A dual-key fluorogenic probe for DNA and proteins imaging via excimer disaggregation and viscosity-activated molecular rotor.

Fluorescence imaging is pivotal for elucidating biomolecular dynamics, yet the development of probes enabling specific and high-contrast imaging of multiple targets remains challenging. Herein, we designed and synthesized a novel cyanovinylene-based fluorogenic probe, CV-N3, which synergistically integrates a dual-response mechanism combining excimer disaggregation with a viscosity-activated molecular rotor. The excimer formation and its CuAAC induced disaggregation are directly supported by dynamic light scattering and time-resolved fluorescence lifetime measurements. The probe enables specific covalent labeling of alkyne-tagged DNA and SNAP-tag proteins in living cells via copper-catalyzed azide-alkyne cycloaddition, with labeling specificity further confirmed by competitive binding assays. In solution, we validated the dual mechanism: covalent binding to targets triggers excimer disaggregation, causing a blueshift in emission from ∼630 nm to 535 nm, while the restricted local microenvironment of biomacromolecules activates the rotor character, significantly enhancing fluorescence intensity. Thus, CV-N3 achieves high signal-to-background, wash-free imaging of both DNA and proteins in living cells, while exhibiting good photostability and low cytotoxicity. Its utility for dynamic cellular studies is further demonstrated by tracking cell turnover and multi-color imaging compatibility. This work establishes a versatile strategy for constructing multi-responsive probes, offering a promising platform for multiplexed biomolecular imaging and dynamic studies in complex biological systems.