Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules
Long-Lived Intracellular Single-Molecule Fluorescence Using Electroporated Molecules
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DOI：
10.1016/j.bpj.2013.09.057
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发表时间：
2013-12-03
影响因子：
3.4
通讯作者：
Kapanidis, Achillefs N.
中科院分区：
文献类型：
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作者：
Crawford, Robert;Torella, Joseph P.;Kapanidis, Achillefs N.
生物分子在体内的研究对于了解它们在自然生物环境中的功能至关重要。一种强大的方法涉及将感兴趣的分子与荧光蛋白融合以研究它们的表达、定位和作用;然而，通过使用有机荧光团，这种研究的范围将大大增加，有机荧光团比荧光蛋白更小，更耐光。在这里，我们描述了一个简单的，多功能的，高通量的方法来内化的DNA片段和蛋白质标记的有机荧光团到活大肠杆菌通过采用电穿孔。我们研究了体内单分子水平上内化分子的拷贝数、扩散曲线和结构，与绿色荧光蛋白相比，能够将单分子观察时间延长两个数量级，从而可以连续监测从几秒到几分钟发生的分子过程。我们还利用有机荧光团的理想特性，在活细菌的细胞质中进行单分子福斯特共振能量转移测量，包括DNA和蛋白质。最后，我们证明了标记的蛋白质和DNA内化到酵母酿酒酵母，一个模型真核系统。我们的方法将拓宽单分子荧光在微生物中可解决的生物学问题的范围。
Studies of biomolecules in vivo are crucial to understand their function in a natural, biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins to study their expression, localization, and action; however, the scope of such studies would be increased considerably by using organic fluorophores, which are smaller and more photostable than their fluorescent protein counterparts. Here, we describe a straightforward, versatile, and high-throughput method to internalize DNA fragments and proteins labeled with organic fluorophores into live Escherichia coli by employing electroporation. We studied the copy numbers, diffusion profiles, and structure of internalized molecules at the single-molecule level in vivo, and were able to extend single-molecule observation times by two orders of magnitude compared to green fluorescent protein, allowing continuous monitoring of molecular processes occurring from seconds to minutes. We also exploited the desirable properties of organic fluorophores to perform single-molecule Forster resonance energy transfer measurements in the cytoplasm of live bacteria, both for DNA and proteins. Finally, we demonstrate internalization of labeled proteins and DNA into yeast Saccharomyces cerevisiae, a model eukaryotic system. Our method should broaden the range of biological questions addressable in microbes by single-molecule fluorescence.