Protein-induced fluorescence enhancement (PIFE) is an increasingly used approach to investigate DNA-protein interactions at the single molecule level. The optimal probe for this type of application is highly photostable, has a high absorption extinction coefficient, and has a moderate fluorescence quantum yield that increases significantly when the dye is in close proximity to a large macromolecule such as a protein. So far, the green-absorbing symmetric cyanine known as Cy3 has been the probe of choice in this field because the magnitude of the increase observed upon protein binding (usually 2-4 -fold) is large enough to allow for the analysis of protein dynamics on the inherently noisy single-molecule signals. Here, we report the characterization of the photophysical properties of the red-absorbing hemicyanine dye Dy-630 in the context of its potential application as a single-molecule PIFE probe. The behavior of Dy-630 in solution is similar to that of Cy3; the fluorescence quantum yield and lifetime of Dy-630 increase with increasing viscosity, and decrease with increasing temperature indicating the existence of an activated nonradiative process that depopulates the singlet state of the dye. As in the case of Cy3, the results of transient spectroscopy experiments are consistent with the formation of a photoisomer that reverts to the ground state thermally in the microsecond timescale. Unfortunately, experiments with DNA samples paint a more complex scenario. As in the case of Cy3, the fluorescence quantum yield of Dy-630 increases significantly when the dye interacts with the DNA bases, but in the case of Dy-630 attachment to DNA results in an already long fluorescence lifetime that does not provide a significant window for the protein-induced enhancement observed with Cy3. Although we show that Dy-630 may not be well-suited for PIFE, our results shed light on the optimal design principles for probes for PIFE applications.

中文翻译：

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半花菁Dy-630的光物理性质及其作为生物物理应用中的单分子荧光探针的潜力。

蛋白质诱导的荧光增强（PIFE）是一种在单分子水平上研究DNA-蛋白质相互作用的越来越广泛的方法。这种类型的应用的最佳探针具有高度的光稳定性，高的吸收消光系数和适中的荧光量子产率，当染料与大分子（例如蛋白质）非常接近时，荧光量子产率会显着提高。到目前为止，被称为Cy3的吸收绿色对称花青素已成为该领域的首选探针，因为蛋白质结合后观察到的增加幅度（通常为2-4倍）足够大，可以进行蛋白质动力学分析固有噪声的单分子信号。这里，我们报道了在其作为单分子PIFE探针的潜在应用的背景下，吸收红色的半菁染料Dy-630的光物理性质的表征。Dy-630在溶液中的行为类似于Cy3。Dy-630的荧光量子产率和寿命随着粘度的增加而增加，而随着温度的升高而降低，表明存在活化的非辐射过程，该过程使染料的单重态减少。与Cy3的情况一样，瞬态光谱实验的结果与光异构体的形成一致，该光异构体在微秒级的时间内会热恢复为基态。不幸的是，使用DNA样本进行的实验描绘了一个更为复杂的场景。与Cy3一样 当染料与DNA碱基相互作用时，Dy-630的荧光量子产率会显着增加，但是如果Dy-630与DNA附着，则荧光寿命已经很长，不能为蛋白质诱导的增强提供重要的窗口用Cy3观察到。尽管我们证明Dy-630可能不适合PIFE，但我们的结果揭示了PIFE应用探头的最佳设计原理。