Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes.
Visualization of nonengineered single mRNAs in living cells using genetically encoded fluorescent probes.
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DOI：
10.1021/ac2009405
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发表时间：
2011-06
影响因子：
7.4
通讯作者：
Toshimichi Yamada;Hideaki Yoshimura;Asumi Inaguma;T. Ozawa
中科院分区：
文献类型：
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作者：
Toshimichi Yamada;Hideaki Yoshimura;Asumi Inaguma;T. Ozawa
活细胞中的单 mRNA 成像是阐明其精确定位和动态的有用技术。我们开发了一种使用基因编码探针以单分子敏感性可视化活细胞中内源 mRNA 的方法。使用人PUMILIO1的RNA结合蛋白(PUM-HD)来识别目标mRNA、β-肌动蛋白mRNA的碱基序列。两个 PUM-HD 通过氨基酸突变进行修饰，以特异性结合目标 mRNA 的串联 8 碱基序列。由于每个 PUM-HD 均与增强型绿色荧光蛋白 (EGFP) 的氨基端和羧基端片段连接，因此探针在与 β-肌动蛋白 mRNA 结合后通过重建 EGFP 片段来发射荧光。用全内反射荧光显微镜监测在 mRNA 上重建的 EGFP。结果表明，活细胞中的每个荧光点代表单个 β-肌动蛋白 mRNA，并且各个 β-肌动蛋白 mRNA 的不同空间和时间运动是可视化的。我们还估计了活细胞中单个 mRNA 沿微管运动的平均速度。该方法广泛适用于追踪活细胞天然状态下的各种感兴趣的 mRNA，具有单 mRNA 敏感性。
Single mRNA imaging in live cells is a useful technique to elucidate its precise localization and dynamics. We developed a method for visualizing endogenous mRNAs in living cells with single molecule sensitivity using genetically encoded probes. An RNA-binding protein of human PUMILIO1 (PUM-HD) was used for recognizing base sequences of a target mRNA, β-actin mRNA. Two PUM-HDs were modified by amino acid mutations to bind specifically to tandem 8-base sequences of the target mRNA. Because each PUM-HD was connected with amino- and carboxyl-terminal fragments of enhanced green fluorescent protein (EGFP), the probes emit fluorescence by reconstitution of EGFP fragments upon binding to β-actin mRNAs. The EGFP reconstituted on the mRNAs was monitored with a total internal reflection fluorescence microscope. Results show that each fluorescent spot in live cells represented a single β-actin mRNA and that distinct spatial and temporal movement of the individual β-actin mRNAs was visualized. We also estimated the average velocity of the movement of the single mRNAs along microtubules in live cells. This method is widely applicable to tracking various mRNAs of interest in the native state of living cells with single-mRNA sensitivity.