A Label-Free Rapid Fluorescence Screening Approach for Identifying Cell-Penetrating Peptides Using ANS as an...

Labeling peptides with fluorophores remains the dominant approach for assessing their cellular uptake, yet this process is time-intensive, costly, and can modify peptide structure and biological behavior. Here a label-free fluorescence-based screening method is presented that exploits the environmental sensitivity of 1-anilino-8-naphthalene sulfonate (ANS) to monitor peptide-membrane interactions in real time. ANS shows negligible emission in water but undergoes a characteristic blue shift and intensity enhancement upon association with hydrophobic regions. These features were used to distinguish penetrating from non-penetrating peptides in both plant protoplasts and mammalian HEK 293 T cells. Classical cationic cell-penetrating peptides (CPPs), poly-arginine (R9) and TAT (49-57), produced distinct ANS responses within minutes, while the non-penetrating mutant mTAT showed no detectable effect. The ANS-based assay provides a cost-efficient, label-free, and high-throughput tool for screening native peptides and offers new insight into the hydrophobic transitions that accompany peptide internalization.