NEW YORK (GenomeWeb) – Researchers from Wake Forest University have built a solid-state nanopore that can distinguish between single-stranded and double-stranded nucleic acids and have demonstrated its ability to identify specific microRNAs. The device could eventually have applications in biomarker detection, particularly cancer-associated markers, according to the researchers.
The team is now working to move beyond the proof of principle, which was described recently in Nano Letters, to enable the detection of multiple biomarkers as well as bacterial nucleic acids, and hopes to partner with a commercial entity to build a robust, scalable device, Adam Hall, a principal investigator in nanobiotechnology at Wake Forest, told GenomeWeb.
The work builds off an earlier study also published in Nano Letters in 2014 by the group that showed how streptavidin proteins bound to a short biotinylated double-stranded DNA molecule could be detected within a silicon nitride nanopore. In that study, Hall and his team demonstrated that neither the double-stranded DNA molecule nor the streptavidin proteins individually elicited a signal when they passed through the pore, but the complex elicited a detectable event.
Now, Hall said the group has tweaked that initial demonstration in order to detect specific sequences.
"It's an efficient way to electrically tell the difference between single-stranded and double-stranded DNA or RNA, and we used that principal to identify a specific sequence within a mixture," he said.
He said the researchers chose to demonstrate the technique on microRNA because of their role in regulation and because some are known biomarkers of cancer. In addition, microRNAs exist cell-free in blood, which would enable easy, noninvasive collection.
The team decided to focus on miR155, a 23-base single-stranded RNA molecule that has been established as a marker of lung cancer. In order to detect the molecule, the researchers designed an oligonucleotide that included a biotin and the complementary sequence to miR155 to act as a bait for miR155. Although neither molecule alone elicited an electric signal as they passed through the pore, when the DNA and RNA molecules were bound, the molecule could be detected.
Next, the team wanted to make sure that their method would detect only the specific molecule and not other microRNAs. To test that, they created a mixture of single-stranded DNA molecules with 25 percent homology to the target to act as "non-specific decoy sequences," the authors wrote. They then combined those decoys with the bait molecule. When the mixture was in the presence of the nanopore and voltage was applied, they observed very few translocation events, indicating that the bait was selective for the target.
When a mixture of decoys and targets were combined with bait molecules and an electric field was applied, they observed translocation events consistent with the levels of targets present.
Although there were still a number of translocation events due to the decoy, the "bait-target coupling was marked by a relative enhancement of about an order of magnitude under applied voltages of greater than150 millivolts," the authors wrote. "This result demonstrated that our assay could be used to discriminate a single sequence of interest from a heterogeneous mixture with high specificity."
The method being developed by Hall and his team differs from Oxford Nanopore Technologies' MinIon, in that the goal is not to sequence long strands of DNA or RNA, but rather to detect the presence of short pieces of nucleic acids. In that sense, Hall said that it is more similar to a PCR assay rather than a sequencing device.
In addition, he said it could fill an important gap for fast identification of hard-to-detect small molecules, like microRNAs, that could indicate a disease like cancer. Detecting microRNAs has proven challenging for other technologies, like quantitative PCR, Hall added. "Because microRNAs are so small, it's difficult to design a PCR probe that's selective for them," he said.
Aside from microRNAs, Hall said the device could have applications for other types of biomarkers, for instance, in detecting conserved sequences of ribosomal RNA indicative of pathogenic bacteria. Such an assay could then be used to assess water contamination or even to diagnose infections like sepsis, he said.
The technique is also fast, he said. Even when miR155 was present at a low concentration, data collection only took 10 minutes, he said.
Hall said the team is now trying to demonstrate that the method can work from actual biological samples. In addition, he said they are working on designing a protocol that would measure rRNA from bacteria.
One feature that would be useful to have would be the ability to detect multiple biomarkers, an aspect Hall said that the researchers are interested in pursuing. "Multiplexing would be very important," he said, "because while one marker might not be the best surrogate for disease, a panel would be a much stronger indicator."
He said the group is exploring two methods for multiplexing. One would be running multiple targets through separate nanopore detectors simultaneously. Alternatively, the team is also exploring whether they can analyze a sample for multiple targets serially using the same device.
Going forward, Hall said he anticipates that a major hurdle will be building the device to scale. Solid state nanopores have been studied for a long time, he said, and their biggest issues have always been around stability and reproducibility. "Even if you make two pores that are the same size, they don't necessarily act the same because of microscopic differences," he said. But reproducibility will be necessary, particularly if the device will eventually be used clinically.
Another common problem is that pores often become clogged because they are so small. Hall said that one nice thing about their approach is that clogging has not been a huge problem, most likely because they are analyzing very small pieces of RNA and DNA, rather than long strands.