Epigenetic Quantification of 5-Hydroxymethylcytosine Signatures via Regulatable DNAzyme Motor Triggered by Strand Displacement Amplification

DNA methylation predominantly occurs within the CpG dinucleotide, which is the main epigenetic form of gene expression regulation in mammals. Genomic DNA with CpG sites has different sequence lengths and complex secondary structures, resulting in the complexity and diversity of the samples. Therefore, highly efficient quantification of DNA methylation in complex samples remains challenging. Herein, the regulatable DNAzyme motor triggered by strand displacement amplification (SDA) was designed to quantify 5-hydroxymethylcytosine (5hmC) signatures as a model. Briefly, the 5hmC sites as primary target were specifically labeled with DNA primers and converted into a large number of single-stranded DNA (secondary target) via the SDA reaction which could activate the DNAzyme motor. With the increase of secondary target, the DNAzyme motor gradually recovered its activity and could continuously cleave the track strands labeled quenching probes, causing electrochemiluminescence signal recovery and detection limit down to 0.49 fM for 5hmC. This strategy provides a new route to quantify natural base modifications in DNA and would hold promising potential for the early diagnosis of cancer and other diseases related to 5hmC.