Physical and Synthetic Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152, Planegg-Martinsried, Germany.
Plant Molecular Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, Großhadernerstr. 2-4, 82152, Planegg-Martinsried, Germany.
Chemphyschem. 2021 Aug 4;22(15):1566-1583. doi: 10.1002/cphc.202000935. Epub 2021 Jun 29.
The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.
荧光技术的应用对包括成像、生化分析、DNA 测序和医疗技术在内的各个研究领域都产生了巨大的影响。这得益于具有优化的光物理和化学性质的众多商业染料的发展。然而，关于染料的化学结构以及用于生物偶联的连接子的信息通常仍然是一个秘密。这可能会给研究应用带来问题，因为在这些应用中，需要了解染料结构才能预测或理解（不期望的）染料-靶标相互作用，或者建立染料-靶标复合物的结构模型。在这里，我们使用光学光谱学、质谱、NMR 光谱和分子动力学模拟相结合的方法，研究了 Alexa Fluor（Alexa Fluor 555 和 647）和 AF 系列（AF555、AF647、AFD647）染料的分子结构和光谱性质。基于可用的数据和已发表的 AF 和 Cy 染料结构，我们提出了 Alexa Fluor 555 的结构，并对 AF555 的结构进行了改进。我们还解决了关于 Alexa Fluor 647 马来酰亚胺连接子组成的相互矛盾的报告。我们还通过连续波吸收和发射光谱、量子产率测定、自由和蛋白质结合染料的荧光寿命和各向异性光谱，对 Alexa Fluor 和 AF 染料进行了全面比较。所有这些数据都支持这样一种观点，即 Alexa Fluor 和 AF 染料具有菁染料核心，是 Cy3 和 Cy5 的衍生物。此外，我们在单分子 FRET 应用中比较了 Alexa Fluor 555 和 Alexa Fluor 647 与其结构类似物 AF555 和 AF(D)647。在使用交替激光激发的基于溶液的 smFRET 实验中，这两对染料都表现出了优异的性能。通过分子动力学模拟研究了染料-蛋白质相互作用的微小差异。我们的研究结果清楚地表明，在 smFRET 研究或其他荧光应用中，AF 荧光团是 Alexa 和 Cy 染料的一种有吸引力的替代品。
