Parallel Screening Using the Chloroalkane Penetration Assay Reveals Structure-Penetration Relationships

The efficiency with which polycationic peptides penetrate the cytosol depends on the number and overall patterning of arginine residues. While general trends and unusually penetrant patterns of arginine residues have been discovered, prior work has not effectively leveraged high-throughput screens to measure cytosolic penetration rather than total cell uptake. In this work, we adapted the chloroalkane penetration assay, which exclusively measures cytosolic penetration, to screen peptide libraries in a high-throughput, quantitative, and automation-ready manner. We demonstrate the usefulness of the screening platform by efficiently exploring how the number, patterning, and stereochemistry of arginine residues affect the cytosolic penetration of a model 10-residue peptide.