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

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.