Using solid-state nanopores, a group from the University of Illinois' Beckman Institute and the Mayo Clinic has developed a single-molecule detection method that can distinguish methylated DNA from unmethylated DNA and can coarsely quantify the extent of methylation on a DNA molecule.
The group is now working to refine the resolution of the assay, which uses methyl binding domain proteins to change the diameter of a DNA molecule at its methylated sites. This diameter change can then be detected — due to changes in current — as the molecule passes through a nanopore.
Currently, the approach can detect the difference between an unmethylated DNA molecule and a methylated DNA molecule, even if only a single protein is attached to it. The group also demonstrated that the assay can crudely quantify the degree of methylation, discriminating between the presence of one, five, and thirty protein-bound methylation sites, publishing the results in a paper in Scientific Reports this month.
Rashid Bashir, one of the study's lead authors, told In Sequence that he and his team believe the method could be useful for studying epigenetics in human disease, but before the assay can be commercialized, the group will have to work on increasing its resolution, and will also have to develop a way for the technique to measure the position of methylated sites on a DNA molecule.
"With the paper we showed the potential [of this approach]," Bashir said, adding that for it to be useful, "we would need better resolution potentially, but also [the] mapping ability."
The assay, as described by the group, currently relies on the use of 75 amino acids of the methyl DNA binding protein MBD1, which binds symmetrically to methylated CpG dinucleotides with high affinity, according to the study authors.
This binding changes the diameter of the DNA molecule by several nanometers, and as the protein-bound molecule passes through a solid-state nanopore, it causes a three-fold change in the ionic current relative to unmethylated and unbound DNA, the group wrote. By measuring this current change, the researchers could detect the presence or absence of even a single methylated site.
Bashir said the use of these MBD proteins to detect methylation is not new. But previous efforts have used fluorescent labels and optical detection methods to measure the proteins' binding activity to methylated DNA.
Using nanopores, the group hopes it will be able to create a method that can work with very small sample sizes, without the need for amplification or bisulfite-based methods. In the paper, the authors write that the assay could potentially be used for early disease detection, monitoring, and prognosis in diseases characterized by DNA methylation.
In the group's recent paper, Bashir and his colleagues described their current version of the assay, using nanopores drilled in a 20 nanometer thick silicone membrane. The group tested different sizes of pores —corresponding to the presence of either single or multiple bound MBD1 proteins, finding that pores between 9 nm and 12 nm were required for the molecules to slip through.
To test the assay, the researchers bound MDB1 fragments to a target DNA fragment — an 827 base pair region of the gene DLX1 containing 36 CpG dinucleotides that were methylated in vitro.
The team was able to detect the presence of even a single methylation-protein complex with "high fidelity," the authors wrote, and could also coarsely determine the number of methylation sites on each molecule — between one site, five sites, and thirty sites, Bashir said — based on the number of bound proteins that change the speed of the molecules' transit through a pore.
To increase the assay's resolution so that it could distinguish, for example, between one and two methylated sites, Bashir said the group will need to create nanopores in thinner material – down to a thickness at least as small as a single MBD1 protein fragment.
He said the team is looking at using graphene to create such thinner membranes. "The holy grail would be, using solid state, to have a membrane thin enough to get single protein resolution," he said.
Additionally, he said the group recognizes that for the assay to be really useful it should also give information about the location of methylated sites. This could potentially be achieved by coupling the methylation detection with nanopore-based sequencing.
"Right now, we can't tell which position the protein is in," Bashir said. "But we'd like to do that. Then, if you know if it is on the top or the bottom, for example, and you know the sequence of the DNA, you can determine [where it is bound]."
Bashir said he and his colleagues, like many other groups, have also been working on sequencing approaches using solid-state nanopores for several years.
Finally, he said that the team is also working on some more practical aspects of making the assay commercially and clinically useful — mainly looking at ways to couple the nanopore detection method with compatible sample extraction steps for isolating small volumes of DNA.