Protein-activated and FRET enhanced excited-state intermolecular proton transfer fluorescent probes for high-resolution imaging of cilia and tunneling nanotubes in live cells

Abstract

Excited-state intermolecular proton transfer (inter-ESPT) fluorescent probes responsive to specific bioactive molecules should be greatly promising for protein sensing, DNA mutation simulating and cellular process regulating. However, the inter-ESPT molecules are recessive ESPT fluorophores, which need the assistance of other molecules with both hydrogen-bond accepting and donating abilities to turn on the tautomeric fluorescence. Valid design strategies to create powerful inter-ESPT fluorescent probes are poorly developed, particularly for proteins as targets. We recently reported a unique supramolecular strategy to trigger the inter-ESPT process based on the probe-protein recognition by H-bonding and to image protein-based subcellular structures in live cells. Herein, we found that our inter-ESPT probes (inter-ESPT-01) bearing a 2-amino-3-cyanopyridine scaffold can anchor proteins and light up the “invisible” ESPT state, so as to image the proteins or the protein-based subcellular organelles. More importantly, the inter-ESPT emission of inter-ESPT-01 can be significantly enhanced by the FRET process between amino and imino tautomers, endowing the inter-ESPT-01 probes with super-bright tautomeric fluorescence. The expressed proteins Ecallantide and MarTX were selected as the models to light up the inter-ESPT fluorescence of the probes and revealed that the inter-ESPT process can be triggered by the specific probe-protein recognition events. In the use of the super-bright inter-ESPT fluorescence, not only the proteins, but also the protein-based cilia and tunneling nanotubes (TNTs) can be specifically visualized in living cancer cells. Furthermore, such recognition-driven strategy allows us to construct a unique inter-ESPT probe to track and image specific endogenous proteins in live cells, highlighting the potential of inter-ESPT fluorogens as novel intelligent biomaterials.