A Fluorogenic System for Measuring Cytosolic Localization

用于测量胞质定位的荧光系统

基本信息

批准号：
10378514
负责人：
Bryan J Lampkin
金额：
$ 6.97万
依托单位：
TUFTS UNIVERSITY MEDFORD
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10378514
关键词：
Address Animals Binding Biological Assay Biological Products Biological Response Modifier Therapy Cell Culture Techniques Cell Line Cell Nucleus Cell membrane Cells Charge Chemicals Combinatorial Optimization Complex Cytosol Detection Development Drug Kinetics Dyes Endosomes Environment Enzymes Eukaryotic Cell Evaluation Evolution Fellowship Flow Cytometry Fluorescence Future Genes Goals Hela Cells In Vitro Incubated Industrialization Kinetics Libraries Literature Mammalian Cell Measurement Measures Messenger RNA Methods Molecular Evolution Monitor National Research Service Awards Outcome Output Penetration Peptides Property Protein Engineering Proteins RNA Reaction Recombinants Reporting Resolution Signal Transduction Sorting - Cell Movement System Testing Therapeutic Time Translations Work Yeasts aerobic respiration control protein base biological development combinatorial design drug development fluorophore human disease improved in vivo interest knock-down macromolecule novel therapeutics prevent protein protein interaction rational design screening success

项目摘要

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.

项目概要生物分子正在成为一类重要的治疗方法，它可以实现令人兴奋的新疗法。包括针对肽和微型蛋白的蛋白质间相互作用、Cas9 的基因编辑方法复合物，以及使用化学修饰的 RNA 将这些大分子递送至 mRNA 的敲除。然而，细胞渗透的定量评估仍然具有挑战性。 Kritzer 实验室报告了 CAPA（氯烷渗透测定法）来应对这一挑战。氯烷接头连接到感兴趣的生物分子并与稳定的 HaloTag 表达一起孵育 HeLa 细胞系。当生物分子到达细胞质时，氯烷连接基会与 HaloTag 共价结合。然后将细胞与氯代烷染料一起孵育，洗涤后与任何剩余的 HaloTag 发生反应。除去未反应的染料，然后使用流式细胞术分析细胞，荧光强度为与生物分子渗透细胞的量成反比。尽管 CAPA 取得了成功，但该测定的范围最终受到限制。胞质定位和荧光强度限制了灵敏度测定，并且必要的洗涤步骤防止为了克服这些缺点，我们建议开发一种荧光CAPA系统。在该测定中，荧光染料将与感兴趣的生物分子结合，并且荧光将在之前很少有 HaloTag 与 HaloTag 发生共价反应的报道。或类似的系统，并且这些染料表现出低荧光开启和/或缓慢的缀合动力学 – 使它们不适合基于细胞的测定的参数，我们将首次应用分子分析。进化以优化 HaloTag-染料对，目标是生产一个可将光通量提高 1000 倍的系统 HaloTag 染料反应发出荧光，反应动力学类似于 WT HaloTag 成功完成。拟议项目的成果将产生荧光 HaloTag 系统，该系统将大大提高吞吐量，从长远来看，荧光染料-HaloTag 对将能够提高 CAPA 方法的灵敏度和时间分辨率。体内测定，例如测量活体动物中的胞质递送和药代动力学，以及方法可以直接测量时间分辨的细胞渗透率。拟议的项目将提供新的方法。有利于药物开发渠道，推进生物分子作为人类疾病的治疗方法。