Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, United Kingdom.
Biophys J. 2013 Dec 3;105(11):2439-50. doi: 10.1016/j.bpj.2013.09.057.
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 Förster 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.
在体内研究生物分子对于理解它们在自然生物环境中的功能至关重要。一种强大的方法是将感兴趣的分子融合到荧光蛋白中，以研究它们的表达、定位和作用；然而，如果使用比荧光蛋白小且更稳定的有机荧光染料，此类研究的范围将大大增加。在这里，我们描述了一种简单、通用且高通量的方法，通过电穿孔将带有有机荧光染料标记的 DNA 片段和蛋白质内化到活大肠杆菌中。我们在体内从单分子水平研究了内化分子的拷贝数、扩散分布和结构，并能够将单分子观察时间延长两个数量级，与绿色荧光蛋白相比，从而能够连续监测从几秒钟到几分钟的分子过程。我们还利用有机荧光染料的理想特性，在活细菌的细胞质中进行了 DNA 和蛋白质的单分子Förster 共振能量转移测量。最后，我们证明了标记的蛋白质和 DNA 可以内化到酵母 Saccharomyces cerevisiae 中，这是一种模型真核系统。我们的方法应该拓宽通过单分子荧光在微生物中可解决的生物学问题的范围。
