DNA Nanotechnology platform for high-throughput cryo-EM studies of small proteins

 描述（由申请人提供）：单粒子低温电子显微镜（cryo-EM）正在成为一种强大的技术，用于可视化大分子结构，而不需要X射线晶体学和NMR等替代技术通常所需的大量样品。最近的技术进步有助于实现近原子结构测定的大分子的冷冻EM。然而，用于小分子（<200 kDa）结构测定的冷冻-EM是相当有限的。Cryo-EM图像数据对比度低，并且小颗粒通常缺乏3D重建的图像对准步骤所需的明确定义的结构特征。此外，该方法在技术上具有挑战性，低通量和昂贵-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，以克服冷冻EM的大小和吞吐量的限制。DNA纳米技术使我们能够创造出前所未有的空间分辨率和化学多功能性相结合的可溶性纳米结构。原则上，我们可以将任何部分连接到我们的设备上，只要它具有DNA结合结构域或它可以与DNA偶联。首先，我们将设计和优化DNA“框架”纳米结构，将结合和定向小DNA结合蛋白，并作为高对比度的基准标记冷冻EM成像和断层扫描。我们还将构建DNA“条形码”纳米结构，并将其连接到DNA框架上以进行样品复用。我们将通过确定一种先前已结晶的称为TALE的特征良好的DNA结合蛋白的结构来验证我们的方法。然后，我们将与我们的合作者合作，首次确定称为LRH-1的核受体的结构，该受体已知在干细胞分化，发育和许多其他重要的细胞过程中发挥重要作用。其次，我们将设计和优化DNA“棱镜”纳米结构，以连接重组抗原结合片段（Fab），这些片段将结合和定向不自然结合DNA的小蛋白质。为了将Fab连接到DNA棱柱，我们将Fab与DNA偶联，然后将其结合到DNA棱柱。我们将把DNA条形码连接到DNA棱镜上，用于样品的多重检测。我们将通过确定一种被称为EGFP的绿色荧光蛋白变体的结构来验证我们的方法。这项技术将极大地提高我们以高通量方式解决小蛋白质的近原子分辨率cryo-EM结构的能力。我们将应用我们的方法来研究与人类疾病相关的蛋白质，并期望我们的努力最终将增强基于结构的药物设计工作，以对抗这些疾病。

 DESCRIPTION (provided by applicant): Single particle cryo-electron microscopy (cryo-EM) is emerging as powerful technique for visualizing structures of macromolecules without the need for large sample quantities often required for alternative techniques such as X-ray crystallography and NMR. Recent technological advances have helped to achieve near-atomic structure determination of macromolecules by cryo-EM. However, cryo-EM for structure determination of small (<200 kDa) macromolecules is quite limited. 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 has a DNA binding domain or it can be coupled to DNA. First, we will design and optimize DNA "frame" nanostructures that will bind and orient small DNA binding proteins 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 frames for sample multiplexing. We will validate our methods by determining the structure of a well-characterized DNA-binding protein called TALE that has been previously crystalized. We will then work with our collaborators to determine first time the structure of nuclear receptor called LRH-1 that is known to play essential roles in stem cell differentiation, development, and many other vital cellular processes. Second, we will design and optimize DNA "prism" nanostructures to attach recombinant antigen-binding fragments (Fabs) that will bind and orient small proteins that do not naturally bind to DNA. To attach Fabs to DNA prisms, we will couple Fabs to DNA, which will then bind to DNA prisms. We will attach DNA barcodes to DNA prisms for sample multiplexing. We will validate our approach by determining the structure of a well- characterized green fluorescent protein variant called EGFP. This technology will hugely improve our ability to solve near-atomic resolution cryo-EM structures of small proteins in a high-throughput manner. We will apply our method to study proteins with relevance to human diseases, and expect that our efforts will ultimately enhance structure-based drug design efforts to combat those diseases.