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Wake Forest Team Developing Nanopore Sensor for Detection of DNA Epigenetic Modifications

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NEW YORK (GenomeWeb) – Building on a previous nanopore sensor method for biomarker detection, researchers from Wake Forest University have shown that their nanopore sensing technique can detect epigenetic modifications.

The technique could be used to identify epigenetic modifications that are difficult to detect by other methods, and would have applications in both research to discover new epigenetic modifications as well as in the clinic to identify epigenetic modifications relevant to cancer.

Adam Hall, senior author of the proof-of-principal study published this month in Scientific Reports, told GenomeWeb that in the future he envisions a handheld device that includes a consumable chip with the nanopore configured with a microfluidic infrastructure. The device could be used to noninvasively monitor cancer patients for disease progression or recurrence and eventually may even be used for early diagnosis, he said.

The university holds intellectual property on the methods and Hall said that the group has spoken with several interested industrial partners to potentially commercialize the technology.

In the most recent study, the group demonstrated that the nanopore sensor could identify 5-hydroxymethylated cytosine in genomic mouse DNA.

Hall's team previously described the nanopore sensor technique — where short single molecules are labeled in such a way that the construct can be detected as it passes through a nanopore — and showed that it could detect specific micro RNAs.

The technique relies on attaching a streptavidin protein to biotinylated DNA, which is detectable within a silicon nitride nanopore. To selectively detect 5-hmC, the researchers use the T4 bacteriophage enzyme beta-glycosyltransferase to attach a glucose molecule specifically to 5-hmc, which can then be biotinylated. The group first demonstrated that it could do that on a 156-bp synthetic DNA molecule with a known 5-hmC nucleotide and that the construct could then be detected in its nanopore system.

Next, the researchers tested whether the approach could detect 5-hmC on mouse DNA from brain tissue because 5-hmC is known to be abundant there and is thought to play a role in neurological diseases.

The nanopore sensor relies on short pieces of double-stranded DNA, so the team first fragmented the DNA into pieces with an average length of 75 bp. Next they performed the 5-hmC biotinylation process — attaching the glucose and biotin to locations with 5-hmC.

To validate the measurement of 5-hmC using the nanopore, the researchers compared it to the established technique of liquid chromatography-tandem mass spectrometry. That method is very sensitive, but is time consuming, expensive, and complex. "Achieving similar results with a rapid, low-cost system would be a tremendous advance," the authors wrote.

Compared with the liquid chromatography-tandem mass spectrometry method, the researchers found that their nanopore method gave comparable results for the abundance of 5-hmC. The team assed the 5-hmC content using two nanopore devices, finding abundances of 0.46 percent and 0.55 percent, while the mass spec-based method calculated an abundance of 0.56 percent.

The devices gives a "quantification of how much of the modification was in the DNA to begin with," Hall said. Levels of 5-hmC have been shown to be important markers for cancer, and are significantly reduced in cancer. Unlike methylated cytosine, Hall said, which is sometimes upregulated and sometimes downregulated in cancer, 5-hmC is "striking in that in nearly every cancer, it is always decreased in the patient" which could make it a "much more straightforward biomarker."

Hall said the next steps are to reproduce the results shown in this study, but with many more platforms. "This paper was a proof of principle that showed that we can measure 5-hmC and that the levels we get are consistent with mass spec," he said. But going forward, he said, the group wants to demonstrate that it can reproducibly "show that we can detect 5-hmC in a fast and meaningful way with our system."

Hall, who is also affiliated with Wake Forest's Comprehensive Cancer Center, said that an important next step would be to use the method on patient samples to determine whether or not measuring global content of 5-hmC would be a good way to diagnose disease.

For instance, he said, he envisions a potential future clinical application as using the method to noninvasively assess patients' 5-hmC levels to monitor them post treatment or surgery. The ultimate goal would be to design a test for early detection of cancer, Hall said. For instance, one that could be used to screen people who are at a high risk due to a family history, and routinely check their 5-hmC levels in cell-free DNA from blood. That application is still far away, since researchers would first have to determine a threshold of 5-hmC that distinguishes between normal and cancer.

In addition, Hall said that his group is working on expanding the method to be able to detect other types of epigenetic modifications like methyl adenine. He would also like to develop a method that not only quantifies the modifications but can determine their position relative to genes, which would require a different type of assay then the one described in the recent study, Hall said. Although a similar biotin labeling scheme could be employed to identify the modifications, to determine their location a longer piece of DNA would be needed, so that the positions of the modifications could be mapped, Hall said.

Hall has also collaborated with Quantapore, a startup developing a nanopore-based sequencing technology, and sits on the firm's scientific advisory board. However, this recent study in Scientific Reports is not related to the Quantapore collaboration, he said. While Quantapore is focused on nanopore sequencing, Hall said his lab focuses on non-sequencing nanopore-related applications.

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