A Fluorogenic System for Measuring Cytosolic Localization

项目摘要 生物分子正在成为一类重要的治疗药物。生物分子使令人兴奋的新疗法成为可能 包括用肽和微型蛋白质靶向蛋白质-蛋白质相互作用，用Cas9进行基因编辑的方法， 复合物和使用化学修饰的RNA的mRNA敲低。将这些大分子递送至 胞质溶胶是关键。然而，细胞渗透的定量评估仍然具有挑战性。近日 Kritzer实验室报告了CAPA（氯代烷烃渗透试验），以解决该挑战。在该测定中， 将氯代烷烃接头连接至感兴趣的生物分子，并与稳定的HaloTag表达载体一起孵育。 HeLa细胞系。当生物分子到达胞质溶胶时，氯代烷烃接头共价结合HaloTag。 然后将细胞与氯代烷烃染料一起孵育，该染料与任何剩余的HaloTag反应。洗涤后 除去未反应的染料，然后使用流式细胞术分析细胞。使用这种方法，荧光强度是 与生物分子的细胞渗透量成反比。 尽管CAPA取得了成功，但试验的范围最终受到限制。之间的反比关系 胞质定位和荧光强度限制了测定灵敏度， 体内应用。为了克服这些缺点，我们建议开发一个荧光CAPA系统。在 在该测定中，荧光染料将与感兴趣的生物分子缀合， 与细胞溶质中的HaloTag共价反应。很少有荧光染料先前已报道HaloTag 或类似系统，并且这些染料显示低荧光开启和/或慢缀合动力学。 这些参数使得它们不太适合基于细胞的测定。这是我们第一次将分子 进化以优化HaloTag-染料对，目标是产生具有1000倍增加的 在HaloTag-染料反应后的荧光和类似于WT HaloTag的反应动力学。成功完成 所提出的项目将产生一个荧光HaloTag系统，将大大提高吞吐量， CAPA方法的灵敏度和时间分辨率。从长远来看，荧光染料-HaloTag对将使 体内测定，例如测量活动物中的胞质递送和药代动力学，以及方法 它可以直接测量时间分辨的细胞渗透。拟议的项目将提供新的方法， 有利于药物开发管道，推进生物分子作为人类疾病的治疗方法。

Project Summary Biomolecules are emerging as an important class of therapeutics. Biomolecules enable exciting new therapies including targeting protein-protein interactions with peptides and mini-proteins, gene editing methods with Cas9 complexes, and mRNA knockdown using chemically modified RNA. Delivery of these macromolecules to the cytosol is critical. However, quantitative assessment of cell-penetration remains challenging. Recently, the Kritzer lab reported CAPA, the Chloroalkane Penetration Assay, to address this challenge. In this assay, a chloroalkane linker is attached to a biomolecule of interest and incubated with a stable HaloTag-expressing HeLa cell line. As the biomolecule reaches the cytosol, the chloroalkane linker covalently binds to HaloTag. The cells are then incubated with a chloroalkane-dye that reacts with any remaining HaloTag. After washing away unreacted dye, cells are then analyzed using flow cytometry. Using this method, fluorescence intensity is inversely proportional to the amount of cellular penetration of the biomolecule. Despite the success of CAPA, the assay is ultimately limited in scope. The inverse relationship between cytosolic localization and fluorescence intensity limits assay sensitivity and the necessary wash steps prevent in vivo applications. To circumvent these shortcomings, we propose developing a fluorogenic CAPA system. In this assay a fluorogenic dye will be conjugated to a biomolecule of interest and fluorescence will turn-on upon covalent reaction with HaloTag in the cytosol. Few fluorogenic dyes have been previously reported for HaloTag or similar systems, and these dyes display low fluorescence turn-on and/or slow conjugation kinetics – parameters that make them poorly suited for cell-based assays. For the first time, we will apply molecular evolution to optimize HaloTag-dye pairs, with the goal of producing a system with 1000-fold increase in fluorescence upon HaloTag-dye reaction, and reaction kinetics similar to WT HaloTag. Successful completion of the proposed project will yield a fluorogenic HaloTag system that will greatly improve the throughput, sensitivity, and time resolution of the CAPA method. Long-term, a fluorogenic dye-HaloTag pair will enable in vivo assays such as measuring cytosolic delivery and pharmacokinetics in live animals, as well as a methods that can measure directly time-resolved cellular penetration. The proposed project will provide new methods to benefit the drug development pipeline, advancing biomolecules as treatments for human diseases.