Single molecule approaches for studying spliceosome assembly and catalysis.
Single molecule approaches for studying spliceosome assembly and catalysis.
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研究剪接体组装和催化的单分子方法。
DOI：
10.1007/978-1-62703-980-2_17
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Hoskins,AaronA
中科院分区：
文献类型：
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作者：
Anderson,EricG;Hoskins,AaronA
剪接和剪接体组装的单分子测定可以提供对前 mRNA 加工的独特见解，从而补充其他技术。这些实验的关键是为每个实验制备荧光分子（前体 mRNA 和剪接体成分）和钝化载玻片。在这里，我们描述了如何通过夹板 RNA 连接产生荧光 RNA，并通过细胞裂解物中的 SNAP 标签蛋白产生荧光剪接体亚基。然后，我们描述了如何用聚乙二醇钝化载玻片，以便在具有基于物镜的全内反射荧光 (TIRF) 光学器件的倒置显微镜上使用。最后，我们描述了如何将前 mRNA 固定在钝化的载玻片表面上，并引入 SNAP 标记的细胞裂解物，通过单分子荧光分析剪接体组装。
Single molecule assays of splicing and spliceosome assembly can provide unique insights into pre-mRNA processing that complement other technologies. Key to these experiments is the fabrication of fluorescent molecules (pre-mRNAs and spliceosome components) and passivated glass slides for each experiment. Here we describe how to produce fluorescent RNAs by splinted RNA ligation and fluorescent spliceosome subunits by SNAP-tagging proteins in cell lysate. We then depict how to passivate glass slides with polyethylene glycol for use on an inverted microscope with objective-based total internal reflection fluorescence (TIRF) optics. Finally, we describe how to tether the pre-mRNA onto the passivated slide surface and introduce the SNAP-tagged cell lysate for analysis of spliceosome assembly by single molecule fluorescence.