Nanopore-BasedAssayforDetectionof Methylation in Double-Stranded DNA

DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogen- esis, can be assessed in various biological fluids and potentially can be usedasmarkersfordetectionofcancer.Analyticallysensitiveandspecific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Build- ingon our prior work,we usedmethyl-binding proteins (MBPs), whichselectively labelthe methylated DNA.The nanopore-based assayselectively detects methylatedDNA/MBPcomplexesthrougha19nmnanoporewithsignificantlydeeperandprolongednanoporeioniccurrentblocking,whileunmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the CpG dyad in DNA fragments and could someday profile the position of methylated CpG sites on DNA fragments.