EAGER: Towards DNA-based Tools for Single-particle Cryo-electron Microscopy of Membrane Proteins

这项研究将开发分子工具，使膜蛋白(MPS)的研究成为可能，膜蛋白是细胞中最重要但最鲜为人知的成分之一。所有细胞都被几乎不透水、盐或细胞所需营养物质的脂膜所包围。因此，许多膜蛋白(MPS)被插入到控制细胞功能的膜中，如物质运输、传感、细胞间通信、细胞黏附和能量转换。MPS也是许多治疗药物分子的靶标。了解MPS的分子结构对于了解其功能的潜在分子机制是必要的，并可以指导许多常见疾病的治疗药物的开发。然而，MPS很难研究，因此大多数MPS的分子结构仍然未知。该项目的目标是利用DNA纳米技术开发广泛适用的新工具，以便于用冷冻电子显微镜解决MP结构。该项目将通过几个既定的项目，为来自代表不足的少数群体的研究生、本科生和高中生提供研究机会，让他们了解实验研究和潜在的职业机会。最后，将通过开发强调物理原理的跨学科方面和应用的单元来加强入门物理课程，并将开发跨学科的分子生物物理学课程。这些努力将培养STEM研究和技术领域的下一代不同的、跨学科的领导者。单粒子冷冻电子显微镜(Cryo-EM)正在成为确定MP结构的标准方法，但到目前为止，一些实验挑战阻碍了解决超过1%的人类MP结构。该项目的总体目标是建立DNA-脂质纳米盘(DLN)，作为一种全新的、可定制的纳米级脂质双层模拟物，用于MPS的单粒子冷冻-EM。这种基于DNA纳米技术的方法将克服现有双层模拟技术的现有局限性，并对可能改变MP研究的结构、化学和物理设计参数提供前所未有的控制。这种范式转变涉及重大风险，需要进行大量的探索性开发工作。首先，将制定增加合成产率和防止脂质双层聚集的策略。接下来，需要建立MP在DLN中的表达、纯化和共重组。然后，需要反复优化冷冻条件、添加剂和其他样品制备参数，以生成高质量的成像网格。预计可以实现与现有的双层模拟相同或更好的分辨率，同时提供新的功能和前所未有的可编程性。预计DNA-脂质纳米盘将在结构生物学、药理学、病毒学和生物催化方面开启新的研究，从而增进对常见疾病的理解。这项研究中开发的方法和模型将公开提供，以造福于研究药物和疫苗作用机制的更大的科学界。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This research will develop molecular tools that allow the study of membrane proteins (MPs), which are among the most important, but least understood components of cells. All cells are surrounded by lipid membranes that are almost impermeable to water, salts or nutrients that cells need. For this reason, many membrane proteins (MPs) are inserted into the membranes that control cellular functions such as material transport, sensing, intercellular communication, cell adhesion, and energy conversion. MPs are also the targets for many therapeutic drug molecules. Knowledge of the molecular structure of MPs is necessary to understand the underlying molecular mechanisms of their function and can guide the development of therapeutic drugs for many common diseases. However, MPs are difficult to study and therefore the molecular structure of most MPs is still unknown. The goal of this project is to develop broadly applicable new tools using DNA nanotechnology that will facilitate solving MP structures with cryo-electron microscopes. This project will provide research opportunities for graduate, undergraduate and high school students from underrepresented minority groups through several established programs, which will inform them about experimental research and potential career opportunities. Finally, introductory physics courses will be strengthened by developing modules that underline interdisciplinary aspects and applications of physical principles, and an interdisciplinary molecular biophysics course will be developed. These efforts will train the next generation of diverse, interdisciplinary leaders in STEM research and technology.Single-particle cryo-electron microscopy (cryo-EM) is becoming the standard method for MP structure determination, but several experimental challenges have prevented solving more than 1% of human MP structures so far. The overall goal of this project is to establish DNA-lipid nanodiscs (DLNs) as a radically new customizable nanoscale lipid bilayer mimetic for single-particle cryo-EM of MPs. This DNA nanotechnology-based approach will overcome existing limitations of established bilayer mimetics and offer unprecedented control over structural, chemical and physical design parameters that could transform MP research. Such a paradigm shift involves significant risks and requires substantial exploratory development efforts. First, strategies will be developed to increase synthesis yields and to prevent lipid bilayer aggregation. Next, MP expression, purification and co-reconstitution of MPs in DLNs need to be established. Then, freezing conditions, additives and other sample preparation parameters need to be iteratively optimized to produce high quality grids for imaging. It is expected that the same or better resolutions can be achieved than with established bilayer mimetics, while providing new functionalities and unprecedented programmability. It is expected that DNA-lipid nanodiscs will initiate new research in structural biology, pharmacology, virology and bio-catalysis and therefore enhance the understanding of common diseases. The methods and models developed in this research will be made publicly available to benefit the larger scientific community researching the mechanisms of actions of drugs and vaccines.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.