Live-cell intracellular immunofluorescence

摘要 免疫荧光是基础生物学研究和临床标本检测的重要工具之一。 由于抗体的广泛可获得性以及荧光成像的分辨率和敏感度，因此可用于成像。 尽管IF自最初发明以来在过去的60年里产生了大量的数据，IF遭受了 来自一个基本的限制：不能询问活细胞内的靶标(细胞必须是 首先修复，从而仅提供动态小区信令过程的快照)。考虑到的3D结构 细胞内具有生物学意义的靶标比细胞膜靶标要多得多， 但免疫制剂，如抗体、抗体片段、多肽、纳米抗体和单链抗体，是 高度亲水性的大分子，不能自发地穿过细胞膜。克服障碍 细胞膜和内吞作用在防止完整的生物分子进入细胞内极其有效 胞质，我们建议开发一种基于一个独特的概念的细胞内蛋白质递送技术：非 共价胆固醇标记。与传统交付技术相比，传统交付技术主要基于 内吞作用(不适合于活体细胞成像，因为被捕获的显像剂产生的高本底 在内体中)，我们的小分子标签使蛋白质能够穿透细胞膜。更多 重要的是，这是在不会在膜上产生导致细胞毒性的气孔的情况下实现的。如果成功，这将是 平台技术应该为分子生物学、药物开发和细胞开辟一个全新的维度 工程学。

ABSTRACT Immunofluorescence (IF) is one of the most important tools for both basic biology research and clinical sample imaging due to the broad availability of antibodies and the resolution and sensitivity of fluorescence imaging. Despite the tremendous amount of data produced by IF in the past ~60 years since its initial invention, IF suffers from a fundamental limitation: incapability of interrogating intracellular targets in live cells (cells have to be fixed first, thus only providing a snapshot of the dynamic cell signaling process). Considering the 3D structure of cells, there are far more intracellular targets with biological significance than their cell-membrane counterparts, but immunological agents such as antibodies, antibody fragments, peptides, nanobodies, and scFvs, being highly hydrophilic macromolecules, cannot spontaneously cross the cell membranes. To overcome the barriers of cell membrane and endocytosis that are extremely effective in preventing intact biomolecules to enter the cytosol, we propose to develop an intracellular protein delivery technology building on a unique concept: non- covalent cholesterol tagging. In contrast to conventional delivery technologies that are mostly based on endocytosis (inappropriate for live cell imaging due to the high background generated by imaging agents trapped in endosomes), our small-molecule tag enables proteins to permeate through the cell membrane. More importantly, this is achieved without generating pores in the membrane that cause cytotoxicity. If successful, this platform technology should open a whole new dimension for molecular biology, drug development, and cell engineering.