Department of Chemical and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden and Department for Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Nucleic Acids Res. 2013 Oct;41(19):e184. doi: 10.1093/nar/gkt755. Epub 2013 Aug 23.
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.
常用荧光染料 YOYO-1（YOYO）已被证明可以在亚化学计量浓度下不均匀地染色 DNA，使用体相技术。我们在这里使用纳米流道和荧光显微镜在单 DNA 分子水平上研究不均匀染色，并证明染料分布是连续的。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，我们展示了如何将不均匀染色用作工具，详细而高效地研究荧光染料如何影响 DNA 的物理性质。我们表明，随着结合的 YOYO 量的增加，DNA 的相对延伸增加在低盐离子强度下更高，并且还推断出天然 DNA 的延伸。我们的研究揭示了 YOYO 如何影响 DNA 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）如何在强约束下影响 DNA。其次，使用不均匀染色的策略对于与蛋白质或配体相互作用的 DNA 的单分子研究具有普遍适用性。
