The goal of the proposed research is to develop Affinity-Mediated Covalent Conjugation (AMCC) reactions that directly modify specific cell types while leaving other cells untouched. The AMCC will target specific receptors on the cell surface and create a permanent covalent link. The ability to directly modify cell membranes with designer chemical functionalities would enable a host of new technologies, such as addressing questions of basic cell biology, increasing therapeutic efficacy, capturing single cells for analysis, to building entirely new self-assembled 3D cell systems from differentiated stem cells. The AMCC reaction starts with an initial, specific non-covalent association, followed by a permanent covalent bond formation induced by irradiation. To promote the non-covalent interaction, the PIs will utilize affibodies that show modest target affinity but are highly robust to modification and incorporation into fusion proteins. In Prior Work (see Research Strategy), the PIs showed that if a specific amino acid on an EGFR-binding affibody was replaced with a benzophenone group, photocrosslinking to EGFR was obtained. This result was validated in both 2D culture and 3D spheroid models, using both UV light and near infrared (NIR) light with upconverting nanoparticles. In this research, the PIs propose to study how the AMCC approach could be utilized to attach specific protein subunits to existing cell receptors using a light-activated, single-step modification. Because affibodies are robust to modification, fusion proteins could be designed with both the photocrosslinkable affibody and an active species (e.g. enzyme, green fluorescent protein (GFP), or streptavidin). Photoconjugation to the cell would create a permanent bond to a cell receptor, followed by long-term expression of this active species on the surface of the cell. As a result, specific cells may be modified at multiple locations simultaneously and orthogonally, leading to numerous advances in areas of model tissue design, in vivo tissue modification, and high throughput cell isolation from complex mixtures. A current barrier to success in this approach is ensuring that the ligated functionalities (e.g., GFP or enzymes) will remain active and intact on the cell membrane over necessary time scales. We hypothesize that covalently binding the ligand to the cell receptor will prolong this lifetime by disguising the conjugated portion as part of the receptor, thereby preventing or hindering receptor-mediated endocytosis and subsequent proteolysis. However, it is not currently known what the long-term fate of the modified receptor will be. Therefore, it is critical to study the effect of photocrosslinking affibodies to cell receptors on the aforementioned biochemical and cellular responses. These questions will be addressed by examining the effects of this approach on cell health, dynamics of the EGFR-affibody complex, and activity of the conjugated segment on the cell surface.

R21GM135668

2020-01-01

2021-12-31