Molecular arrows: DNA markers for electron cryotomography

细胞过程由称为蛋白质和核酸的生物大分子的复杂协调和动力学控制。它们不是孤立地起作用，而是相互作用并组装形成细胞复合物。了解复合物的结构不仅是了解基础细胞生物学的重要一步，也是了解疾病的重要一步，因为病毒等入侵病原体的蛋白质与宿主细胞蛋白质之间也会形成此类复合物。事实上，要确定病毒的功能，不仅需要了解其自身蛋白质的分子排列，还需要了解它们在病毒生命周期中与宿主细胞的相互作用。特别令人感兴趣的是进入过程，即周期中感染的最早阶段，当病毒首次与宿主细胞接触并将病毒物质引入细胞时。我们项目的目标是获得一个结构视图的进入过程中的一个最大和最复杂的家庭病毒感染人类-疱疹病毒。由这些病毒引起的疾病包括唇疱疹、生殖器溃疡、水泡、失明和危及生命的疾病，包括致命的脑炎、脑膜炎和癌症。由于其在世界范围内的流行、易于传播和相关症状的严重性，该家族构成了一个主要的公共卫生问题。了解病毒进入的最好方法之一是拍摄病毒和细胞的高倍“快照”。在过去的十年中，低温电子显微镜和断层摄影术已成为观察生物复合体的重要工具。利用这些技术，样品使用低温液体快速冷冻，然后用电子轰击，产生许多二维样品的图像，这些图像可以组合成更清晰的三维图像。在断层扫描中，这些数据可以提供细胞和组织的整体组织，并可以在细胞入侵期间捕获病原体。它们在天然环境中含有许多不同的大分子复合物。虽然这些技术已经带来了许多有趣的发现，但提取所有潜在信息可能具有挑战性，因为图像拥挤且对比度较低。为了帮助识别病毒和细胞的特定部分，我们将开发一个“分子箭头”工具包，专门结合一个感兴趣的生物分子。这些分子箭头将由紧密排列的核酸形成，因此它们在蛋白质的背景下脱颖而出。我们将在快速冷冻之前将箭头添加到病毒和细胞中，然后使用电子断层扫描来确定它们的三维结构。这项工作将提供病毒表面进入细胞所需的蛋白质图谱，并允许我们尝试阻止它们进入。利用这些结果，我们将改进目前人类疱疹病毒进入过程的模型，这是确定药物靶点的关键一步。我们的新分子箭头方法将适用于许多生物系统，并将为细胞生物学，医学和其他领域提供重要工具。

Cellular processes are governed by the intricate coordination and dynamics of biological macromolecules called proteins and nucleic acids. These do not act in isolation but rather interact with each other and assemble to form cellular complexes. Understanding the structures of complexes can be an important step not only in understanding basic cell biology but also disease, as such complexes are also formed between the proteins of invading pathogens like viruses and the host cell proteins. Indeed, to determine how a virus functions, knowledge is needed not only about the molecular arrangement of its own proteins but also about their interactions with the host cell over the course of the viral life cycle. Particularly interesting is the entry process, the earliest stage of infection in the cycle, when the virus comes into first contact with the host cell and introduces viral material into the cell. The goal of our project is to gain a structural view of the entry process in one of the largest and most complex families of viruses that infect humans - the Herpesviruses. The conditions caused by these viruses range from cold sores, genital ulcers, and blisters to blindness and life-threatening conditions including fatal encephalitis, meningitis and cancer. This family constitutes a major public health concern due to their worldwide prevalence, ease of spread, and severity of the associated symptoms. One of the best ways to understand viral entry to is to take high-magnification 'snapshots' of viruses and cells. In the last decade, cryogenic electron microscopy and tomography have become important tools for observing biological complexes. With these techniques, samples are rapidly frozen using cryogenic liquids and then bombarded with electrons, yielding many images of the 2-dimensional sample that can be combined into a clearer 3-dimensional picture. In tomography, such data can provide the overall organization of cells and tissues, and can capture pathogens during cell invasion. They contain many different macromolecular complexes in their native environment. Though these techniques have led to many interesting discoveries, extracting all of the potential information can be challenging, as the images are crowded and the contrast is low.To help identify specific parts of the virus and cell, we will develop a toolkit of 'molecular arrows' that bind specifically to one biomolecule of interest. These molecular arrows will be formed from tightly packed nucleic acids, so they stand out against the background of protein. We will add our arrows to viruses and cells before rapid freezing, and then use electron tomography to determine their 3-dimensional structure. This work will provide a map of the proteins on the virus surface needed for entry into cells, and allow us to try to block their entry. Using these results, we will improve current models of the human herpsevirus entry process, a crucial step towards identifying drug targets. Our novel molecular arrow approach will be applicable to many biological systems and will provide an important tool for cell biology, medicine and beyond.