Multicyclic Ligation-Driven Self-Assembly of Magnetic Nanobundles for Sensitive Profiling of Multiple Repair Glycosylases in Cancer Cells

Base excision repair (BER) enzymes are essential for maintaining genomic stability and contribute significantly to disease pathogenesis, but simultaneous monitoring of multiple BER enzymes remains a challenge due to their mechanistic diversity and lack of versatile detection platforms. Herein, we demonstrate for the first time multicyclic ligation-driven self-assembly of magnetic nanobundles for sensitive profiling of multiple repair glycosylases in cancer cells. In this assay, we design two programmable dsDNA substrates with the incorporation of 8-oxo-7,8-dihydroguanine (8-oxoG) and uracil (U) base lesions, which can be cleaved by Fpg and UDG to generate nicking sites, respectively. Endonuclease IV can cleave these nicking sites to produce the two corresponding downstream substrates. The resultant two downstream substrates can hybridize with the corresponding ligation probes, triggering cyclic ligation cascades with the incorporation of signal probes into the magnetic nanobundles. After magnetic isolation and exonuclease digestion, FAM and TAMRA fluorophores embedded within the magnetic nanobundles are liberated into the solution. The released FAM and TAMRA fluorophores can be determined by fluorescence spectroscopy, with FAM indicating UDG activity and TAMRA indicating Fpg activity. This assay exhibits a detection limit of 7.70 × 10^-6 U/μL for UDG and 7.09 × 10^-6 U/μL for Fpg. In addition, the proposed method is applicable to enzymatic kinetic analysis, inhibitor screening, and simultaneous monitoring of UDG and Fpg glycosylases in diverse cancer cells. Furthermore, this assay enables the simultaneous measurement of diverse DNA repair enzymes through programmable dsDNA substrate recognition site modification, offering a new platform for biomedical study and clinical diagnostics.