DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules

用于小大分子高通量冷冻电镜研究的 DNA 纳米结构

• 批准号: 10115755
• 负责人: Shawn M Douglas
• 金额: $ 40.38万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115755
• 关键词: 3-Dimensional; Adoption; Amino Acids; Bar Codes; Binding; Biological Process; Chemicals; Complex; Computer software; Coupled; Cryoelectron Microscopy; Crystallization; DNA; DNA Binding; DNA-Binding Proteins; Data; Devices; Dimensions; Disease; Drug Design; Excision; Health; Human; Image; Individual; Location; Methods; Microscope; Nanostructures; Nanotechnology; Play; Proteins; Resolution; Sampling; Side; Structure; Techniques; Technology; Visualization; Work; X-Ray Crystallography; base; combat; computerized data processing; design; flexibility; human disease; improved; macromolecule; nanometer; novel; particle; reconstruction; tomography; tool

DNA Nanostructures for High-Throughput Cryo-EM Studies of Small Macromolecules Single particle cryo-electron microscopy (cryo-EM) is an approach for visualizing structures of macromolecules and their complexes at near-native conditions without the need for large sample quantities or removal of flexible regions often required for alternative techniques such as X-ray crystallography. Recent improvements in microscope hardware and data-processing software have helped to achieve near-atomic structure determination of macromolecules by cryo-EM allowing, for example, the visualization of individual amino acid side chains of protein targets. However, cryo-EM is quite limited for structure determination of small (<100 kDa) macromolecules. Cryo-EM image data are low contrast, and small particles often lack well-defined structural features required for the image alignment step of 3D reconstruction. Additionally, the method is technically challenging, low-throughput, and expensive—further hindering its widespread adoption. We propose to use DNA nanotechnology to develop a novel suite of tools to overcome the size and throughput limitations of cryo-EM. DNA nanotechnology allows us to create soluble nanostructures with an unprecedented combination of spatial resolution and chemical versatility. In principle, we can attach any moiety to our devices as long as it can be coupled to DNA, or to a DNA-binding molecule. We can build structures with dimensions ranging from 10 nanometers to 1 micrometer in size, but still create structures in which the location of every atom is defined with atomic or near-atomic resolution. We will design and optimize megadalton-sized DNA “hinge” nanostructures that will bind and orient small macromolecules and serve as high-contrast fiducial markers for cryo-EM imaging and tomography. We will also construct DNA “barcode” nanostructures and attach them to the DNA hinges for sample multiplexing. We will validate our methods by determining the structure of a well-characterized DNA-binding protein that has been previously crystalized. We will then work with collaborators to study several macromolecules of unknown structure. This technology will hugely improve our ability to solve near-atomic resolution cryo-EM structures of small macromolecules in a high-throughput manner. We will apply our method to study macromolecules with relevance to several human diseases, and expect that our efforts will ultimately enhance structure-based drug design efforts to combat those diseases.

DNA纳米结构用于小分子的高通量冷冻电镜研究 单粒子低温电子显微镜（cryo-EM）是一种用于可视化大分子结构的方法 在接近天然的条件下，不需要大量的样品或去除 灵活的区域通常需要替代技术，如X射线晶体学。最近有所改善 在显微镜硬件和数据处理软件的帮助下， 通过冷冻EM测定大分子，例如，允许单个氨基酸的可视化 蛋白质靶点的侧链。然而，冷冻电镜对小分子（<100 kDa）大分子。Cryo-EM图像数据对比度低，小颗粒往往缺乏明确的 3D重建的图像对准步骤所需的结构特征。此外，该方法 技术上具有挑战性、低吞吐量和昂贵-进一步阻碍了其广泛采用。 我们建议使用DNA纳米技术开发一套新的工具，以克服尺寸和吞吐量 冷冻电镜的局限性。DNA纳米技术使我们能够创造出前所未有的可溶性纳米结构， 空间分辨率和化学多功能性的组合。原则上，我们可以将任何部分连接到我们的设备上， 只要它能与DNA或DNA结合分子偶联。我们可以建造有维度的结构 从10纳米到1微米的大小，但仍然创造了结构，其中每个位置 原子以原子或近原子分辨率定义。 我们将设计和优化兆道尔顿大小的DNA“铰链”纳米结构， 高对比度的基准标记物，用于冷冻EM成像和断层扫描。我们将 还构建DNA“条形码”纳米结构，并将它们附着到DNA铰链上用于样品复用。我们 将通过确定一种具有良好特征的DNA结合蛋白的结构来验证我们的方法， 之前已经结晶。然后，我们将与合作者一起研究几种未知的大分子， 结构 这项技术将极大地提高我们解决近原子分辨率的低温EM结构的能力， 以高通量的方式对大分子进行纯化。我们将应用我们的方法来研究大分子， 与几种人类疾病的相关性，并期望我们的努力将最终提高基于结构的药物 努力对抗这些疾病。