Friday, March 14, 2025
3/14/2025
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.
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