Intracellular Strand Break End Extension-Encoded Amplification for In Situ Visualization of Diverse DNA Damages

DNA damage refers to chemical alterations in genomic DNA arising from endogenous or exogenous factors, and delineating the types and levels of these lesions is essential for elucidating the molecular mechanisms and regulatory networks underlying DNA repair. However, current methods are limited to one specific type of damage and often rely on DNA extraction and in vitro processing, which fails to capture the spatial distribution of diverse DNA damages within cellular context. Here we present intracellular strand break end extension-encoded amplification (ISBEA), an in situ imaging strategy that enables simultaneous visualization of multiple DNA lesion types. ISBEA uses a sequential enzymatic workflow in which glycosylases, endonucleases and exonucleases selectively recognize oxidized purines, AP sites and strand breaks, converting them stepwise into uniform 3′-hydroxyl termini. These activated termini undergo programmable extension and serve as primers to initiate DNA-encoded amplification, ultimately generating fluorescence signals present to each damage type. With spatially resolved visualization of diverse DNA damages within cells, ISBEA provides a robust platform for investigating the origins, repair pathways and biological consequences of DNA damage, offering valuable methodological support for advancing the understanding of genome stability maintenance and the development of related diseases.