Exploring RNA transcription and turnover in vivo by using click chemistry
Exploring RNA transcription and turnover in vivo by using click chemistry
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DOI：
10.1073/pnas.0808480105
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发表时间：
2008-10-14
影响因子：
11.1
通讯作者：
Salic, Adrian
中科院分区：
文献类型：
--
作者：
Jao, Cindy Y.;Salic, Adrian
我们描述了一种检测细胞中 RNA 合成的化学方法，该方法基于尿苷类似物 5-乙炔尿苷 (EU) 生物合成掺入新转录的 RNA，平均每 35 个尿苷残基总 RNA 掺入一次。通过使用铜 (I) 催化的环加成反应（通常称为“点击”化学）与荧光叠氮化物，然后进行显微成像，可以快速、高灵敏度地检测 EU 标记的细胞 RNA。我们展示了这种方法在培养细胞中的应用，其中我们检查了不同长度的 EU 脉冲后大量 RNA 的周转率。我们还使用 EU 来测定整个动物中各种组织的转录率，包括切片和整体染色。我们发现不同组织和器官内不同细胞类型之间的总转录率差异很大。
We describe a chemical method to detect RNA synthesis in cells, based on the biosynthetic incorporation of the uridine analog 5-ethynyluridine (EU) into newly transcribed RNA, on average once every 35 uridine residues in total RNA. EU-labeled cellular RNA is detected quickly and with high sensitivity by using a copper (I)-catalyzed cycloaddition reaction (often referred to as "click" chemistry) with fluorescent azides, followed by microscopic imaging. We demonstrate the use of this method in cultured cells, in which we examine the turnover of bulk RNA after EU pulses of varying lengths. We also use EU to assay transcription rates of various tissues in whole animals, both on sections and by whole-mount staining. We find that total transcription rates vary greatly among different tissues and among different cell types within organs.