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

用于高通量低温电子显微镜研究小分子的DNA纳米结构 单粒子冷冻电子显微镜(Cryo-EM)是一种显示大分子结构的方法 和它们的络合物在接近自然的条件下，不需要大量的样品或去除 X-射线结晶学等替代技术通常需要灵活的区域。最近的改进 在显微镜中，硬件和数据处理软件帮助实现了近原子结构 用低温电子显微镜测定大分子，例如，允许单个氨基酸的可视化 蛋白质靶标的侧链。然而，低温电子显微镜对Small(&lt；100)的结构测定是相当有限的 Kda)大分子。Cryo-EM图像数据对比度低，小颗粒往往缺乏清晰的定义 三维重建的图像对齐步骤所需的结构特征。此外，该方法是 技术挑战、低吞吐量和昂贵--进一步阻碍了它的广泛采用。 我们建议使用DNA纳米技术来开发一套新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创造出前所未有的可溶纳米结构 空间分辨率和化学多功能性的结合。原则上，我们可以在我们的设备上附加任何部分 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 大小从10纳米到1微米不等，但仍然创造出每一个 原子是用原子或近原子分辨率来定义的。 我们将设计和优化百万吨大小的DNA“铰链”纳米结构，这种结构将结合和定位小分子 作为低温电磁成像和层析成像的高对比度基准标记物。我们会 还可以构建DNA“条形码”纳米结构，并将其连接到DNA铰链上进行样本多路传输。我们 将通过确定具有良好特性的DNA结合蛋白的结构来验证我们的方法 之前已经结晶过了。然后我们将与合作者一起研究几个未知的大分子 结构。 这项技术将极大地提高我们解决小分子近原子分辨低温电磁结构的能力。 大分子以高通量的方式。我们将应用我们的方法来研究大分子 与几种人类疾病相关，并期待我们的努力最终将加强基于结构的药物 设计抗击这些疾病的努力。

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.