Construction of HaloTag-based macromolecular probe for multiple logic gates and photoactivatable bioimaging

Protein bioconjugation has emerged as one of the most valuable tools for the development of protein-based biochemical assays. Herein, we report a fluorescent macromolecular probe RF12_POI, in which the coumarin derivative RF12 is specifically conjugated onto the HaloTag fused protein of interest (POI) to achieve a dual stimuli-mediated fluorescence response. RF12 is first obtained by installing a photo-cleavable 1-ethyl-2-nitrobenzyl group onto the C7 hydroxy moiety of coumarin fluorophore with a HaloTag ligand attaching to the acid-labile 1,3-dioxane moiety. Upon stimulation, RF12_Halo exhibits a sequential fluorescence response to photon/H⁺ on both liquid and solid interfaces. Through the conjugation of RF12 onto the GFP_Halo protein, RF12_GFP_Halo presents a fluorescence resonance energy transfer (FRET) from photo-cleaved RF12 to GFP in the protein complex. Furthermore, by utilizing the stimuli-responsive fluorescence characteristics of coumarin derivatives RF12 (photon/H⁺) and RF16 (H₂O₂/H⁺), we construct RF12/RF16_POI based protein films and achieve multiple applications of logic circuits, including AND, OR, XOR, INHIBIT, Half-adder or Half-subtractor. In these circuits, the output value of I/I₀ is dependent on the input sequence of photon, H₂O₂, and H⁺. Additionally, we evaluate the fluorescence labeling ability of RF12 to intracellular IRE1_Halo protein and demonstrate that RF12 containing the HaloTag ligand could be precisely retained in cells to track IRE1_Halo protein. Hence, we provide a unique structural design strategy to construct fluorescence dual-responsive macromolecules for information encryption and cellular protein visualization.