DARPins as proximity inducing agents for the investigation of interactomes and as labels for long-term live cell fluorescence microscopy

• Applicants: Professor Dr. Volker Dötsch; Professor Dr. Mike Heilemann
• Subject Area: Biochemistry ; Bioinformatics and Theoretical Biology ; Cell Biology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 573586139

Designed Ankyrin Repeat Proteins – DARPins – can be created as highly selective binders that can target basically any folded protein domain. They consist of 1 to 3 internal modules, each comprising two α-helices that are connected by a loop. An N-terminal and a C-terminbal module provide high stability for these proteins. In each internal module, 6 positions located in the loop but some also in the helices can be randomized to create a large library of different DARPins. Those ones that bind tightly to a given target domain can be selected out of this pool using ribosome- and phage- display techniques. The small size of DARPins and the lack of disulfide bonds make them ideally suited as binding modules in the reducing environment of living cells. Their high affinity and selectivity can be used to target enzymatic functions to selected target proteins. Such enzymatic functions can be for example E3 ligases, thus creating BioPROTACS. Here we propose to develop protocols for the combination of DARPins with biotin ligases to investigate the interactome of target proteins. Using the DARPins to guide the biotin ligase to the target protein eliminates the need for genetic manipulation of the cells by fusing the biotin ligase directly to the target protein, thus enabling the investigation of interacomes also in primary cells. Using mass spectrometry following initiation of biotinylation enables the identification of the interactome. Similarly, we propose to fuse the DARPins to modified Halo-tags that bind to ligands in a non-covalent manner. Due to the exchange of the fluorescent labels, the problem of photo bleaching can be addressed in live cell imaging experiments. Again, no genetic manipulation of the investigated cells is necessary to visualize the localization of the protein of interest. In each case the mRNA of the DARPin fusion proteins will be transfected into cells using transfection lipids. We will develop these methods with two different isoforms of p63 that play different biological roles in the maintenance of the basal compartment of epithelial tissues and in the genetic quality control in oocytes. While these methods will be developed with model cell lines, for the future we plan to use these methods in the relevant epithelial cells and oocytes to characterize interactome and localization of p63.

• DFG Programme: Research Grants