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.

用于小型大分子的高通量冷冻EM研究的DNA纳米结构 单粒子冷冻电子显微镜（Cryo-EM）是一种可视化大分子结构的方法 以及它们在近本条件下的复合物，而无需大量样品或去除 X射线晶体学等替代技术通常需要的柔性区域。最近的改进 在显微镜硬件和数据处理软件中有助于实现近原子结构 通过冷冻EM测定大分子，例如，单个氨基酸的可视化 蛋白质靶标的侧链。但是，冷冻EM在结构测定小（<100）方面非常有限（<100） KDA）大分子。冷冻EM图像数据的对比度很低，而小颗粒通常缺乏明确的明确的对比度 3D重建的图像对齐步骤所需的结构特征。另外，该方法是 从技术上讲，挑战，低通量和昂贵 - 造成阻碍其宽度采用的采用。 我们建议使用DNA纳米技术来开发一套新颖的工具来克服大小和吞吐量 冷冻EM的局限性。 DNA纳米技术使我们能够以前所未有的方式创建坚固的纳米结构 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附加到我们的设备上 只要可以与DNA耦合或DNA结合分子。我们可以建立具有尺寸的结构 尺寸从10纳米到1千米，但仍会创建结构，其中每个位置的位置 原子用原子或接近原子分辨率定义。 我们将设计和优化MEGADALTON大小的DNA“铰链”纳米结构，这些纳米结构将绑定和正向小 大分子并用作冷冻成像和层析成像的高对比度基准标记。我们将 还构建DNA“条形码”纳米结构，并将其连接到DNA铰链上，以进行样品多路复用。我们 将通过确定具有良好表征的DNA结合蛋白的结构来验证我们的方法 然后，我们将与合作者合作研究几种未知的大分子 结构。 这项技术将大大提高我们解决小型分辨率的小型EM结构的能力 高通量方式的大分子。我们将应用我们的方法研究大分子 与几种人类疾病有关，并期望我们的努力最终将增强基于结构的药物 设计努力来打击这些疾病。