权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

项目总结