Determination of DNA Methyltransferase 1 in Cells Using a RG108-Fluorescein Conjugate to Monitor the Fluorescent Ratio with a Microplate Reader

Abstract

DNA methyltransferase 1 (DNMT1) is one of the most essential proteins for the development and maintenance of DNA methylation patterns. Developing a simple and accurate method to assess DNMT1 levels is crucial because its overexpression is associated with abnormalities in DNA methylation and disease development. In our previous work, a series of RG108-fluorescein conjugates were constructed as DNMT1 detectors and confirmed their membrane permeability, binding site, and detection ability in cells through a confocal laser-scanning microscopy assay. In the present study, the fluorescence microplate reader was applied to further validate DNMT1 detection ability of probe 1a in cells due to its accuracy for fluorescence detection. The results show that the fluorescent intensity of probe 1a in HeLa cells correlated with both the probe concentration and cell number. By introducing the concept of relative fluorescent unit ratio (RFU ratio), the detection capability of probe 1a was comparable with anti-DNMT1 Ab in a series of cell lines, suggesting the considerable potential of probe 1a as a replacement for anti-DNMT1 Ab with better cell membrane permeability. Furthermore, probe 1a was confirmed to be applicable for DNMT1 quantification in different types of cell lines, including normal cells, cancer cells, and DNMT1 down-regulated HeLa cells, with the ability being verified by western blotting or quantitative real-time polymerase chain reaction (qRT-PCR).