Ultrasmall Chemogenetic Tags with Group‐Transfer Ligands

Abstract Chemogenetic tags facilitate exploration of activities of a protein‐of‐interest (POI) that lacks small‐molecule ligands; however, most tags are too large for several POIs. Here, we report two ultrasmall chemogenetic tags (mgTag and cTag) of 36 and 50 amino acids (aa) that, to the best of our knowledge, are the smallest. These tags exhibit transferase ‐type reactivity with their ligands, allowing the attachment of any moiety‐of‐interest to the tag. cTag utilizes an engineered C1 domain‐bearing cysteine that undergoes a group‐transfer reaction with its ligand. Likewise, mgTag utilizes an engineered zinc‐finger domain‐bearing cysteine that undergoes a group‐transfer reaction with its molecular glue ligand in the presence of cereblon (CRBN). While the fusion of HaloTag (297 aa) or SNAPTag (182 aa) to the KRAS G12D (188 aa) disrupted its growth‐signaling pathway, fusion of mgTag or cTag did not, pointing to the importance of tag size. Group‐transfer of BRD4 binder to tags appended to Abelson kinase (ABL) induced proximity between ABL and BRD4, resulting in the latter's phosphorylation. Deletion of the transferase ‐type reactivity reduced phosphorylation levels, suggesting that proximity‐inducing chimeras with group‐transfer design may be more efficacious. We envision these ultrasmall tags to have wide‐ranging applications, including in basic science, biotechnology, and medicine.