Small Fluorogenic Amino Acids for Peptide-Guided Background-Free Imaging.

Area of Science:

• Biochemistry
• Microscopy
• Molecular Biology

Background:

• Super-resolution microscopy requires advanced labeling techniques for peptide imaging.
• Existing fluorescent reporters often have limitations like large size and non-specific binding.
• Minimally invasive strategies are crucial for accurate peptide-guided fluorescence imaging.

Purpose of the Study:

• To develop novel, minimally invasive fluorescent reporters for peptide imaging.
• To create background-free imaging agents for super-resolution microscopy.
• To assess the utility of new reporters in biological systems.

Main Methods:

• Synthesis of a library of benzodiazole amino acids.
• Systematic examination of benzodiazole derivatives as fluorescent reporters.
• Solid-phase synthesis of fluorescent peptides using identified amino acids.
• Application in peptide-PAINT imaging of protein nanoclusters.

Main Results:

• Identified amine-derivatized benzoselenadiazoles as scalable and photostable amino acids.
• Demonstrated straightforward solid-phase synthesis of fluorescent peptides.
• Benzodiazole amino acids retain peptide binding capabilities and offer high signal-to-background ratios.
• Successfully applied the reporters for peptide-PAINT imaging of postsynaptic density protein-95 nanoclusters.

Conclusions:

• Benzodiazole amino acids are effective reporters for background-free fluorescence microscopy.
• These novel reporters facilitate minimally invasive peptide imaging with super-resolution techniques.
• The developed strategy enables precise visualization of biomolecular targets in complex biological tissues.