin
生物素标记的RNA在化学、生物和生物医学等领域有着广泛的应用。在这个方案中，我们描述了一种简单的一步体外转录标记5‘RNA生物素的方法。化学合成了生物素-单磷酸腺苷(AMP)偶联物生物素-HDAAMP(腺苷5‘-(6-氨基己基)磷酰胺；其中HDA为1，6-己二胺)。生物素-HDAAMP在T7phi 2.5启动子作用下启动转录，产生5‘标记的RNA，标记效率高。该方法对于大于60个核苷酸的RNA的生物素标记特别有用。此外，该方案提供了一种有吸引力的替代化学合成生物素标记的小于60个核苷酸的小RNA，特别是当所需的RNA量较低时。整个过程，从化学合成到分离生物素标记的RNA，可以在两周内完成。
Biotin-labeled RNA has found broad applications in chemistry, biology and biomedicine. In this protocol, we describe a simple procedure for 5' RNA biotin labeling by one-step in vitro transcription. A biotin-AMP (adenosine 5'-monophosphate) conjugate, biotin-HDAAMP (adenosine 5'-(6-aminohexyl) phosphoramide; where HDA is 1,6-hexanediamine), is chemically synthesized. Transcription initiation by biotin-HDAAMP under the T7 phi 2.5 promoter produces 5' biotin-labeled RNA with high labeling efficiency. The procedure is especially useful for biotin labeling of RNA that is larger than 60 nucleotides. In addition, the protocol provides an attractive alternative to chemical synthesis of biotin-labeled small RNA of less than 60 nucleotides, particularly when the desired quantity of RNA is low. The whole procedure, from chemical syntheses to isolated biotin-labeled RNA, can be completed within 2 weeks.