Biolayer interferometry for DNA-protein interactions.
Biolayer interferometry for DNA-protein interactions.
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DOI：
10.1371/journal.pone.0263322
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发表时间：
2022
期刊：
影响因子：
3.7
通讯作者：
Van Dyke MW
中科院分区：
文献类型：
--
作者：
Barrows JK;Van Dyke MW
生物层干涉测量（BLI）是一种广泛应用的用于真实的实时测定大分子相互作用动力学的技术。利用从生物传感器尖端反射的白色光的干涉图案的变化，BLI可以确定蛋白质-蛋白质的结合参数（例如，抗体-底物动力学）或蛋白质-小分子（例如，药物发现）相互作用。然而，BLI分析的一个不太受重视的应用是DNA-蛋白质相互作用。DNA结合蛋白在细胞生物学中起着巨大的作用，控制着包括转录、DNA复制和DNA修复在内的关键过程。了解蛋白质如何与DNA相互作用往往提供了重要的洞察其生物学功能，和新的技术来测定DNA-蛋白质相互作用的广泛兴趣。目前，缺乏一个详细的协议，利用BLI的DNA-蛋白质相互作用。在下面的方案中，我们描述了使用BLI和生物素化DNA探针来确定转录因子与特定DNA序列的结合动力学。实验步骤包括生物素化DNA探针的产生、BLI实验的执行以及通过科学绘图和统计软件（例如，GraphPad Prism）。尽管本方案中使用的示例性实验涉及原核转录因子，但该技术可以容易地翻译为任何DNA结合蛋白。还提出了BLI研究DNA结合蛋白的陷阱和潜在的解决方案。
Biolayer interferometry (BLI) is a widely utilized technique for determining macromolecular interaction dynamics in real time. Using changes in the interference pattern of white light reflected off a biosensor tip, BLI can determine binding parameters for protein-protein (e.g., antibody-substrate kinetics) or protein-small molecule (e.g., drug discovery) interactions. However, a less-appreciated application for BLI analysis is DNA-protein interactions. DNA-binding proteins play an immense role in cellular biology, controlling critical processes including transcription, DNA replication, and DNA repair. Understanding how proteins interact with DNA often provides important insight into their biological function, and novel technologies to assay DNA-protein interactions are of broad interest. Currently, a detailed protocol utilizing BLI for DNA-protein interactions is lacking. In the following protocol, we describe the use of BLI and biotinylated-DNA probes to determine the binding kinetics of a transcription factor to a specific DNA sequence. The experimental steps include the generation of biotinylated-DNA probes, the execution of the BLI experiment, and data analysis by scientific graphing and statistical software (e.g., GraphPad Prism). Although the example experiment used throughout this protocol involves a prokaryotic transcription factor, this technique can be easily translated to any DNA-binding protein. Pitfalls and potential solutions for investigating DNA-binding proteins by BLI are also presented.
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影响因子：
3.7
作者：
Van Dyke MW;Beyer MD;Clay E;Hiam KJ;McMurry JL;Xie Y
通讯作者：
Xie Y
影响因子：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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