Development of an OPTogenetic InteractoMics Assay (OPTIMA)

项目摘要/摘要 在过去的十年里，越来越清楚的是，对人类疾病的更全面的了解 需要在系统生物学的背景下来看待它们，特别是通过对 细胞内发生的分子相互作用的网络。大量成功的应用(例如，疾病基因 优先排序)建立在分子网络结构的局部和全球结构上，分子网络结构主要基于蛋白质- 蛋白质相互作用(PPI)，提供关键的生物信息。随着几部大型电影的发布 自从我们小组制作了第一张人类二元组相互作用组的蛋白质组比例图以来，系统的PPI数据集 2005年，基因和临床数据与交互组信息的集成成为可能，并提供了 对更深入地理解疾病及其潜在的病理生理学的有意义和批判性的见解 给精准医学带来革命性的变化。然而，我们还没有达成一张全面的PPI网络图 任何模型系统或在人类中，因此临床应用将大大受益于更深和更广泛的 人类互动组的探索。 已经开发了高通量方法来在全球范围内确定许多生物体的PPI，但是 这些化验方法仍然存在内在的局限性和劳动密集型。筛查的一个主要瓶颈是确定 二元交互伙伴的身份。该Optima项目旨在消除这一瓶颈并填补 通过开发一种新的颠覆性技术来实现高性能交互操作，从而使En 大规模筛选PPI，以提供全面的二进制PPI图。这一创新的制度将产生于 集成了两种最近得到验证的技术，一方面是基于PPI的生物发光检测 关于Split-Nanoluc报告的互补性，另一方面，在光遗传学上最敏感的之一 驱动DNA条码融合的程序化启动子。通过利用整体二进制PPI检测，这一新的 二元交互检测策略将极大地提高蛋白质组规模的整体覆盖率 互动组地图。这条新的高通量流水线将与现有的蛋白质组规模的二进制文件正交 交互作图平台，如酵母双杂交后进行验证，从而能够显著 增强可用的工具，以扩展现有的互动。

Project Summary/Abstract Over the last decade, it has become increasingly clear that a more complete understanding of human diseases requires viewing them in the context of systems biology, in particular through a comprehensive understanding of a network of molecular interactions that occur in a cell. Numerous successful applications (e.g. for disease gene prioritization) build on the fact that the local and global structures of molecular networks, mostly based on protein- protein interactions (PPIs), provide critical biological information. With the release of several large-scale systematic PPI datasets since our group produced the first proteome-scale map of the human binary interactome in 2005, integration of genetic and clinical data with interactome information has become possible and provides meaningful and critical insights towards a deeper pathophysiological understanding of diseases and the potential to revolutionize precision medicine. However, we have not reached a comprehensive map of the PPI network in any model system or in humans yet, and thus clinical applications would greatly benefit from deeper and wider explorations of the human interactome. High-throughput approaches have been developed to determine PPIs on a global scale for many organisms, but these assays remain intrinsically limited and labor intensive. A major bottleneck in screening is determining the identities of binary interacting partners. This OPTIMA project aims to eliminate that bottleneck and fill the gap in current networks by developing a novel disruptive technology for high-performance interactomics allowing en masse screening of PPIs to provide comprehensive binary PPI maps. This innovative system will result from the integration of two recently validated technologies, on the one hand the bioluminescent detection of PPIs based on complementation of a split-Nanoluc reporter and, on the other hand, one of the most sensitive optogenetically programmed promoters driving DNA barcode fusion. By leveraging en masse binary PPI detection, this new binary interaction detection strategy will dramatically enhance the overall coverage of proteome-scale interactome maps. This new high-throughput pipeline will be orthogonal to existing proteome-scale binary interaction mapping platforms, such as yeast two-hybrid followed by validation, and thus able to significantly enhance the available tools to expand existing interactomes.