Methyltransferase Detection Based on Target-Responsive Transcription Amplification of Light-Up Aptamer

The sensitive and accurate evaluation of DNA methyltransferases (MTases) is essential for biomedical and synthetic biology applications. However, existing approaches have limited applications because they are labor-intensive with low sensitivity, require sophisticated instruments, and involve complicated processes. Here, we developed a simple, label-free, and versatile strategy called <u>M</u>Tase-responsive <u>T</u>ranscription <u>A</u>mplification (MTA) assay for sensitive and specific MTase detection. We designed a DNA template encoding a Broccoli RNA light-up aptamer, followed by a kleptamer (Kb) sequence with MTase and endonuclease recognition sites located between them. Methylation of the recognition site by an MTase results in selective cleavage or protection of the methylated DNA from specific endonucleases, thus allowing the regulation of MTase-mediated transcription amplification by switching ON or OFF. Leveraging this transcription amplification process and the high signal-to-background ratio of the light-up aptamer/fluorophore complex, the MTA assay demonstrated remarkable sensitivity in detecting Dam and M. SssI MTase, with detection limits of 0.022 and 0.038 U/mL, respectively. Furthermore, the modular design enabled the implementation of logic gate-based strategies, allowing multitarget detection and discrimination of MTases. The MTA assay offers a label-free, programmable, and transcription-driven approach for highly sensitive and specific detection of MTase, thus revealing various potential biomedical applications for MTase analysis.