A new super-resolution proximity assay to probe RNA transcription condensates

虽然每个细胞在细胞核中以基因组DNA的形式具有相同的遗传信息，但在给定细胞中使用（“转录”）这些信息的哪一部分来制造蛋白质是受到密切调控的，从而使细胞能够实现大量不同的功能。这种转录调控的基础机制尚未完全理解，尽管它们对每个生物体都具有根本的重要性。最近发现，在细胞核内有专门的微观区域，参与遗传转录的分子集中在那里。为了确定这些微观区域或“凝聚物”的作用，我们将研究哪些分子在其中相互作用，以及当细胞暴露于应力时这些相互作用如何变化。由于微观凝聚物的多样性和非常小的尺寸，使用传统方法很难检测和量化微观凝聚物中的相互作用。因此，我们将开发和应用一种新的显微镜技术，以非常高的空间分辨率解剖分子相互作用，以便我们可以直接看到（i）哪些分子相互作用，（ii）这些分子相互作用的位置以及（iii）有多少分子以这种方式相互作用，所有这些都在微观凝聚物中。为了达到这些严格的要求，我们将使用纳米技术的方法，采用合成DNA序列来测试两个分子是否彼此接近（比如说在10 nm以内），并以一系列称为“光学超分辨率显微镜”的显微镜技术提供的非常高的分辨率对其成像，该显微镜技术允许在~该项目将侧重于通过使用DNA纳米技术和超分辨率成像的原理来调整和增强称为ePD-PAINT（增强型邻近依赖性PAINT）的新技术。ePD-PAINT将首先用合成测试样品进行验证，该样品由DNA本身制成，并被称为“DNA折纸”，类似于纸张折叠技术，将DNA分子折叠成定义的分子模式。然后，改进的ePD-PAINT方法将在生物细胞中使用测定法进行验证，其中可以操纵蛋白质以诱导分子相互作用，以响应小化学品雷帕霉素的类似物，雷帕霉素也可用作免疫抑制剂。为了完成验证，将使用ePD-PAINT技术进行的测量与经典的生物化学蛋白质相互作用测定进行比较。我们将使用ePD-PAINT直接在显微浓缩物中研究参与转录的几个关键蛋白质之间的相互作用，并测试当转录受损时这些相互作用如何变化，或者通过在转录期间进行的步骤中进行干预，或者通过将细胞暴露于外部应激，已知所述外部应激在细胞响应并适应外部约束时改变转录。这些发现将对基础细胞生物学产生重大影响，并使人们能够更深入地了解基因转录机制集中区域的蛋白质-蛋白质相互作用。此外，它们可能会成为基因表达调控的一个重要新机制的基础。这不仅对所有细胞生物学都很重要，而且还可能为疾病期间发生错误时操纵该过程的新策略提供信息。值得注意的是，许多已知在冷凝物中积累的蛋白质（包括本文将研究的BRD 4）与癌症和神经退行性疾病有关。

Although each cell has the same genetic information in the cell nucleus in the form of genomic DNA, which parts of this information are used ("transcribed") in a given cell to make proteins is closely regulated, allowing cells to fulfil a large number of different functions. The mechanisms that underlie the regulation of this transcription are incompletely understood despite their fundamental importance for every organism. Recently it has been discovered that there are specialised microscopic regions within the cell nucleus where molecules involved in genetic transcription are concentrated. To establish the roles of these microscopic regions or "condensates" we will study which molecules interact in them and how these interactions change when the cell is exposed to stress.Detecting and quantifying interactions within the microscopic condensates is difficult using traditional methods due to their multiplicity and very small size. We will therefore develop and apply a new microscopy technology to dissect the molecular interactions with very high spatial resolution so that we can directly see (i) which molecules interact, (ii) where these molecules interact and (iii) how many molecules interact in this way, all within the microscopic condensates. To achieve these stringent requirements we will use methods of nanotechnology that employ synthetic DNA sequences to test if two molecules are close to each other (say within 10 nm) and image this with the very high resolution provided by a range of microscopy techniques termed "optical super-resolution microscopy" which allow detection and localisation of such protein pairs to within ~ 10 nm.This project will focus on adapting and enhancing the new technology, termed ePD-PAINT (enhanced proximity-dependent PAINT), by using the principles of DNA nanotechnology and super-resolution imaging. ePD-PAINT will be first validated with synthetic test samples, made of DNA itself, and termed "DNA origami" in analogy with the art of paper folding, folding DNA molecules to defined molecular patterns. The improved ePD-PAINT approach will then be validated in biological cells using assays where proteins can be manipulated to induce molecular interactions in response to an analogue of the small chemical rapamycin, which is also used as an immune-suppressant. To complete validation, the measurements with the ePD-PAINT technology will be compared to classical biochemical protein interaction assays.We will investigate the interactions between several key proteins involved in transcription using ePD-PAINT directly in the microscopic condensates and test how these interactions change when transcription is impaired, either by interventions in the steps performed during transcription or by exposing the cell to external stress which is known to change transcription as the cell responds and adapts to the external constraints.These findings will have significant impact on basic cell biology and enable a deeper understanding of protein-protein interactions in regions where the gene transcription machinery is concentrated. Furthermore, they may come to underpin a major new mechanism underlying the regulation of gene expression. Not only is this important for all of cell biology, but it may also inform novel strategies to manipulate the process when errors occur during disease. Of note, many of the proteins known to accumulate in condensates (including BRD4 as will be studied here) are implicated in cancer and neurodegeneration.