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Cambridge Team Demos DNA Origami Nanopores; Oxford Nanopore Licenses Technology

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Researchers from the University of Cambridge have demonstrated that they can build tunable nanopores — controlling both the size of the pore and the number of binding sites within it — using hybrid DNA origami structures on glass nanocapillaries, technology that Oxford Nanopore has licensed.

The work was published this month in ACS Nano and builds on a previous publication that the Cambridge group published last year, demonstrating that such a hybrid pore could be built. In the most recent publication, they showed that they could control the size of the pore, that DNA could translocate through the pore, and that by controlling the binding sites within the pore they could speed up or slow down translocation.

Ulrich Keyser, senior author of the study and a professor in the physics department at the University of Cambridge, told In Sequence that he has a pending patent application on the technique and has been working with Oxford Nanopore to further optimize the pores for label-free molecular analysis, which could eventually include sequencing.

Gordon Sanghera, Oxford Nanopore's CEO, told IS via email that the company is looking to incorporate DNA origami into nanopore design for a broad range of applications.

DNA origami nanopores "can give us even more tools for modifying the structure of these pores," he said. "We expect to be able to design pores that can give us orders of magnitude enhancements in sensitivity, specificity, and speed for many molecular sensing applications."

Aside from sequencing, Oxford Nanopore has been developing its GridIon system for protein analysis, and Sanghera said that industrializing the process of nanopore design is essential to the company's goal of developing "different pores for different applications for deployment on a single electronic platform."

Before the DNA origami nanopore could be used for sequencing, Keyser said that much more work would need to be done. For one thing, "we need to demonstrate much more control over translocation," he said.

Nevertheless, the DNA origami hybrid pore could have advantages over other types of pores being developed, he said.

"The biggest drawback of standard nanopores is you can't make them different sizes or different surface compositions," Keyser said. Purely solid-state nanopores have adjustable sizes, but not adjustable chemistries. Meanwhile, the surface chemistry of protein nanopores can be adjusted, but not their size. There's a "huge push for developing artificial systems for making pores that are different sizes and have controllable surface chemistries," he said.

The team built its nanopore using self-assembling DNA and open-source software from Cadnano to design the structure. In this way, they were able to create structures with pores of varying sizes. The software calculates the sequences that are needed based on the structure, which can then be ordered, Keyser explained.

The DNA origami structure is then fitted to a glass nanocapillary by applying voltage, which was verified with fluorescence and ionic current detection. The team then demonstrated that the process was reversible by applying negative voltage, and showed that they could repeatedly bind and disengage the DNA origami and nanocapillary.

"The demonstrated reversibility and reproducibility indicate the feasibility of combining DNA origami platforms with solid-state nanopores for highly sensitive and robust analyte detection," the authors wrote.

Next, they built nanopores of two different sizes, a 14-nanometer pore and a 5-nanometer pore. After building the pores, double-stranded DNA of 48.5 kilobases was added to the pores and voltage was applied, which started translocation of the DNA through the pore.

Similar to other nanopore sequencing systems, as DNA translocates through the pore it blocks the current flowing through the pore, producing measurable peaks.

When DNA translocated through the smaller pore, it went through in a linear fashion, but when it translocated through the larger pore, the DNA folded as it entered the pore, which showed up as two peaks.

Next, the team tweaked the structure of the pore itself, adding single-stranded DNA overhangs to the entrance of the pore in order to detect specific sequences. To either side of the pore, they attached a different overhang and then added single-stranded pieces of DNA 50 bases in length.

If the piece of DNA passing through the pore contained the complementary sequence to the overhang, it would interact with the overhang, which could be detected by a "switching of the ionic current." When the DNA is bound to the overhang, the current is blocked.

Increasing the length of the overhang increased translocation time, due to stronger interactions between the overhang and the DNA passing through the pore. Modifying the sequence overhangs will also allow for the detection of different types of analytes.

Sanghera said that this "bottom-up approach" for nanopore design "gives us more choices for geometrical design and the introduction of specific chemistry at targeted positions on the structure."

Aside from sequence detection, Keyser said this type of pore could have many other applications in analyte detection. Researchers could "think about incorporating RNA structures or other proteins, or any polymer that binds to DNA," he said.

Additionally, the system could be used to create artificial ion channels that could be embedded into cell membranes for biomedical applications.

For Oxford Nanopore's purposes, Sanghera said that the DNA origami nanopores have "potential advantages in terms of the pore's interaction with the proprietary membranes that we work with as a replacement for the traditional lipid bilayer."

"These nanopores can be turned into smart designer nanopores," Keyser added.

Nevertheless, DNA origami nanopores still have a way to go before they catch up to development of protein and solid-state nanopores, and while they offer advantages in terms of the flexibility in design, Keyser said the main disadvantage is the amount of noise the pore currently generates. "I think there are benefits to both technologies," he said.

Going forward, aside from continuing to optimize the pore, his team is also working to multiplex the DNA origami nanopores and demonstrated in a recent paper that measurements from single molecules could be taken simultaneously across 16 nanopores.

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