Madariaga-Marcos Julene, Aldag Pierre, Kauert Dominik J, Seidel Ralf
Molecular Biophysics Group, Peter Debye Institute for Soft Matter Physics, Universität Leipzig, Leipzig, Germany.
Methods Mol Biol. 2024;2694:421-449. doi: 10.1007/978-1-0716-3377-9_20.
Combining force spectroscopy and fluorescence microscopy provides a substantial improvement to the single-molecule toolbox by allowing simultaneous manipulation and orthogonal characterizations of the conformations, interactions, and activity of biomolecular complexes. Here, we describe a combined magnetic tweezers and total internal reflection fluorescence microscopy setup to carry out correlated single-molecule fluorescence spectroscopy and force/twisting experiments. We apply the setup to reveal the DNA interactions of the CRISPR-Cas surveillance complex Cascade. Single-molecule fluorescence of a labeled Cascade allows to follow the DNA association and dissociation of the protein. Simultaneously, the magnetic tweezers probe the DNA unwinding during R-loop formation by the bound Cascade complexes. Furthermore, the setup supports observation of 1D diffusion of protein complexes on stretched DNA molecules. This technique can be applied to study a vast range of protein-DNA interactions.
将力谱学与荧光显微镜相结合，通过同时操纵和对生物分子复合物的构象、相互作用及活性进行正交表征，极大地改进了单分子工具箱。在此，我们描述了一种结合磁镊和全内反射荧光显微镜的装置，用于进行相关的单分子荧光光谱和力/扭转实验。我们应用该装置揭示CRISPR-Cas监测复合物Cascade与DNA的相互作用。标记的Cascade的单分子荧光能够追踪蛋白质与DNA的结合和解离。同时，磁镊探测结合的Cascade复合物在R环形成过程中DNA的解旋。此外，该装置支持观察蛋白质复合物在拉伸的DNA分子上的一维扩散。这种技术可应用于研究广泛的蛋白质-DNA相互作用。
