Characterizing the interaction between DNA and GelRed fluorescent stain
Characterizing the interaction between DNA and GelRed fluorescent stain
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DOI：
10.1007/s00249-014-0995-4
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发表时间：
2015-02-01
影响因子：
2
通讯作者：
Rocha, M. S.
中科院分区：
文献类型：
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作者：
Crisafuli, F. A. P.;Ramos, E. B.;Rocha, M. S.
我们进行了单分子拉伸和动态光散射（DLS）实验来表征DNA分子与荧光染色剂GelRed之间的相互作用。单分子拉伸结果表明，当配体浓度达到临界浓度时，DNA-GelRed复合物的持续长度增加，当配体浓度更高时，持续长度减小。另一方面，配合物的轮廓长度作为GelRed浓度的函数单调增加，表明插层是主要的结合机制。为了表征相互作用的物理化学性质，我们使用mcgee -von Hippel结合等温线从轮廓长度数据中提取相互作用的物理化学数据。这样的分析使我们得出结论，GelRed染色实际上是一个双插入物。此外，通过DLS实验研究了DNA-GelRed配合物的有效尺寸随配体浓度的变化，以流体动力学半径测量。通过比较流体动力半径和旋转半径的行为，我们观察到两种技术得到的结果在定性上是一致的，因为后者可以表示为拉伸实验确定的力学性能的函数。
We have performed single-molecule stretching and dynamic light-scattering (DLS) experiments to characterize the interaction between the DNA molecule and the fluorescent stain GelRed. The results from single-molecule stretching show that the persistence length of DNA-GelRed complexes increases as the ligand concentration increases up to a critical concentration, then decreases for higher concentrations. The contour length of the complexes, on the other hand, increases monotonically as a function of GelRed concentration, suggesting that intercalation is the main binding mechanism. To characterize the physical chemistry of the interaction, we used the McGhee-von Hippel binding isotherm to extract physicochemical data for the interaction from the contour length data. Such analysis enabled us to conclude that the GelRed stain is, in fact, a bis-intercalator. In addition, DLS experiments were performed to study the changes of the effective size of the DNA-GelRed complexes, measured as the hydrodynamic radius, as a function of ligand concentration. We observed qualitative agreement between the results obtained from the two techniques by comparing the behavior of the hydrodynamics radius and the radius of gyration, because the latter quantity can be expressed as a function of mechanical properties determined from the stretching experiments.