Ultrasensitive and Accurate Assay of Human Methyltransferase Activity at the Single-Cell Level Based on a Single Integrated Magnetic Microprobe

Human DNA methyltransferase (MTase) activity expression patterns and inhibition response are linked to related cancer initiation, progression, and therapeutic responses. Sensitive and accurate human MTase activity assay in cancer cells, especially at the single-cell level, is essential for biological study, clinical diagnosis, and therapy. Here, we developed an ultrasensitive and accurate DNA (cytosine-5)-methyltransferase 1 (Dnmt1) activity assay at the single-cell level based on a single integrated magnetic microprobe of functionalized double-stranded DNA (dsDNA) anchored to a single magnetic microbead surface. Functionalized dsDNA is designed with a hemimethylated DNA site for Dnmt1 recognition and a single-stranded tail to trigger in situ rolling circle amplification (RCA). Under the action of Dnmt1, hemimethylated dsDNA could be recognized and catalyzed to fully methylated dsDNA, which would protect them from the cleavage of BssHII. However, the dsDNA without full methylation would be cut by BssHII, making single-stranded tail separated from the single integrated microprobe. Subsequently, full methylation-protected in situ RCA could be performed, and multiple signal probes were hybridized to the single integrated microprobe for amplified signal accumulation. Finally, Dnmt1 activity could be evaluated by reading the fluorescence of the single integrated microprobe. Meanwhile, to minimize matrix interferences, magnetic separation was performed in the process. In this strategy, the single integrated magnetic microprobe was provided with integrated capacities of target recognition, signal amplification, signal accumulation, and matrix isolation. Therefore, an ultralow detection limit of 0.007 U/mL Dnmt1 was obtained, and accurate Dnmt1 activity assays in multiple cell lysates at the single-cell level were achieved. Furthermore, the inhibition effect of RG108 was evaluated conveniently. These results indicate that the single integrated magnetic microprobe-based strategy is an excellent candidate for sensitive monitoring of Dnmt1 activity and screening of anticancer drugs.