Knowledge-based design of reagentless fluorescent biosensors from a designed ankyrin repeat protein

Area of Science:

• Protein engineering
• Bioconjugation chemistry
• Biosensor development

Background:

• Designed ankyrin repeat proteins (DARPins) are highly stable, soluble, and expressible protein scaffolds.
• Fluorescent conjugates of DARPins could enable sensitive antigen detection for nano-analytical sciences.

Purpose of the Study:

• To explore the creation of reagentless fluorescent biosensors from DARPins.
• To investigate the design rules for DARPins that exhibit fluorescence variation upon antigen binding.

Main Methods:

• Site-specific mutagenesis of a DARPin (Off7) to introduce cysteine residues.
• Chemical coupling of fluorophores to engineered cysteine residues.
• Characterization of fluorescence changes upon antigen (MalE) binding.

Main Results:

• Engineered DARPin-fluorophore conjugates showed a >1.7-fold increase in fluorescence intensity upon antigen binding.
• The best conjugate maintained parental DARPin affinity and exhibited a linear, specific signal increase with antigen concentration.
• A low limit of detection (0.3 nM) was achieved, with fluorescence variation attributed to antigen-induced shielding of the fluorophore.

Conclusions:

• Novel reagentless fluorescent biosensors can be designed from DARPins.
• Specific design principles for DARPin-based fluorescent biosensors were elucidated.
• This approach holds promise for micro- and nano-analytical applications.