Nanopore sequencing entails the passage of biomolecules, suspended in an ionic solution, through a tiny pore in an ultra-thin membrane. As each molecule traverses the pore, it partially obstructs the aperture, permitting the passage of varying quantities of ions. Researchers typically analyze changes in the ionic current to derive structural information during the sequencing process.
These nanopores are on the order of magnitude of similar pores we have in the cells that facilitate the transport of different size molecules from cell to cell and subcellular components to their neighboring compartments. The potential for advancing applications utilizing this newly developed nanopore system, as it may relate to various biological applications is significant. Presently the published work explored the potential of the nanopore system for DNA sequencing.
The ability to control and monitor the movement of molecules through these pores has opened up new avenues for research in the last two decades, according to Marija Drndić, a physicist at the University of Pennsylvania. The field of synthetic nanopores, where pores on the scale of nanometers are made out of materials like graphene and silicon, has already led to significant advances in DNA sequencing.
In a paper published in Nature Nanotechnology, Drndić and Dimitri Monos, her longtime collaborator at the Perelman School of Medicine and the Children’s Hospital of Philadelphia, demonstrated a new kind of nanopore platform, a dual-layer nanopore system. This design consists of two or more nanopores stacked just nanometers apart, allowing for more precise detection and control of molecules like DNA as they wiggle through.
Their joint efforts have yielded a platform that advances nanopore technology and opens up new possibilities for clinical applications by eliminating the need for proteins on the sequencing platform that guide and unwind the DNA, as it is in a currently and commercially available nanopore sequencing platform, creating a purely solid-state system for DNA sequencing.
“With current platforms, when molecules like DNA are placed near the nanopores, it’s sort of like having spaghetti in a pot—tangled and difficult to work with, let alone guiding through one hole,” explains Monos. So, typically, researchers need to use proteins to capture, unwind, and straighten it, which, while effective, has many limitations due to nanopore degradation that leads to reduced sensitivity and shorter operational lifespans.
“But with this new design,” Monos says, “we’re essentially guiding molecules through two coupled nanopores in the material, providing a controlled, smoother passage of molecules for proper detection and characterization.”
The researchers call this new platform GURU to denote that it’s a “guiding and reusable” device for studying molecules. This confers a few key benefits, most notably the sensitivity to molecule length and conformation as they pass through the GURU device.
“Because we know the precise distance between the two nanopores, we can use it as a kind of ruler,” says Drndić, “decoding the signals to determine the velocity of the DNA passing through.” This system offers researchers an unprecedented level of spatial control, enabling them to capture the position of molecules, down to nanometers, in real time more accurately.
Unlike traditional single-pore systems, GURU works like a speed bump, slowing down DNA before it enters the sensing zone, enhancing the detection process. One of the most intriguing outcomes of this slowing down is the discovery of unique signal patterns shaped like the letters “W” and “T,” a characteristic that first authors of the paper, Yung-Chien (Killian) Chou and Chih-Yuan (Scottie) Lin, first observed.
“These patterns correspond to how molecules interact with the dual-pore system,” Lin explains. “When we measure signals that look like a ‘W,’ the first drop of the W indicates DNA enters the bottom pore, whereas the second drop of W tells us the moment when the same DNA enters the pore on top.” Chou adds that the T-shaped signal occurs when a molecule is long enough and able to block both nanopores simultaneously, reflecting the portion of the molecules that is under characterization.
This pattern details when and how the molecule passes through the GURU system, revealing its interactions with each layer.
As the teams continue to refine their system, they believe it could lead to more efficient, accurate, and cost-effective sequencing technologies that overcome the limitations of current protein-based nanopore systems.
“What truly cemented our collaboration was the shared goal of improving sequencing technology, particularly for applications like human leukocytic antigen (HLA) genes that require long DNA reads,” Monos says. As the Director of the Immunogenetics Laboratory at CHOP, Monos works extensively with HLA genes, crucial for assessing immune system compatibility in transplantation.
“The Major Histocompatibility Complex a 4Mb region on chromosome 6 that harbors the HLA genes is among the most complex regions of the human genome, and accurate long-read sequencing is essential to understanding their variations,” he adds. “That’s where nanopore technology like GURU comes in—it offers the potential for more precise and comprehensive sequencing in this challenging area and certainly others around our genome.”
“The problems we’re trying to solve with nanopores, like DNA sequencing and molecular detection, require expertise from people all over the place,” Drndić says, “it’s not just about the physics or materials science. We need input from biologists to understand the molecules, chemists to help with the reactions, and medical professionals to see the real-world applications.”
Marija Drndić is the Fay R. and Eugene L. Langberg Professor of Physics in the Department of Physics & Astronomy in the School of Arts & Sciences at the University of Pennsylvania.
Dimitri S. Monos is the Evelyn Willing Bromley Endowed Chair in Clinical Laboratories and Pathology at the Perelman School of Medicine, Director of Immunogenetics Laboratory and member of the Division of Genomic Diagnostics at Children's Hospital of Philadelphia.
Yung-Chien (Killian) Chou is a former postdoctoral researcher in the Drndić Laboratory at Penn of Arts & Sciences, now working at IBM Research.
Chih-Yuan (Scottie) Lin is a postdoctoral researcher in the Drndić Laboratory at Penn of Arts & Sciences.
Other authors include Rachael Keneipp of Penn Arts & Sciences, Joshua Chen of the Massachusetts Institute of Technology, Parisa Yasini of Wolfspeed, and Alice Castan of C12 quantum.
