PicoGreen 与 dsDNA 的相互作用特征及其结合后荧光增强的起源。
Characterization of PicoGreen interaction with dsDNA and the origin of its fluorescence enhancement upon binding.
机构信息
Institute of Fluorescence, University of Maryland, Baltimore County, Maryland, USA.
出版信息
Biophys J. 2010 Nov 3;99(9):3010-9. doi: 10.1016/j.bpj.2010.09.012.
PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed.
PicoGreen 是一种荧光探针，与溶液中的游离染料相比，它能与双链 DNA 结合形成高发光复合物。这种独特的探针广泛用于 DNA 定量分析，但由于对其物理性质和 DNA 结合特性的了解有限，其在 DNA 和 DNA-蛋白质系统的生物物理分析中的应用有限。在这里，我们研究了 PicoGreen 与 DNA 的结合，以揭示 PicoGreen/DNA 相互作用的起源和模式，特别是静电和非静电相互作用在复合物形成中的作用，以及证明小沟结合特异性。分析游离 PicoGreen 的荧光性质、PG/DNA 复合物的扩散性质以及结合 DNA 时的激发态寿命变化和溶剂极性变化，以及粘度，表明 PicoGreen 在游离状态下的猝灭是由于其分子内动态波动。与 DNA 结合后，嵌入和静电相互作用使染料分子固定，导致其荧光增强 1000 多倍。基于这项研究的结果，提出了 PicoGreen/DNA 复合物形成的模型。
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