Label-Free and Template-Free Chemiluminescent Biosensor for Sensitive Detection of 5-Hydroxymethylcytosine in Genomic DNA.

5-Hydroxymethylcytosine (5hmC) is a modified base present at low levels in various mammalian cells, and it plays essential roles in gene expression, DNA demethylation, and genomic reprogramming. Herein, we develop a label-free and template-free chemiluminescent biosensor for sensitive detection of 5hmC in genomic DNAs based on 5hmC-specific glucosylation, periodate (IO4+) oxidation, biotinylation, and terminal deoxynucleotidyl transferase (TdT)-assisted isothermal amplification strategy, which we term hmC-GLIB-IAS. This hmC-GLIB-IAS exhibits distinct advantages of bisulfite-free, improved sensitivity, and genome-wide analysis of 5hmC at constant reaction temperature without the involvement of either specially labeled nucleic acid probes or specific templates for signal amplification. This method can sensitively detect 5hmC with a detection limit of 2.07 × 10-13 M, and it can detect 5hmC in the whole genome DNA with a detection limit of 3.92 × 10-5 ng/μL. Moreover, this method can distinguish 5hmC from 5-methylcytosine (5mC) and cytosine (C) and even discriminate 0.1% 5hmC in the mixture of 5hmC-DNA and 5mC-DNA. Importantly, this hmC-GLIB-IAS strategy enables genome-wide analysis without the involvement of either isotope-labeled substrates or specific antibodies, providing a powerful platform to detect 5hmC in real genomic DNA with high reproducibility and accuracy.