Photoelectrochemical detection of 5-hydroxymethylcytosine in genomic DNA based on M. HhaI methyltransferase catalytic covalent bonding

Abstract A simple and selective photoelectrochemical method was reported for 5-hydroxymethylcytosine (5hmC) detection in genomic DNA based on DNA methyltransferase (MTase) catalytic covalent bonding of –CH 2 OH of 5hmC with thiol compounds, where WS 2 nanomaterial was used as a photoactive material, polydopamine (PDA) was employed as a solid-phase electron donor, phos-tag-biotin was used as bridge to link 5hmC and streptavidin. PDA was attached to 4-mercaptophenulboronic acid (MPBA) via the covalent bonding between its vicinal diol and the boracic acid of MPBA. Then, under the catalytic effect of M. HhaI MTase, 5hmC deoxyribonucleotide was captured on electrode surface through the covalent bonding between –CH 2 OH and –SH. Afterwards, phos-tag-biotin, a kind of specific reagent for phosphate group, was further captured through the specific bonding between the phosphate group of 5hmC deoxyribonucleotide and phos-tag-biotin. Subsequently, based on the “bridge” role of phos-tag-biotin, streptavidin was further modified on electrode surface to further block the transfer of photogenerated-electron to electrode surface. The photocurrent shows linear relationship with the logarithm value of 5hmC concentration in the range from 0.01 to 100 nM, and the detection limit of 4.12 pM (3σ). The developed method presents high detection specificity, which can discriminate 5-methylcytosine and 5hmC. Moreover, the applicability of this photoelectrochemical method was evaluated by detecting 5hmC content change in maize seedlings leaves and chicken embryo fibroblast cell.