NEW YORK – A team of researchers from the University of Missouri has developed a new protein nanopore-based sensor that can profile aptamer–small molecule interactions in real time and without the need for labels.
In a study in Proceedings of the National Academy of Sciences in June, they showed that the sensor was able to detect aptamer conformational changes in response to small-molecule binding with millisecond resolution, opening the door to a number of applications.
A family of small, single-stranded DNA or RNA oligonucleotides, aptamers are capable of binding to specific targets, such as small molecules, with high affinity and specificity, similar to antibodies. This can be harnessed to develop biosensors that can detect biologically important small molecules, such as neurotransmitters and hormones, according to Li-Qun Gu, a professor of chemical and biomedical engineering at the University of Missouri and a corresponding author of the study.
In addition, because aptamers are the native structure of riboswitch, an RNA regulatory element that controls gene expression upon binding with target ligands, they can be utilized in synthetic biology to engineer gene circuits to control gene expression and gene editing, Gu said.
Despite the potential utility of small-molecule-binding aptamers, the study authors noted, the field still needs a sensitive, rapid, and low-cost tool to help characterize these molecules and study their conformational dynamics when binding to small molecules.
Previously, biological nanopores have been deployed to help study biomolecular structures at the single-molecule level. However, these nanopore measurements were often limited to providing a conformational "snapshot," Gu said, leaving out the temporal dynamics of the analytes as they change conformation.
"We are extending [nanopore sensing] further, where it is one molecule at a time, but you are also getting this time-dependent information of these conformational changes that are occurring with ligand binding," said Kevin Gillis, a professor of chemical and biomedical engineering at the University of Missouri and the other corresponding author of the study.
To achieve this, the researchers utilized Mycobacterium smegmatis porin A (MspA) pore, a protein nanopore originally developed for DNA sequencing by Jens Gundlach's lab at the University of Washington.
For academic research purposes, Gu said the team does not need to license to the MspA pore, including the M2 mutant pore from UW. For future commercial products in the future, it may require a license to the pore, though. The group has also developed its own version of the pore, M7
Taking advantage of the fact that the MspA pore contains a nanocavity with a positively charged ring on its walls, the researcher designed and optimized a scheme where the aptamer scaffolds can be noncovalently docked inside the pore using a cluster of site-specific charged residues.
As small molecules flow through the pore, they are captured by the aptamer inside, and the resulting conformational changes cause the ion current through the pore to characteristically vary.
"One thing that is very important here is that the pore has to be the right size," Gillis said, adding that the cavity has to accommodate aptamers inside while incurring a deviation in conductance during their conformational changes.
In their proof-of-concept, the researchers tested the sensor with three nucleic acid aptamers targeting different small molecules: dopamine, serotonin, and theophylline. Overall, they reported that the continuous ion current fluctuations through the nanopore can "act as a movie depicting molecular interactions," providing clear signals of the conformational dynamics of the aptamers upon binding to the target small molecules.
In addition, the study demonstrated the sensor's molecular screening capabilities. By introducing small molecules with different structures, the researchers noted, developers can use the sensor to screen for those molecules that can specifically bind to and interact with the target aptamers.
With continued optimization and development, the researchers envision their sensing technology could be used in a wide range of translational applications down the road. These include small-molecule drug discovery, in particular for drugs targeting RNA; tool development in synthetic biology for programming small-molecule-regulated gene expression mechanisms; and sensor development for small molecules, including neurotransmitters, for translational and diagnostic purposes.
"Now that nanopores are on the scene and getting more ubiquitous, finding new applications and extending them beyond sequencing polymers, I think, is pretty interesting," said Jeff Nivala, a professor at the University of Washington who was not involved in the study.
According to Nivala, one of the strengths of the study is that the sensor appears to be pretty generalizable. "Any new platforms that can potentially detect a wide range of different metabolites in a faster, less expensive way, and with less complex instrumentation, would be an important need for the field," he pointed out.
In addition, Nivala applauded the sensor's capability of obtaining a continuous measurement during the aptamer-small molecules interactions.
One hurdle that remains to be overcome for this technology, he said, is its sensitivity. According to the study, the limit of detection (LOD) for dopamine was 100 nM, which is "not low enough" to detect neurotransmitters in tissue that are typically in the 1 nM to 10 nM range, the authors acknowledged.
"It is remarkable that these interactions are observed in real time, so you have the time-resolved analysis of these interactions," said Liviu Movileanu, a professor at Syracuse University who was not involved in the study. "Because we are at the beginning of understanding this process, any type of technology, such as [the one in] this paper, it's good for us, because it teaches us how things work."
Still, the sensor needs to be further developed to improve its detection limit before it can be widely used for real-world applications, Movileanu said.
Acknowledging this limitation, Gillis said he and the team "still have a ways to go to optimize this technology to achieve the sensitivity we'd like to have."
"Part of the limit is how long you have to wait for a binding event of the analyte," he added. "Since you are waiting for a single molecule to bind, if the concentration of the analyte is low, it can take a while."
His team is currently seeking funding to further investigate the biophysics of the pore to improve the system's sensitivity and achieve a low detection limit.
Moving forward, the researchers also plan to further develop the sensor into an automatic system by incorporating microfluidics and other engineering strategies for screening small molecules, he said. As data analysis is currently done "old fashioned by hand," where the researchers manually identify each signal interval, the team is also seeking collaborations to incorporate AI for automated data analysis, Gu added.