Fluorescence Coincidence Spectroscopy for Single-Molecule Fluorescence Resonance Energy-Transfer Measurements
Fluorescence Coincidence Spectroscopy for Single-Molecule Fluorescence Resonance Energy-Transfer Measurements
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DOI：
10.1021/ac8009092
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发表时间：
2008-11-15
影响因子：
7.4
通讯作者：
Kienerman, David
中科院分区：
文献类型：
--
作者：
Orte, Angel;Clarke, Richard W.;Kienerman, David
单分子荧光共振能量转移（FRET）通常用于探测单个生物分子的不同构象和构象动力学。然而，对原始突发轨迹的分析并不总是简单的。“零峰”的存在和邻近比直方图中高和低FRET效率下峰的偏斜使得直方图的准确评估成为一项具有挑战性的任务。将两个荧光团放置在彼此的最佳范围内的困难进一步加剧了这一点。在这里，我们提出了一种替代的分析方法，处理符合FRET光子爆发的基础上，解决这些问题。此外，我们证明了方法，以提高重合水平，从而FRET测定的准确性：使用双色激发，包括受体荧光团的直接激发;添加远程染料的生物分子，不参与FRET过程;或两者的组合。我们通过研究几个标记的双链DNA样品作为FRET模型的双重激发的优势。Ibis方法将单分子FRET的应用扩展到只有一小部分复合物完全组装的更复杂的生物系统。
Single-molecule fluorescence resonance energy transfer (FRET) is commonly used to probe different conformations and conformational dynamics of single biomolecules. However, the analysis of raw burst traces is not always straightforward. The presence of a "zero peak" and the skewness of peaks at high and low FRET efficiencies in proximity ratio histograms make the accurate evaluation of the histogram a challenging task. This is further compounded by the difficulty associated with siting two fluorophores in optimal range of each other. Here we present an alternative method of analysis, based on handling coincident FRET photon bursts, that addresses these problems. In addition, we demonstrate methods to enhance coincidence levels and thus the accuracy of FRET determination: the use of dual-color excitation, including direct excitation of the acceptor fluorophore; the addition of a remote dye to the biomolecule, not involved in the FRET process; or a combination of the two. We show the advantages of dual excitation by studying several labeled double-stranded DNA samples as FRET models. Ibis method extends the application of single-molecule FRET to more complicated biological systems where only a small fraction of complexes are fully assembled.