title: Site-specific immunochemical methylation assessment from genome DNA utilizing a conformational difference between looped-out target and stacked-in nontarget methylcytosines.
abstract: We report the sequence-selective immunochemical discrimination of methylcytosine from genomic DNA that we achieved by utilizing selective antibody binding to a looped-out methylcytosine in a bulge region and without using bisulfite treatment, a methylation-sensitive restriction enzyme, or PCR. First, we investigated the affinity of an anti-methylcytosine antibody for methylcytosine in full match, bulge, mismatch and abasic sites using surface plasmon resonance measurement and a microtiter plate to explore the differences as regards antibody binding to the target methylcytosine. The highest affinity, which was comparable to that in a single strand of DNA, was observed in the bulge region. In particular, no affinity was observed in a full match site. This is because there is no interaction such as hydrogen bond or π-π stacking for the bulged methylcytosine, thus enabling only the target in the bulge to be looped out. Methylated and unmethylated genomic DNA were blended to form a model DNA with which to assess the methylation ratio at a specific site. Fragmented DNA was hybridized with a biotinylated probe DNA, which has a sequence capable of forming a single base bulge at the target. The probe design is simple because it consists solely of the elimination of guanine paired with the target cytosine from a full match sequence. As a result, we successfully obtained a linear relationship (r(2)=0.9962) between the immunoassay signal and the methylation ratio of a specific site within 4 h.
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan. Electronic address: r.kurita@aist.go.jp.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Kurita
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Yanagisawa
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Yoshioka
authors.affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Niwa
mesh_terms: D002874:Chromosome Mapping / Q000379:methods*; D003596:Cytosine / Q000032:analysis* / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D019175:DNA Methylation; D016678:Genome / Q000235:genetics / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D009690:Nucleic Acid Conformation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D003596:Cytosine; D004247:DNA
keywords: DNA methylation; Epigenetics; Immunoassay; Surface plasmon resonance