Illuminating cellular dark matter through the development of novel chemical tools

项目总结 在细胞内有大量的物种，我们对它们的功能和 生物分子相互作用。这些物种可以被称为生物学中的“暗物质”，因为它们的机制 在常规的观察中是隐藏的。我们的实验室试图阐明细胞“暗”的功能 物质“通过新的化学技术的发展。拟议的研究计划将进行两项 主要的研究成果。首先，我们计划开发针对特定位置的RNA修饰工具，并应用这些工具 用于操作、成像和分离与疾病相关的RNA蛋白质复合体。我们将创建工具，用于 在活细胞中使用，并开发出将蛋白质共价招募到RNA，形成RNA-蛋白质的能力 大分子偶联物。这项技术将被应用于研究与疾病有关的RNA。具体来说， 我们对C9orf72核苷酸重复扩增致病途径的特征很感兴趣 被认为在遗传性肌萎缩侧索硬化症(ALS)中起主要作用的RNA。在第二次冲刺中， 我们将在活细胞内进行脂质物种的原位合成，目的是揭示分子 神秘的类脂物种影响细胞行为的机制。我们计划开发方法，使 选择性和生物正交性地将鞘磷脂输送到活细胞。以以前的技术为基础 在我们实验室开发的，我们将提供细胞通透性脂质前体，这些前体将自发组装成 细胞内的功能性脂类。利用这种方法，我们将创建光亲和探针，用于下拉 鞘磷脂相互作用蛋白，目的是阐明非规范的 脱氧鞘磷脂1-脱氧二氢神经酰胺，具有细胞毒性，与多种疾病有关。实现 我们的研究计划目标将提高我们对细胞生物学的知识，并导致新的 用于询问核糖核酸和类脂物种的工具。我们的长期愿景是创造和应用能够实现 改进了对生物分子相互作用的机理理解，从而增加了对 人类疾病，并加快开发可能的治疗干预措施。

PROJECT SUMMARY There is a vast repertoire of species within cells for which we have a poor understanding of their function and biomolecular interactions. These species can be referred to as the “dark matter” of biology, as their mechanism of action is hidden from conventional observation. Our laboratory seeks to illuminate the function of cellular “dark matter” through the development of new chemical technology. The proposed research program will pursue two major research thrusts. First, we plan to develop tools for site-specific RNA modification, and apply these tools for the manipulation, imaging, and isolation of disease relevant RNA protein complexes. We will create tools for use in live cells and develop the ability to covalently recruit proteins to RNA, forming RNA-protein macromolecular conjugates. The technology will be applied to study RNAs implicated in disease. Specifically, we are interested in characterizing the pathways of pathogenicity for the C9orf72 nucleotide repeat expansion RNA, which is thought to play a major role in genetic amyotrophic lateral sclerosis (ALS). In the second thrust, we will carry out the in situ synthesis of lipid species within living cells, with the goal of uncovering the molecular mechanism by which enigmatic lipid species affect cell behavior. We plan to develop approaches enabling the selective and bioorthogonal delivery of sphingolipids to living cells. Building upon technology previously developed in our lab, we will deliver cell permeable lipid precursors which will spontaneously assemble into functional lipids within the cell. Leveraging this approach, we will create photoaffinity probes for the pulldown of sphingolipid-interacting proteins, with the goal of elucidating the protein partners of the non-canonical deoxysphingolipid 1-deoxydihydroceramide, which is cytotoxic and implicated in several diseases. Realization of our research program goals would improve our knowledge of cell biology and lead to the development of new tools for interrogating RNA and lipid species. Our long-term vision is to create and apply technology that enables improved mechanistic understanding of biomolecular interactions, leading to an increased understanding of human disease, and accelerating the development of possible therapeutic interventions.