Dye Dynamics in Three-Color FRET Samples
Dye Dynamics in Three-Color FRET Samples
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DOI：
10.1021/jp3064273
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发表时间：
2012-09-06
影响因子：
3.3
通讯作者：
Kumke, Michael U.
中科院分区：
文献类型：
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作者：
Gehne, Soeren;Flehr, Roman;Kumke, Michael U.
使用先进的分布分析模型仔细分析了两个三色福斯特共振能量转移 (tc-FRET) 系统的时间分辨发射数据（荧光衰减和荧光去偏振），该系统由喹诺酮供体 (D)、作为中继染料的钌络合物 (Ru) 和 Cy5 衍生物 (Cy) 或任选的蒽醌猝灭剂 (Q) 组成。由此，可以获得有关与双链（ds）DNA结合的发色团的灵活性和移动性的信息。特别是基于荧光去偏振分析的距离分布是补充荧光衰减时间分析数据的一种有吸引力的方法。从实验数据中提取的距离分布与可访问体积（AV）模拟确定的距离分布非常一致。此外，研究表明，对于 tc-FRET 系统，在涉及 DNA 等大分子的情况下，在不同时间尺度（例如纳秒与微秒）发射的染料组合对于时间分辨发光数据的分布分析非常有益。在这里，短寿命可以产生有关染料分子本身旋转的信息，而长寿命可以深入了解大分子的整体动力学。
Time-resolved emission data (fluorescence decay and fluorescence depolarization) of two three-color Forster resonance energy transfer (tc-FRET) systems consisting of a carbostyril donor (D), a ruthenium complex (Ru) as relay dye, and a Cy5 derivative (Cy) or, optionally, an anthraquinone quencher (Q) were carefully analyzed using advanced distribution analysis models. Thereby, it is possible to get information on the flexibility and mobility of the chromophores which are bound to double stranded (ds) DNA. Especially the distance distribution based on the analysis of the fluorescence depolarization is an attractive approach to complement data of fluorescence decay time analysis. The distance distributions extracted from the experimental data were in excellent agreement with those determined from accessible volume (AV) simulations. Moreover, the study showed that for tc-FRET systems the combination of dyes emitting on different time scales (e.g., nanoseconds vs microseconds) is highly beneficial in the distribution analysis of time-resolved luminescence data in cases where macromolecules such as DNA are involved. Here, the short lifetimes can yield information on the rotation of the dye molecule itself and the long lifetime can give insight in the overall dynamics of the macromolecule.