Shehzad Sadaf, Kim HyeongJun
Department of Physics and Astronomy, University of Texas Rio Grande Valley, Edinburg, Texas 78539, USA.
BMB Rep. 2025 Jan;58(1):41-51. doi: 10.5483/BMBRep.2024-0177.
Single-molecule techniques allow researchers to investigate individual molecules and obtain unprecedented details of the heterogeneous nature of biological entities. They play instrumental roles in studying DNA-protein interactions due to the ability to visualize DNA or proteins and to manipulate individual DNA molecules by applying force or torque. Here, we describe single-molecule DNA-flow stretching assays as hybrid tools that combine forces with fluorescence. We also review how widely these assays are utilized in elucidating working mechanisms of DNA-binding proteins. Additionally, we provide a brief explanation of various efforts to prepare DNA substrates with desired internal protein-binding sequences. More complicated needs for DNA-protein interaction research have led to improvements in single-molecule DNA flow-stretching techniques. Several DNA flow-stretching variants such as DNA curtain, DNA motion capture assays, and protein-induced fluorescence enhancement (PIFE) are introduced in this mini review. Singlemolecule DNA flow-stretching assays will keep contributing to our understanding of how DNA-binding proteins function due to their multiplexed, versatile, and robust capabilities. [BMB Reports 2025; 58(1): 41-51].
单分子技术使研究人员能够研究单个分子，并获得有关生物实体异质性的前所未有的细节。由于能够可视化DNA或蛋白质，并通过施加力或扭矩来操纵单个DNA分子，它们在研究DNA-蛋白质相互作用中发挥着重要作用。在这里，我们将单分子DNA流拉伸分析描述为一种将力与荧光相结合的混合工具。我们还回顾了这些分析在阐明DNA结合蛋白工作机制方面的广泛应用。此外，我们简要解释了为制备具有所需内部蛋白质结合序列的DNA底物所做的各种努力。对DNA-蛋白质相互作用研究更复杂的需求推动了单分子DNA流拉伸技术的改进。本综述介绍了几种DNA流拉伸变体，如DNA幕、DNA运动捕获分析和蛋白质诱导荧光增强（PIFE）。单分子DNA流拉伸分析将因其多重、通用和强大的能力，继续有助于我们理解DNA结合蛋白的功能。[《BMB报告》2025年；58(1):41-51]
