PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy
PEGylated surfaces for the study of DNA-protein interactions by atomic force microscopy
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用于通过原子力显微镜研究 DNA-蛋白质相互作用的聚乙二醇化表面
DOI：
10.1101/680561
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Akpinar B
中科院分区：
文献类型：
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作者：
Akpinar B
DNA-蛋白质相互作用对于细胞功能至关重要，在基因表达调节和基因组维护中发挥着关键作用。原子力显微镜 (AFM) 能够在近生理缓冲液中以纳米分辨率可视化 DNA-蛋白质相互作用，但它要求 DNA 粘附在固体基质的表面。当在生物学相关的蛋白质浓度下工作时，这会带来一个问题，其中蛋白质可能大量过量地存在于溶液中；因此，许多生物物理相关信息可能被与底层底物结合的非特异性蛋白质所遮挡。在这里，我们探索使用 PLLx-b-PEGy 嵌段共聚物实现 DNA 在云母表面的选择性吸附，用于 AFM 研究。通过改变嵌段共聚物中赖氨酸和乙二醇残基的数量，我们在含有高浓度链霉亲和素的溶液中通过 AFM 成像观察到，DNA 在用 PLL10-b-PEG113/PLL1000-2000 混合物功能化的云母上选择性吸附。我们通过使用生物素化 DNA 和链霉亲和素证明，这种选择性吸附延伸至 DNA-蛋白质复合物，并且尽管溶液中存在过量的未结合链霉亲和素，但仍可以明确地区分 DNA 结合的链霉亲和素。最后，我们将其应用于核酶 PARP1，通过液体 AFM 解析单个 PARP1 分子与 DNA 的结合。
DNA–protein interactions are vital to cellular function, with key roles in the regulation of gene expression and genome maintenance. Atomic force microscopy (AFM) offers the ability to visualize DNA–protein interactions at nanometre resolution in near-physiological buffers, but it requires that the DNA be adhered to the surface of a solid substrate. This presents a problem when working in biologically relevant protein concentrations, where proteins may be present in large excess in solution; much of the biophysically relevant information can therefore be occluded by non-specific protein binding to the underlying substrate. Here we explore the use of PLLx-b-PEGy block copolymers to achieve selective adsorption of DNA on a mica surface for AFM studies. Through varying both the number of lysine and ethylene glycol residues in the block copolymers, we show selective adsorption of DNA on mica that is functionalized with a PLL10-b-PEG113/PLL1000–2000 mixture as viewed by AFM imaging in a solution containing high concentrations of streptavidin. We show – through the use of biotinylated DNA and streptavidin – that this selective adsorption extends to DNA–protein complexes and that DNA-bound streptavidin can be unambiguously distinguished in spite of an excess of unbound streptavidin in solution. Finally, we apply this to the nuclear enzyme PARP1, resolving the binding of individual PARP1 molecules to DNA by in-liquid AFM.