NEW YORK (GenomeWeb) – Researchers from the National Institutes of Standards and Technology have come up with a design for nanopore sequencing that relies on Watson-Crick base pairing and straining a thin membrane. The study, which was published recently in ACS Nano, builds on previous work by the group where they looked to use graphene as the membrane.
In the ACS Nano study, funded in part by the US's Materials Genome Initiative, the researchers described a molybdenum disulfide-based nanopore. Lead author Alex Smolyanitsky, a materials scientist at NIST, told GenomeWeb that the group is now applying for patents on the method and is interested in collaborating with industry on prototype design.
The NIST group's previous study relied on specific properties of graphene to record an electronic signal, but Smolyanitsky said that the group wanted to see if alternative materials could be used since DNA has a tendency to stick to graphene.
A number of researchers have looked for ways to use graphene to design a solid-state nanopore sequencer because the material is only one atom thick — ideal for sequencing since only one nucleotide would be present in the pore at a time, which could enable for more precise base calling. Researchers from the Kavli Institute of Nanoscience in the Netherlands were the first to demonstrate that DNA could translocate through a graphene nanopore in 2010. However, there have been a number of challenges with using graphene as a nanopore, including the tendency for DNA molecules to stick to it.
Smolyanitsky said that in the NIST team's first paper, the researchers thought that one option would be to coat the graphene so that DNA would not stick. However, they also decided to test other materials, and in the most recent study, they demonstrated that molybdenum disulfide (MoS2) may in fact be more suitable.
In the study, the researchers describe a device that includes a MoS2 nanoribbon that has been functionalized with a nucleobase over a solid electrode sensor. By functionalizing the nanoribbon with a cytosine base, as DNA passes through the pore, guanine will form a Watson-Crick base pair with the cytosine. The DNA will continue translocating, straining the MoS2. Eventually the G-C bond will break, and the MoS2 will snap back into place. The strain and subsequent release generates a signal that can be read out electrically, Smolyanitsky said.
Aside from not sticking to the DNA, another advantage of MoS2 may be an improved signal-to-noise ratio. The material is slightly more rigid than graphene, the authors wrote, so there is less noise as DNA translocates through. However, it is still flexible enough such that the Watson-Crick base pairing causes enough bend to be detected.
There are two options for designing a device that can read all four bases, according to the authors. The device can include a set of four MoS2 sensors, each functionalized with a different base, stacked on top of each other. Then, a DNA molecule would translocate through the four sensors, each of which would generate a different signal. A second option is to send four identical DNA molecules through four different sensors simultaneously.
Using known properties of the MoS2 ribbon and Watson-Crick base pair splitting forces, the researchers simulated the translocation of a DNA strand through a pore functionalized with C bases. They show that, in theory, the device would be able to distinguish G bases separated by two A bases, as well as distinct G bases in a string of repeats. In their tests, the researchers found that they could detect 11 distinct signals out of an expected 14.
Smolyanitsky said that the group has two main next steps. First, he said, the researchers need to validate the design — demonstrating that they can construct a MoS2 nanoribbon functionalized with each of the four bases. "It has to be proper in terms of the orientation of the functional group," he said. In addition, he said the researchers plan to test the electric signal generated from the binding of base pairs and stretching of the MoS2 membrane. "Once we confirm those things and see that the signal is able to stand out from the noise, we will be ready to build a prototype," he said.
The NIST team is not the only group looking to design a nanopore sequencing platform based on MoS2. Researchers at the Swiss Federal Institute of Technology in Lausanne demonstrated they could drill holes in the material and translocate double-stranded DNA through the pore and that the DNA did not stick as it did to graphene.
Smolyanitsky said that the NIST team plans to move forward with development of the MoS2 membrane as opposed to graphene because of that benefit. In addition, he said the researchers would test other configurations of the material, not just a nanopore. Rather than creating a functionalized nanopore, he said, the team plans to test the design of a functionalized edge of MoS2. In that case, the DNA would still have to pass through an aperture in order to keep the molecule in the correct position. Then it would translocate perpendicular to the functionalized edge of a MoS2 layer.