Probing DNA-protein interactions using single-molecule diffusivity contrast.
Probing DNA-protein interactions using single-molecule diffusivity contrast.
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DOI：
10.1016/j.bpr.2021.100009
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发表时间：
2021-09-08
期刊：
影响因子：
--
通讯作者：
Wang, Quan
中科院分区：
文献类型：
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作者：
Wilson, Hugh;Lee, Miles;Wang, Quan
蛋白质-核酸相互作用的单分子荧光研究需要强大的手段来实时识别单个底物分子的结合状态。在这里，我们展示了广泛用于整体水平的荧光相关光谱和单个(但缓慢扩散的)物种的单粒子示踪的扩散率对比，可以作为一般读数来确定单个DNA分子与溶液中未标记蛋白质的结合状态。我们首先描述了在反布朗电动陷阱中无漂移测量单分子扩散系数的技术基础。然后，我们用蛋白质诱导的荧光增强来交叉验证我们的方法，这是一种流行的技术，可以用单分子灵敏度来检测核酸底物上的蛋白质结合。我们扩展了现有的流体动力学模型框架，将测量的扩散系数与特定的DNA-蛋白质结构联系起来，并获得了测量和预测的扩散系数值之间的良好一致性。最后，我们表明，结合扩散对比和蛋白质诱导的荧光增强，允许同时映射结合的化学计量比和单个DNA-蛋白质复合体上的位置，潜在地增强了相关生物物理过程的单分子视角。
Single-molecule fluorescence investigations of protein-nucleic acid interactions require robust means to identify the binding state of individual substrate molecules in real time. Here, we show that diffusivity contrast, widely used in fluorescence correlation spectroscopy at the ensemble level and in single-particle tracking on individual (but slowly diffusing) species, can be used as a general readout to determine the binding state of single DNA molecules with unlabeled proteins in solution. We first describe the technical basis of drift-free single-molecule diffusivity measurements in an anti-Brownian electrokinetic trap. We then cross-validate our method with protein-induced fluorescence enhancement, a popular technique to detect protein binding on nucleic acid substrates with single-molecule sensitivity. We extend an existing hydrodynamic modeling framework to link measured diffusivity to particular DNA-protein structures and obtain good agreement between the measured and predicted diffusivity values. Finally, we show that combining diffusivity contrast with protein-induced fluorescence enhancement allows simultaneous mapping of binding stoichiometry and location on individual DNA-protein complexes, potentially enhancing single-molecule views of relevant biophysical processes.