Identification of Senescence‐Associated β‐Galactosidase With Single‐Molecule Resolution

Abstract

ABSTRACT The majority of human senescent cells exhibit overexpression of senescence‐associated β‐galactosidase (SA‐β‐gal), rendering this enzyme the most extensively utilized biomarker of senescence and a key prodrug target for intervening in aging. However, strategies for the authentic in situ identification of SA‐β‐gal with single‐molecule resolution have not been established, limiting the accuracy of senescence targeting. Here we present an unprecedented molecular approach to identify SA‐β‐gal in a manner that avoids dissociation from the target enzyme, which integrates the atomic hybridization of SA‐β‐gal substrates with protein‐environment‐sensitive fluorescence technology. Using this design principle, we created nine fluorescent probes with distinct working modes for the single‐molecule (target enzyme)‐resolved identification of SA‐β‐gal. Five of them formed crystal complexes with the wild‐type β‐galactosidase by binding at the active site, clearly evidencing their single‐molecule resolution capability. Our strategy, capable of both identifying and locating SA‐β‐gal, facilitated its application in dynamic single‐molecule localization microscopic imaging, achieving the in situ tracking of SA‐β‐gal in living cells at the nanoscale. Significantly, the probing of SA‐β‐gal with high fidelity enabled the precise evaluation of the aging degree in mice. As such, our research provides a promising method for the authentic in situ identification of this senescence‐associated protein with single‐molecule resolution.