Sequential Assessment of Multiple Epigenetic Modifications of Cytosine in Whole Genomic DNA by Surface Plasmon Resonance.

Abstract

Since the discovery of the active DNA demethylation pathway in mammals, numerous efforts have been made to distinguish epigenetic cytosine variants, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). However, the rapid discrimination of multiple cytosine variants in DNA remains challenging because the conventional assays require time-consuming DNA pretreatments, such as enzymatical digestion and chemical conversion. Here we demonstrated the high-throughput discrimination of four cytosine variants in DNA by using a sequential surface-plasmon-resonance (SPR)-based immunochemical assay. The target DNAs were biotinylated in one step with a bifunctional linker 1 and robustly immobilized on a streptavidin-coated sensor surface to hold them in place during an alkali washing designed to remove residual antibodies. By repeating the injection of antibodies and washing, we achieved a sequential assessment of cytosine variants in identical DNA and identified the yield of in vitro 5mC oxidation in genomic DNA by the ten-eleven translocation 1 (TET1) enzyme. These results demonstrated that our sequential SPR-based immunochemical assay was effective for evaluating multiple epigenetic modifications in a whole genome with a single row operation without time-consuming DNA pretreatments.