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

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Traditional peptide labeling for cellular uptake studies is time-consuming, expensive, and can alter peptide properties.
• Developing efficient, label-free methods is crucial for accurate peptide behavior assessment.

Purpose of the Study:

• To present a novel, label-free fluorescence-based assay for real-time monitoring of peptide-membrane interactions.
• To establish a cost-efficient and high-throughput screening method for peptide cellular uptake.

Main Methods:

• Utilized the environmental sensitivity of 1-anilino-8-naphthalene sulfonate (ANS) fluorescence.
• Monitored changes in ANS emission spectra (blue shift, intensity enhancement) upon interaction with hydrophobic regions during peptide internalization.
• Tested the assay in plant protoplasts and mammalian HEK 293T cells using known cell-penetrating peptides (CPPs) like poly-arginine (R9) and TAT (49-57), and a non-penetrating mutant (mTAT).

Main Results:

• ANS fluorescence exhibited distinct changes correlating with peptide membrane interaction and internalization.
• The assay successfully differentiated between penetrating (R9, TAT) and non-penetrating (mTAT) peptides within minutes.
• Demonstrated applicability in both plant and mammalian cell systems.

Conclusions:

• The ANS-based assay offers a rapid, cost-effective, and label-free alternative for screening peptide uptake.
• This method provides insights into the hydrophobic changes associated with peptide internalization.
• Enables high-throughput screening of native peptides without structural modification.