title: Designing DNA interstrand lock for locus-specific methylation detection in a nanopore.
abstract: DNA methylation is an important epigenetic regulation of gene transcription. Locus-specific DNA methylation can be used as biomarkers in various diseases including cancer. Many methods have been developed for genome-wide methylation analysis, but molecular diagnotics needs simple tools to determine methylation states at individual CpG sites in a gene fragment. In this report, we utilized the nanopore single-molecule sensor to investigate a base-pair specific metal ion/nucleic acids interaction, and explored its potential application in locus-specific DNA methylation analysis. We identified that divalent Mercury ion (Hg²⁺) can selectively bind a uracil-thymine mismatch (U-T) in a dsDNA. The Hg²⁺ binding creates a reversible interstrand lock, called MercuLock, which enhances the hybridization strength by two orders of magnitude. Such MercuLock cannot be formed in a 5-methylcytosine-thymine mismatch (mC-T). By nanopore detection of dsDNA stability, single bases of uracil and 5-methylcytosine can be distinguished. Since uracil is converted from cytosine by bisulfite treatment, cytosine and 5'-methylcytosine can be discriminated. We have demonstrated the methylation analysis of multiple CpGs in a p16 gene CpG island. This single-molecule assay may have potential in detection of epigenetic cancer biomarkers in biofluids, with an ultimate goal for early diagnosis of cancer.
authors.affiliation: Department of Bioengineering and Dalton Cardiovascular Research Center.
authors.forename: Insoon
authors.initials: I
authors.lastname: Kang
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Corbin
authors.initials: C
authors.lastname: Reagan
authors.forename: Yumei
authors.initials: Y
authors.lastname: Fu
authors.forename: Michael X
authors.initials: MX
authors.lastname: Wang
authors.forename: Li-Qun
authors.initials: LQ
authors.lastname: Gu
mesh_terms: D020029:Base Pairing; D015374:Biosensing Techniques; D018899:CpG Islands; D003596:Cytosine; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation; D056426:Genetic Loci; D058608:Nanopores; D014498:Uracil
chemical_list: D014498:Uracil; D003596:Cytosine; D004247:DNA