Development of Covalent Small-Molecule Fluorescent Probes for DNA Methyltransferase 1 Detection in Cancer Cells and...

DNA methyltransferase 1 (DNMT1) overexpression is associated with aberrant methylation and tumorigenesis, making its detection vital for tumor diagnosis. In this study, RG108 derivatives bearing cysteine-targeted covalent moieties were constructed as warheads for the DNMT1 detectors. Following affinity assessment by surface plasmon resonance, warheads containing a 2-fluoroacrylamido moiety were selected for preparing fluorescein-labeled probes 20a and 20b. In-gel fluorescence scanning and competitive assays confirmed that the probes can covalently bind to DNMT1 at the S-adenosyl-l-homocysteine site. Probe 20b showed concentration- and time-dependent fluorescence in HeLa cells and demonstrated detection performance comparable to DNMT1 antibody with superior nuclear membrane permeability across diverse cell lines. Notably, the relative fluorescence unit ratios of probe 20b to 4',6-diamidino-2'-phenylindole in clinical cervical exfoliated cells showed significant differences among normal cells, low-grade squamous intraepithelial lesion cells, high-grade squamous intraepithelial lesion cells, and cancer cells, indicating its great potential as a tumor diagnostic agent.