Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in-situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present Proximity-Dependent PAINT (PD-PAINT), a method for sub-diffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA labels anchored to the target species. Labeled protein pairs are then imaged with high contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution and the ability to image quantitatively.

中文翻译：

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通过接近依赖性超分辨率显微镜检测蛋白质对的纳米级分布

生物分子（例如蛋白质）之间的相互作用是大多数细胞过程的基础。直接在细胞内可视化这些分子相互作用复合物，精确显示这些相互作用发生的位置并因此改善我们对细胞调节的理解至关重要。用于此类相互作用的原位成像的当前可用的接近敏感测定法产生衍射受限的信号，因此排除了关于相互作用复合物的纳米级分布的信息。相比之下，光学超分辨率成像可提供有关具有纳米分辨率的分子分布的信息，这极大地增进了我们对细胞生物学的理解。然而，当前的超分辨率荧光成像的共定位分析很容易产生假阳性信号，因为蛋白质接近度的检测直接取决于局部光学分辨率。在这里，我们介绍邻近度依赖的PAINT（PD-PAINT），一种对蛋白质对进行亚衍射成像的方法，其中接近度检测与光学分辨率脱钩。通过锚定在靶标物种上的两个DNA标记高度依赖于距离的相互作用来检测邻近度。然后使用DNA-PAINT的超分辨率方法以高对比度和纳米级分辨率对标记的蛋白质对成像。通过粗粒度分子模拟对新技术的基础机制进行了分析，并通过对分离的心肌细胞中的DNA折纸和心脏蛋白表位进行成像实验证明了这一新技术的作用。