Two-Dimensional Design Strategy to Construct Smart Fluorescent Probes for the Precise Tracking of Senescence

The tracking of cellular senescence usually depends on the detection of senescence-associated β-galactosidase (SA-β-gal). Previous probes for SA-β-gal with this purpose only cover a single dimension: the accumulation of this enzyme in lysosomes. However, this is insufficient to determine the destiny of senescence because endogenous β-gal enriched in lysosomes is not only related to senescence, but also to some other physiological processes. To address this issue, we introduce our fluorescent probes including a second dimension: lysosomal pH, since de-acidification is a unique feature of the lysosomes in senescent cells. With this novel design, our probes achieved excellent discrimination of SA-β-gal from cancer-associated β-gal, which enables them to track cellular senescence as well as tissue aging more precisely. Our crystal structures of a model enzyme E. coli β-gal mutant (E537Q) complexed with each probe further revealed the structural basis for probe recognition.