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Theoretical Study Points Toward Thermal Control of DNA Translocation Through Solid-State Nanopores

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A Japanese-led team has performed a theoretical analysis suggesting it may be feasible to use temperature to both denature DNA and to translocate the resulting single-stranded molecule through solid-state nanopores at speeds approaching those needed for sequencing.

With that analysis in hand, the investigators are now attempting to apply the method in proof-of-principle experiments in the lab.

"[W]e decided to first give some theoretical evaluation based on the current experiments, and now are trying [it] with the real experiments," Osaka University's Masateru Taniguchi, the study's co-senior author, told In Sequence in an email message.

In a paper appearing online this month in ACS Nano, researchers from Osaka University's Institute of Scientific and Industrial Research, along with collaborators from Uppsala University and Peking University, presented details on this so-called thermophoretic approach to DNA translocation through solid-state nanopores, arguing that it may eventually offer an effective alternative to approaches that drive DNA through these nanopores using electrical current.

The approach is expected to curb the electrical cross talk created by voltages applied on either side of the membrane during tunneling conductance, Taniguchi noted.

Moreover, he and his colleagues explained, the high temperature used to establish the thermal gradient that moves DNA from one side of an insulated membrane to the other achieves another goal: spurring the dissociation of double-stranded DNA into a single-stranded form that tends to resist self-hybridization.

Based on their models of DNA capture rate and translocation speed, the investigators estimated that thermophoresis would slow DNA translocation through a solid-state pore by two orders of magnitude compared to existing electrophoresis methods.

Despite the theoretical dip in DNA translocation speeds that may be achieved using thermal manipulation, though, the authors' models indicate that thermophoresis alone probably can't slow DNA down enough to create a situation where a single base passes through the pore each millisecond — a speed suspected of being ideal for nanopore-based sequencing methods.

For that, they speculated that it might be necessary to further tweak and tune the system, perhaps by varying the proportion of salt ions in the solution bathing the biomolecule, similar to approaches proposed for electrophoretic systems in the past (IS 1/24/2012).

And there are other hurdles to overcome before researchers can attempt to apply such an approach in a nanopore sequencing system — from finding precise pore dimensions amenable to both thermophoresis and individual base recognition to determining the effects that thermophoretic manipulation might have on the current signals associated with each base.

Even so, Taniguchi and his co-authors argued that the possibility of using thermophoretics is worth exploring, since "an efficient strategy, which can denature [double-stranded DNA], preserve the resulted [single-stranded DNA] from self-hybridization, and then propel the molecules through [the] nanopore with sufficiently low speed would greatly benefit this research field."

As several teams press forward with schemes to use protein or solid-state nanopores as the basis for new DNA sequencing devices, much effort has gone into finding ways to nudge DNA through nanopores at speeds that allow for accurate detection of every nucleotide in a DNA strand.

Generally speaking, the go-to method for moving DNA through nanopores has relied on electrophoresis, using a voltage to manipulate the molecule and drive it into the pore.

The trouble is that the level of voltage needed to achieve that translocation typically sends DNA zipping through the pore too quickly to detect current changes associated with each base.

And that has left researchers looking for a way to slow the translocation. For instance, a team from the University of California, Santa Cruz, has developed a ratcheting system that eases DNA through protein nanopores with help from the DNA polymerase enzyme phi29 (see IS 9/28/2010, IS 2/21/2012, IS 3/27/2012).

And those working with biological nanopore systems have been devising ways to precisely modify pore proteins with an eye to producing pores capable of more refined DNA transit or enhanced base detection (IS 7/3/2012).

For their part, the authors of the current study argued that there might be another solution,at least for solid-state pores housed in insulating material: DNA manipulation based on a temperature rather than a voltage gradient.

From their theoretical models — assuming a pore depth of 50 nanometers and a diameter of 10 nanometers — Taniguchi and his colleagues estimated that an effective thermophoresis system would require a maximum local temperature of 353 K (~80º C or 176º F) on one side of the membrane, the "cis chamber" where DNA denaturation takes place.

From there, single-stranded DNA would be pushed along a temperature gradient, moving through nanopores in an insulated membrane to reach the "trans chamber," where the temperature would be maintained at around 293 K (~20º C or 68º F).

"The temperature difference is about 60 K," Taniguchi said, explaining that the team anticipates that this thermal gradient between the cis and trans chambers will be sufficient to coax DNA through the pore.

The approach is not expected to be immediately compatible with biological nanopores, such as those based on MspA or alpha-hemolysin, Taniguchi explained. That's because pore proteins themselves will denature at sufficiently high temperatures, affecting protein function and, importantly for this application, size and charge properties of the pore.

Moreover, the lipid bilayers surrounding these proteins likely wouldn't provide enough insulation to establish the temperature gradient needed to achieve thermophoretic DNA translocation.

That may change if researchers more regularly turn to hybrid nanopore systems that contain protein nanopores such as alpha-hemolysin that are set within solid-state nanopores, as some have proposed (IS 12/21/2010).

"Currently, our group focuses on experimental and theoretical studies using synthesized (solid-state) nanopores," Taniguchi said. "In the future, our group or others may try inserting [alpha-hemolysin] into solid-state nanopores."

For the time being, though, the Japanese-led group is considering the possibility of creating solid-state nanopores within thin films formed by alternating layers of tungsten and selenium.

"The selection of membrane material is crucial for our thermophoretic design," Taniguchi explained in his email message. "The thermal conductivity … of the membrane should be much smaller than the water solution, in order to obtain the wanted temperature distribution in our nanopore system."

In addition to the type of material used, the theoretical analysis presented in the new ACS Nano paper indicates that a nanopore's size and thickness would likely impact DNA translocation speeds in a thermophoretic system as well.

In particular, Taniguchi noted that relatively deep pores with wide diameters appear most apt to slow DNA translocation speeds. On the other hand, he emphasized that for nanopore sequencing applications, it will be necessary to find a balance between pore features that provide attractive DNA transit speeds with features suitable for reading individual bases within the DNA strand as it moves through the pore.

"[I]n the real experiments a compromise with other requirements of sequencing has to be made," he said. "For example, the nanopore diameter should be kept smaller than some critical value for preventing simultaneous multi-strand translocation."

Going forward, the group hopes to pair the thermophoretic approach with other methods that might slow DNA translocation even more — ideally, bringing this process closer to speeds of around one base per millisecond.

Taniguchi noted that the team is in the midst of proof-of-principle experiments aimed at demonstrating the feasibility of the theoretical thermophoretic principles described in the current study.

If those experiments pan out, the next step would be looking at ways of fine-tuning and further slowing DNA translocation, perhaps through tweaks in the concentrations of potassium chloride, sodium chloride, or lithium chloride salts included in the system.

Because cations and anions tend to have different migration speeds in a temperature gradient such as the one the researchers are proposing, Taniguchi said it should be possible to use them for creating a non-uniform charge background in the system.

And, he explained, past research suggests that the resulting "thermo-built electrical field" can help to control biomolecule movement. "We hope to make use of this fact to further slow down the translocation speed."

Another kink that needs to be ironed out before thermophoretics makes its debut in a nanopore sequencing system is accurate base detection, Taniguchi explained, since rising temperatures sometimes bump up the noise associated with electrical currents.

"Tunneling currents show small dependence on temperature," he said, "while ionic currents show large dependence."

"We are currently working on development of measurement systems to overcome noise," he added.

Though Taniguchi said he believes "world collaboration is a key issue to realize [a] commercial platform for nanopore sequencing," he and his colleagues are not currently working with any groups attempting to develop nanopore sequencing devices.

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