# Dual‐Channel Fluorescence Assays with Supramolecular Host‐Dye Reporter Pairs for Membrane Activity Mapping of Peptides

## TL;DR

This paper introduces a dual-channel fluorescence assay to classify membrane-active peptides based on their ability to translocate or create pores in lipid membranes.

## Contribution

The novel dual-channel assay combines a dye efflux assay with a supramolecular tandem membrane assay for advanced mechanistic characterization of MAPs.

## Key findings

- The assay distinguishes between peptides that translocate and those that form pores.
- Validation with known peptides confirmed their classification as CPPs or AMPs.
- An advanced variant can differentiate between transient and stable pores.

## Abstract

Membrane‐active peptides (MAPs) are a major class of peptides that renders lipid bilayer membranes permeable for hydrophilic compounds. MAPs include cell‐penetrating peptides (CPPs) and pore‐forming antimicrobial peptides (AMPs), which are believed to be mechanistically related. CPPs render the membrane sufficiently permeable to enable their own translocation, while AMPs create membrane damage and induce cell death. We report herein a fluorescence‐based, dual‐channel assay, which combines a classical dye efflux assay based on self‐quenched carboxyfluorescein (CF) and a recently established supramolecular tandem membrane assay based on the supramolecular host‐dye complex of p‐sulfonatocalix[4]arene (CX4) and lucigenin (LCG). The new assay provides a functional classification of MAPs, which distinguishes between their capability to directly translocate across the vesicle membrane or to induce sufficient membrane permeability to allow dye efflux. The assay was validated with melittin, penetratin, Pep‐1, TP10, and various oligoarginine peptides including the TAT peptide, which confirmed their classification as CPPs or pore‐forming peptides. An advanced variant of the tandem membrane assay also allowed to distinguish between the formation of transient pores and stable equilibrium pores. Overall, the established dual‐channel assay provides a simple and easy to implement method for the advanced mechanistic characterization of MAPs and an exploration of their mechanistic landscape.

Membrane‐active peptides (MAPs) such as cell‐penetrating peptides (CPPs) and antimicrobial peptides (AMPs) can translocate across lipid membranes and create membrane pores. The combination of a dye efflux and a supramolecular tandem membrane assay in a dual‐channel format enables an advanced mechanistic characterization and an exploration of the mechanistic landscape of MAPs enabling their straightforward functional classification.