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: 10552488
• 负责人: Shawn M Douglas
• 金额: $ 41.98万
• 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 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10552488
• 关键词: 3-Dimensional; Address; Amino Acids; Antifungal Therapy; Bar Codes; Binding; Binding Sites; Biological Process; Complex; Computer Models; Computer Simulation; Computer software; Cryoelectron Microscopy; Custom; DNA; DNA Binding; DNA-Binding Proteins; Devices; Diabetes Mellitus; Docking; Drug resistance; Engineering; Excision; Future; Generations; Health; Human; Image; Individual; Inflammatory Bowel Diseases; Link; Malignant neoplasm of pancreas; Microscopy; Modification; Molecular; Nanostructures; Nanotechnology; Nuclear Receptors; Play; Preparation; Proteins; Rotation; Sampling; Side; Structure; System; Techniques; Variant; Visualization; X-Ray Crystallography; Yeasts; design; flexibility; human disease; improved; instrument; macromolecule; malignant breast neoplasm; non-alcoholic fatty liver disease; particle; prevent; reconstruction; tool; transcription factor

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 the removal of flexible regions often required for alternative techniques such as X-ray crystallography. Improvements in microscopy hardware and 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, structure determination by cryo-EM remains challenging for many macromolecules. Small (<100 kDa) particles can be especially difficult to study because they lack well-defined structural features required for the image alignment step of 3D reconstruction. To address these challenges, we constructed tiny goniometers—instruments that precisely orient objects—using DNA nanotechnology. Our molecular goniometers can dock a single DNA-binding protein onto a linear double-helix stage. With the power of DNA origami design, we can make several goniometer variants, each with user-defined tilt and rotation angles of the protein-DNA complex. The goniometers include asymmetric barcode domains. Thus, we can bind and precisely orient small DNA-binding proteins, easily identify them on the grid, correctly assign the angle priors, and rapidly solve their structure. We have validated our approach by obtaining a 6.5-Å structure of an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. We propose to apply our DNA origami molecular goniometers to characterize several biomedically important DNA-binding proteins of unknown structure that remain recalcitrant to cryo-EM analysis. We will study nuclear receptors linked to pancreatic and breast cancer, inflammatory bowel disease, diabetes, and nonalcoholic fatty liver disease. We will also study small transcription factors that regulate pleiotropic drug resistance in yeast. That structural understanding may aid in developing future antifungal therapies. We will build custom molecular goniometers for each protein target with the appropriate DNA-binding site and any necessary structural modifications. We will continue to optimize our first-generation designs to address outstanding challenges related to sample preparation and extend the devices for general use with non-DNA binding proteins. These engineering efforts will be aided by computational modeling and simulation tools that our lab has developed and will adapt for each target system.

用于小大分子高通量冷冻电镜研究的 DNA 纳米结构 单粒子冷冻电子显微镜 (cryo-EM) 是一种可视化结构的方法 在接近天然条件下分析大分子及其复合物，无需大样本 X 射线等替代技术通常需要数量或去除柔性区域 显微镜硬件和软件的改进有助于实现接近- 通过冷冻电镜测定大分子的原子结构，例如，可以进行可视化 然而，通过冷冻电镜进行结构测定。 对于许多大分子来说仍然具有挑战性。小（<100 kDa）颗粒尤其困难。 进行研究，因为它们缺乏图像对齐步骤所需的明确的结构特征 3D 重建。 为了应对这些挑战，我们建造了微型测角仪——能够精确定位的仪器 物体——使用 DNA 纳米技术，我们的分子测角仪可以对接单个 DNA 结合。 借助 DNA 折纸设计的力量，我们可以制作多种蛋白质。 测角仪变体，每个变体都具有用户定义的蛋白质-DNA 复合物的倾斜和旋转角度。 测角器包含不对称条形码域，因此，我们可以结合并精确定向小尺寸。 DNA 结合蛋白，在网格上轻松识别它们，正确分配角度先验，并快速 我们通过获得 82-kDa 的 6.5-Å 结构验证了我们的方法。 DNA 结合蛋白的螺旋假对称性妨碍了精确的图像定位 传统冷冻电镜。 我们建议应用我们的 DNA 折纸分子测角仪来表征几种生物医学 结构未知的重要 DNA 结合蛋白仍然难以进行冷冻电镜分析。 我们将研究与胰腺癌、乳腺癌、炎症性肠病、 我们还将研究糖尿病和非酒精性脂肪肝疾病的小转录因子。 调节酵母的多效性耐药性，这种结构理解可能有助于发展。 未来的抗真菌疗法我们将为每个蛋白质靶标构建定制分子测角仪。 适当的 DNA 结合位点和任何必要的结构修饰。 我们将继续优化我们的第一代设计，以解决与以下方面相关的突出挑战： 样品制备并将设备扩展为非 DNA 结合蛋白的通用用途。 工程将得到我们实验室拥有的计算建模工作和模拟工具的帮助 开发并将适应每个目标系统。