Oscillating Probe for Dual Detection of 5'PO4 and 5'OH DNA Breaks in Tissue Sections

Several types of DNA cuts are used as markers of apoptosis for detection of apoptotic cells in situ . We recently introduced a ligase-based in situ assay that is specific for a single type of DNA damage—a double-strand break of DNase I-type, bearing 5′PO4 . Here we describe a vaccinia topoisomerase I-based approach to label another type of DNA damage in situ —a double-strand break of DNase II-type, bearing 5′OH. The assay uses a new type of probe, a molecular oscillator. The probe self-assembles in solution out of a dual-hairpin oligonucleotide and vaccinia topoisomerase I. The enzyme continuously separates and religates two fluorescently labeled hairpins, which can participate in energy transfer. We describe the successful combination of topoisomerase- and ligase-based systems into an in situ assay. The assay uses an oscillating probe for simultaneous detection of two types of DNA cuts in tissue sections.