Protein interference applications in cellular and developmental biology using DARPins that recognize GFP and mCherry

Area of Science:

• Molecular Biology
• Synthetic Biology
• Protein Engineering

Background:

• Protein-protein interactions are vital for cellular functions, but studying intracellular interactions is challenging.
• Antibodies are limited in cytoplasmic applications, necessitating alternative protein-binding tools.
• Non-immunoglobulin scaffolds offer intracellular functionality for biological system engineering.

Purpose of the Study:

• To develop novel intracellular protein binders using the Designed Ankyrin Repeat Protein (DARPin) scaffold.
• To engineer DARPins targeting fluorescent proteins like GFP and mCherry for precise biological manipulation.
• To demonstrate the in vivo application of DARPins for altering protein function and studying developmental processes.

Main Methods:

• Selection of DARPins against GFP and mCherry using ribosome display technology.
• Affinity determination (KD values) and validation of specific binding in cellular environments.
• Application of DARPins to alter fluorescent protein localization and induce degradation in cell cultures and model organisms (Drosophila, Danio rerio).

Main Results:

• High-affinity DARPin binders for GFP (KD down to 160 pM) and mCherry (KD of 6 nM) were successfully generated.
• Specific intracellular binding was confirmed, with an affinity cut-off in the mid-nanomolar range for cellular detection.
• Engineered DARPins effectively altered fluorescent protein localization and facilitated protein degradation in vivo, phenocopying loss-of-function mutations.

Conclusions:

• Designed Ankyrin Repeat Proteins (DARPins) can be selected in vitro to specifically bind intracellular targets.
• These DARPins offer a powerful tool for reprogramming biological systems at the protein level in vivo.
• This approach bypasses limitations of DNA/RNA-based methods, enabling direct protein manipulation for research and synthetic biology.