B CUBE Center for Molecular Bioengineering, TU Dresden, Tatzberg 41, 01307, Dresden, Germany.
Department of Bionanoscience, Delft University of Technology, 2629HZ, Delft, Netherlands.
Nat Commun. 2023 Oct 16;14(1):6511. doi: 10.1038/s41467-023-42232-3.
Single-molecule FRET (smFRET) has become a versatile tool for probing the structure and functional dynamics of biomolecular systems, and is extensively used to address questions ranging from biomolecular folding to drug discovery. Confocal smFRET measurements are amongst the widely used smFRET assays and are typically performed in a single-well format. Thus, sampling of many experimental parameters is laborious and time consuming. To address this challenge, we extend here the capabilities of confocal smFRET beyond single-well measurements by integrating a multiwell plate functionality to allow for continuous and automated smFRET measurements. We demonstrate the broad applicability of the multiwell plate assay towards DNA hairpin dynamics, protein folding, competitive and cooperative protein-DNA interactions, and drug-discovery, revealing insights that would be very difficult to achieve with conventional single-well format measurements. For the adaptation into existing instrumentations, we provide a detailed guide and open-source acquisition and analysis software.
单分子荧光共振能量转移（smFRET）已经成为探测生物分子系统结构和功能动力学的一种通用工具，广泛用于解决从生物分子折叠到药物发现等各种问题。共焦 smFRET 测量是广泛使用的 smFRET 分析方法之一，通常在单孔格式中进行。因此，许多实验参数的采样既费力又耗时。为了解决这一挑战，我们通过将多孔板功能集成到共焦 smFRET 中，将其功能扩展到单孔测量之外，从而能够进行连续和自动的 smFRET 测量。我们展示了多孔板分析在 DNA 发夹动力学、蛋白质折叠、竞争性和合作性蛋白-DNA 相互作用以及药物发现方面的广泛适用性，揭示了用传统的单孔格式测量非常难以获得的见解。为了适应现有的仪器，我们提供了详细的指南和开源采集及分析软件。
