Basic science and clinical medicine researchers at the University of Missouri have collaborated to develop a nanopore-based detection assay that can discriminate single-nucleotide differences between microRNA family members, potentially allowing more precise quantification of disease-associated miRNAs.
Such precision is lacking in current miRNA detection methods for disease marker development and diagnosis, like qRT-PCR and microarrays, Michael Wang, one of the study's two lead authors, told Gene Silencing News this week. While nanopore technology has been around for more than a decade, according to Wang's colleague and co-author Li-Qun Gu, using nanopores to detect miRNAs is relatively new.
Using alpha-hemolysin, a bacterial toxin protein, Gu and colleagues at the university's department of biological engineering created a nanopore sensor that uses programmable oligonucleotide probes to quantitatively detect miRNA at the subpicomolar level and can distinguish between miRNAs of the same family with single-nucleotide differences. The group described the new method — and showed that it could selectively detect microRNAs in plasma samples from lung cancer patients — in Nature Nanotechnology earlier this month.
"It is a single-molecule detector, so each time you can identify a signature event from one miRNA molecule,” Gu said. ”And if there are miRNA that have similar sequence [in a blood sample], we can identify this sequence from the signature signal.”
"We believe that we have a strong[er] ability to do this than other technologies," he added.
Movement of nucleotide strands through the two nanometer pores of alpha-hemolysin has been well studied and has been a particular area of interest for DNA sequencing applications. However as Gu and Wang wrote in their report, the ability to distinguish between different miRNAs moving through nanopores poses a greater challenge than longer molecules like DNA because miRNAs are all very similar to each other in length.
In nanopore detection, a molecule passing through the pore lumen affects the pore's electrical current. The quality of this change distinguishes between different sequences. But with all miRNAs having a very similar length, marking the passage of different molecules through the nanopore structure is challenging.
To overcome this, the researchers designed oligonucleotide probes with signal tags on both ends that would bind to specific miRNAs by Watson-Crick base pairing, creating a detectable difference as the hybrid of miRNA and probe translocate the nanopore.
"When the hybrids are in the lumen of the nanopore, there is a process called unzipping, which means the [probe and miRNA] disconnect from each other,” Wang said.
"One single chain is passed first and the next chain is passed later. This process can generate an electronic signature signal, and that's why the nanopore can specifically detect the miRNAs," he said.
"We engineered this probe so that it can be trapped in the nanopore with a high frequency so we reach a high sensitivity," said Gu.
Using the lung cancer-associated miRNA miR-155, the group measured voltage changes in the nanopores as probe-bound miRNAs moved through and broke apart. The signature signal they achieved, the group wrote in the study, "ensured the high selectivity required for microRNA detection in plasma RNA extract."
Then the team evaluated the sensor's ability to distinguish sequence-similar miRNAs from the same family, choosing the let-7 tumor-suppressing group as a target.
"Different species in the same miRNA family can have just a one- or two-nucleotide difference, and in the clinical case, those [differences] have a significant consequence because each family member indicates a different clinical situation," said Wang.
"For example, in the case of let-7, multiple members in different clinical situations will have different expression. Therefore, to detect those different members is clinically significant," he said.
The researchers measured the areas under receiver operating characteristic curves to evaluate the ability of the nanopore method to distinguish between miRNAs, finding that the area under the curves varied between 0.72 and 0.83.
"We found that that from the signature signal, we can clearly distinguish these miRNAs with similar sequence," Gu said.
Finally, the group then tested their nanopore device in the detection of miRNAs in clinical samples, using peripheral blood samples from six normal controls and six lung cancer patients. They compared their nanopore detection against a SYBR Green-based qRT-PCR assay for the detection of miR-155 in the samples.
In the Nature Nanotechnology paper, the authors wrote that PCR measured a 4.3-fold increase in miR-155 in the lung cancer group, while the nanopore sensor measured a 2.6-fold increase.
"Basically we found a consistent result from those two methods," Wang said. "However, we also found that the nanopore method had a more accurate result because the result from qRT-PCR had a bigger standard deviation."
In their report, the authors write that although both assays "indicated a significant increase of miR-155 in lung cancer patient samples, the nanopore method demonstrated higher accuracy with no requirement for labeling or amplification."
Another group of researchers at the University of Pennsylvania has also been working on using nanopores to detect miRNA, but Gu said their pores are synthetic, rather than protein-based, and their method requires a commercial kit to enrich the miRNA. "In our case, we detect miRNA from the total RNA extraction from patient plasma," he said.
Because the Missouri team's clinical sample size was so small — just six controls and six cancer samples — they are not yet at the point where they have established sensitivity and specificity for the assay, Wang said. But the group is now planning to do a study using a larger cohort.
"We proved the method works, and verified the nanopore tech by RT-PCR … and in the near future we are going to use more samples to get statistical data," said Wang. The group already has lung cancer samples available, he said, so it plans to move forward with those.
"The reason we selected lung cancer this time is because lung cancer does not have a reliable screening method, which results in later-stage diagnosis and a very poor prognosis. So we tried to use lung cancer as an example to demonstrate this method," he said.
However, cancer is not the only diagnostic application for the technology. "In the future I think it can also be used in other cancer, as well as other diseases like diabetes or heart disease, or in autoimmune disease. We know these diseases have some dysregulation of the miRNA," Wang said.
Gu's arm of the group will then try to develop the nanopore method as a "versatile analytical tool," he said.
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