Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA
Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA
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DOI：
10.1093/nar/gkt755
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发表时间：
2013-10-01
影响因子：
14.9
通讯作者：
Westerlund, Fredrik
中科院分区：
文献类型：
--
作者：
Nyberg, Lena;Persson, Fredrik;Westerlund, Fredrik
常用的荧光染料YOYO-1(YOYO)已被证明可以在亚化学计量浓度下对DNA进行异质染色。在这里，我们使用纳米流体通道和荧光显微镜，研究了单DNA分子水平上的异质染色，并证明了染料的连续分布。YOYO在DNA上的平衡非常缓慢，但可以通过增加离子强度和/或温度来加速。此外，我们还演示了如何使用异质染色作为一种工具，详细和省时地研究荧光染料如何影响DNA的物理性质。结果表明，在低离子强度下，DNA延伸量随YOYO结合量的增加而增大，并推测了天然DNA的延伸量。我们的研究揭示了YOYO如何影响DNA的物理性质的重要信息，但它也有更广泛的应用。首先，它揭示了阳离子嵌入剂，如潜在的DNA药物，如何在强限制下影响DNA。其次，使用异质染色的策略对于DNA与蛋白质或配体相互作用的单分子研究是普遍使用的。
The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.