Life Sciences Center, Institute of Biotechnology, Vilnius University, Saulėtekio av. 7, LT-10257 Vilnius, Lithuania.
Langmuir. 2021 Mar 23;37(11):3428-3437. doi: 10.1021/acs.langmuir.1c00066. Epub 2021 Mar 9.
Over the past 20 years, single-molecule methods have become extremely important for biophysical studies. These methods, in combination with new nanotechnological platforms, can significantly facilitate experimental design and enable faster data acquisition. A nanotechnological platform, which utilizes a flow-stretch of immobilized DNA molecules, called DNA Curtains, is one of the best examples of such combinations. Here, we employed new strategies to fabricate a flow-stretch assay of stably immobilized and oriented DNA molecules using a protein template-directed assembly. In our assay, a protein template patterned on a glass coverslip served for directional assembly of biotinylated DNA molecules. In these arrays, DNA molecules were oriented to one another and maintained extended by either single- or both-end immobilization to the protein templates. For oriented both-end DNA immobilization, we employed heterologous DNA labeling and protein template coverage with the antidigoxigenin antibody. In contrast to single-end immobilization, both-end immobilization does not require constant buffer flow for keeping DNAs in an extended configuration, allowing us to study protein-DNA interactions at more controllable reaction conditions. Additionally, we increased the immobilization stability of the biotinylated DNA molecules using protein templates fabricated from traptavidin. Finally, we demonstrated that double-tethered Soft DNA Curtains can be used in nucleic acid-interacting protein (e.g., CRISPR-Cas9) binding assay that monitors the binding location and position of individual fluorescently labeled proteins on DNA.
在过去的 20 年中，单分子方法已成为生物物理研究的重要手段。这些方法与新的纳米技术平台相结合，可以显著促进实验设计并实现更快的数据采集。一种纳米技术平台，利用固定化 DNA 分子的流拉伸，称为 DNA 窗帘，就是这种组合的最佳示例之一。在这里，我们采用了新的策略，使用蛋白质模板指导的组装来制造稳定固定和定向 DNA 分子的流拉伸测定法。在我们的测定中，玻璃载玻片上的蛋白质模板用于生物素化 DNA 分子的定向组装。在这些阵列中，DNA 分子彼此定向，并通过单端或两端固定到蛋白质模板来保持伸展。对于定向的两端 DNA 固定化，我们采用了异源 DNA 标记和抗生物素蛋白模板覆盖。与单端固定化不同，两端固定化不需要持续的缓冲液流来保持 DNA 处于伸展状态，从而允许我们在更可控的反应条件下研究蛋白质-DNA 相互作用。此外，我们使用来自链霉亲和素的蛋白质模板增加了生物素化 DNA 分子的固定化稳定性。最后，我们证明了双束缚 Soft DNA 窗帘可用于核酸相互作用蛋白（例如，CRISPR-Cas9）结合测定，以监测荧光标记蛋白在 DNA 上的结合位置和位置。
