Measuring local conformations and conformational disorder of (Cy3)2 dimer labeled DNA fork junctions using absorbance, circular dichroism and two-dimensional fluorescence spectroscopy
Measuring local conformations and conformational disorder of (Cy3)2 dimer labeled DNA fork junctions using absorbance, circular dichroism and two-dimensional fluorescence spectroscopy
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DOI：
10.1039/c8fd00245b
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发表时间：
2019-07-01
影响因子：
3.4
通讯作者：
Marcus, Andrew H.
中科院分区：
文献类型：
--
作者：
Heussman, Dylan;Kittell, Justin;Marcus, Andrew H.
单链 (ss)-双链 (ds) 连接附近的 DNA 糖磷酸骨架可能在广泛的构象分布范围内波动，以允许在这些位点发挥作用的基因组调节蛋白正确结合。在这项工作中，我们使用吸光度、圆二色性 (CD) 和二维荧光光谱 (2DFS) 来研究发色团标记的 DNA 构建体中的局部构象和构象紊乱。这些构建体采用荧光发色团 Cy3 的二聚体，该二聚体在 ss-ds DNA 叉连接处特异性地掺入 DNA 链的糖磷酸主链中。我们表明，可以分析这些数据以确定 (Cy3)(2) 二聚体的局部构象以及构象无序程度。我们的分析采用了本质状态荷斯坦-弗兰克尔哈密顿模型，该模型考虑了每个 Cy3 发色团内的内部电子振动运动，以及将两个发色团耦合在一起的共振电子相互作用。我们的结果表明，这种方法可以普遍应用于了解 ss-ds DNA 叉连接处的局部主链构象和构象紊乱。
The sugar-phosphate backbone of DNA near single-stranded (ss)-double-stranded (ds) junctions likely fluctuates within a broad distribution of conformations to permit the proper binding of genome regulatory proteins that function at these sites. In this work we use absorbance, circular dichroism (CD), and two-dimensional fluorescence spectroscopy (2DFS) to study the local conformations and conformational disorder within chromophore-labeled DNA constructs. These constructs employ dimers of the fluorescent chromophore Cy3 that are site-specifically incorporated into the sugar-phosphate backbones of DNA strands at ss-ds DNA fork junctions. We show that these data can be analyzed to determine the local conformations of the (Cy3)(2) dimer, and the degree of conformational disorder. Our analysis employs an essential-state Holstein-Frenkel Hamiltonian model, which takes into account the internal electronic-vibrational motions within each Cy3 chromophore, and the resonant electronic interaction that couples the two chromophores together. Our results suggest that this approach may be applied generally to understand local backbone conformation and conformational disorder at ss-ds DNA fork junctions.