# Penta‐ALFA‐Tagged Substrates for Self‐Labelling Tags Allow Signal Enhancement in Microscopy

## TL;DR

Researchers developed a new method using a pentaALFA peptide and fluorescent nanobodies to significantly boost signal intensity in fluorescence microscopy.

## Contribution

The novel pentaALFA-tag system enables multivalent labeling and signal amplification for enhanced imaging.

## Key findings

- PentaALFA peptides covalently react with SNAP- and HaloTag substrates to amplify fluorescence signals.
- HaloTag substrates outperformed SNAP-Tag in signal-to-noise and signal-to-background ratios.
- The system is compatible with super-resolution imaging techniques like STED.

## Abstract

Self‐labelling proteins like SNAP‐ and HaloTag have advanced imaging in life sciences by enabling live‐cell labeling with fluorophore‐conjugated substrates. However, the typical one‐fluorophore‐per‐protein system limits signal intensity. To address this, we developed a strategy using the ALFA‐tag system, a 13‐amino acid peptide recognized by a bio‐orthogonal and fluorescently labelled nanobody, for signal amplification. We synthesized a pentavalent ALFA5 peptide and used an azidolysine for conjugation with a Cy5‐modified SNAP‐ or HaloTag ligand through strain‐promoted click chemistry. In vitro measurements on SDS‐PAGE showed labelling, and the peptides covalently reacted with their respective tag. HEK293 cells expressing SNAP‐ and HaloTag‐mGluR2 fusion proteins were labeled with ALFA5‐Cy5 substrates, and confocal microscopy revealed a significant enhancement in the far‐red signal intensity upon nanobody addition, as quantified by integrated signal density ratios. Comparisons between SNAP‐ and HaloTag substrates showed superior performance for the latter, achieving better signal‐to‐noise and signal‐to‐background ratios, as well as overall signal intensity in plasma membrane‐localized regions. Our results demonstrate the potential of ALFA‐tag‐based systems to amplify SLP fluorescent signals. This strategy combines the photostability of synthetic fluorophores with multivalent labeling, providing a powerful tool for advanced imaging applications including super‐resolution in cells. Its versatility is expandable across diverse protein systems and colors.

A pentaALFA peptide may be reacted with a SNAP‐ or HaloTag and allows for signal enhancement using fluorescently labelled nanobodies. Tagged and peptide/nanobody treated cell surface receptors give generally higher signal intensity using fluorescence microscopy and are amenable for stimulated emission by depletion (STED) super‐resolution imaging.