Single-molecule imaging at high fluorophore concentrations by local activation of dye.
Single-molecule imaging at high fluorophore concentrations by local activation of dye.
复制标题
通过染料的局部激活在高荧光团浓度下进行单分子成像。
DOI：
10.1016/j.bpj.2014.12.019
复制
发表时间：
2015
影响因子：
3.4
通讯作者：
vanOijen,AntoineM
中科院分区：
文献类型：
--
作者：
Geertsema,HylkjeJ;Schulte,AartjeC;Spenkelink,LisanneM;McGrath,WilliamJ;Morrone,SeamusR;Sohn,Jungsan;Mangel,WalterF;Robinson,Andrew;vanOijen,AntoineM
单分子荧光显微镜是一种强大的工具，用于观察生物分子相互作用的高空间和时间分辨率。然而，检测来自高于高水平背景荧光的单个标记蛋白的荧光信号仍然具有挑战性。由于这个原因，体外测定中标记蛋白的浓度通常保持低于其体内浓度。在这里，我们提出了一种新的荧光成像技术，通过该技术可以在高的生理相关浓度下真实的时间观察单个荧光分子。该技术需要用不同的荧光团标记蛋白质及其大分子底物。利用短距离能量转移机制，只有那些与底物结合的蛋白质的荧光被激活。这种方法通过用嵌入染料标记DNA底物，激发染料，并使用来自染料的能量转移来激活仅与DNA结合的那些标记的DNA结合蛋白的荧光来证明。这样的实验设计使我们能够观察Cy5标记的干扰素诱导蛋白16与DNA的序列无关的相互作用，以及在数百纳摩尔Cy5荧光团的背景存在下，Cy5标记的腺病毒蛋白酶在DNA上通过一维扩散的滑动。
Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.