NEW YORK (GenomeWeb) – Researchers from the University of Missouri-Columbia have begun refining a nanopore-based microRNA detection method so that it can identify the expression of multiple miRNAs simultaneously and potentially be used for the diagnosis of cancer and other diseases.
The work is being funded by a three-year grant from the National Institutes of Health worth roughly $300,000 in its first year.
While RT-PCR remains the gold-standard for miRNA detection, that method involves covalent labeling and target amplification, which can introduce variability into study results. To address this, MU's Li-Qun Gu and colleagues developed an approach that combines nanopores and oligonucleotide probes.
As they described in a 2011 publication, they created a nanopore sensor based on the alpha-hemolysin protein. Noting that distinguishing the translocation of different miRNAs in this context has been difficult because the small, non-coding RNAs are short and similar in length, Gu and his team created programmable oligonucleotide probes that bind to specific miRNAs and generate target-specific signature signals as they pass through the nanopore.
Using the approach, the investigators were able to detect miRNAs in the serum of lung cancer patients at subpicomolar levels, and could distinguish single-nucleotide differences between miRNA family members.
Looking to take the technology further, Gu's group later developed a way to "barcode" the probes so that multiple miRNAs could be detected in a single nanopore.
"When the probe is bound with the target, the barcode group polyethylene glycol attached on the probe through click chemistry can specifically modulate nanopore ion flow," the scientists explained in a 2014 paper. "The resulting signature serves as a marker for the encoded target. Therefore, counting different signatures in a current recording allows simultaneous analysis of multiple targets in one nanopore."
While this research was encouraging, Gu and his collaborators were still encountering noise from non-target molecules, he told GenomeWeb this week. However, they were able to fix this by employing a nanopore effect they discovered and dubbed carrier-guided nanopore dielectrophoresis (CND).
Essentially, this effect involves the creation of a highly non-uniform electric field outside a nanopore entrance. When the probe and miRNA hybridize, they form a dipole that is attracted via dielectrophoresis by the electric field. Meanwhile, non-target molecules, which carry a negative charge, are electrophoretically repelled away from the nanopore.
"Consequently, only the signatures for the miRNA/carrier probe complex can be identified [and] any interference signal originating from non-target species is completely eliminated," Gu wrote in the NIH grant's abstract.
This, he noted, has the added advantage of concentrating target miRNAs around the nanopore, facilitating their translocation.
Having received the new NIH funding, Gu is now focused on further improving his miRNA detection method, as well as demonstrating its clinical utility.
The grant money, he said, will be used to elucidate the CND effect and to improve the sensitivity of the probes. Because miRNAs are often expressed at low levels in circulation, improving the method's detection limit is an important consideration. This, he said, may be achieved by manipulating the electrical charges of both the nanopore and probes to enhance their interaction.
Gu also aims to optimize the CND mechanism so that it can be used to achieve single-nucleotide discrimination and with the multiplexed approach described in the earlier publications. Ideally, he added, his team will be able to detect as many as 10 different miRNAs at once.
Seeing the biomedical potential in his technology, Gu will also test how the CND-enabled miRNA detection method stacks up against RT-PCR in patient samples.
In collaboration with researchers at the MU School of Medicine, total RNA will be extracted from blood collected from lung cancer patients and then analyzed for miRNA content using RT-PCR or the nanopore approach.
Should his technology prove as or more effective than RT-PCR, Gu said he envisions its further development as the foundation of a diagnostic assay, not only for cancer but other diseases for which miRNA expression can be informative.