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Hitachi Team Finds Narrow Nanopores Slow DNA; Plans to Use Faster Amplifiers, Improve Pore Chemistry

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NEW YORK (GenomeWeb) − One challenge developers of nanopore sequencing technologies have been facing is DNA passing through the pores too quickly to detect its sequence with single-base resolution. As a result, research groups have come up with various schemes for reducing the molecule's speed, for example, by coupling it to a polymerase.

Researchers from Hitachi in Japan have now shown that single-stranded DNA slows down more than 16 times when a solid-state nanopore is made half as wide, and could potentially be slowed down more by reducing the diameter further. While they say that this is still too fast for sequencing, they plan to combine this approach with chemical changes to the pore to further reduce the speed of the DNA, and plan to use a high-speed amplifier to increase signal resolution.

The team, led by Takashi Anazawa at Hitachi's Central Research Laboratory in Tokyo, published its results – which also found that single-stranded DNA interacts more weakly with the pores than double-strand DNA – in Nanotechnology last month.

It is unclear whether the work is part of a collaboration between Hitachi and UK-based Base4 Innovations to develop a solid-state nanopore sequencer, which Base4 announced a year ago. Anazawa declined to comment on any aspects of the work that are not addressed in the journal article.

The sequencer developed under the partnership over a three-year period would combine solid-state nanopores with optical detection, using gold nanostructures and lasers, Base4 CEO Cameron Frayling said at the time.

Researchers working with solid-state nanopores – unlike groups from Oxford Nanopore Technologies, Genia, or the University of Washington, which are using biological pores – have explored a variety of approaches to slow DNA translocation, including decreasing the voltage across the pore or the temperature, increasing the viscosity of the solution, or using a salt gradient across the pore.

But according to the authors of the Hitachi study, these methods for reducing DNA speed "decrease the sensitivity of ion current measurement," whereas narrowing the nanopore "is a simple approach compatible with DNA sequencing."

Others have also explored that approach, for example, Meni Wanunu's group at Northeastern University, which showed last year that single- and double-stranded DNA passing through narrow hafnium oxide nanopores is slower than DNA traveling through wider pores made from other materials.

For their recent study, the Hitachi researchers used silicon nitride pores, prepared with a transmission electron microscope, which they narrowed to various diameters – ranging from 2.3 nanometers to 10.3 nanometers – using atomic layer deposition.

They fed 1-kilobase double-stranded DNA and 5.3-kilobase single-stranded poly-A DNA through the pores and measured their translocation speed.

Single-stranded DNA, they found, traveled 16 times more slowly through the 2.3-nanometer pore than through the 4.5-nanometer pore; its speed was reduced from 0.011 microseconds per base to 0.18 microseconds per base.

Double-stranded DNA, on the other hand, passed through a 4.4-nanometer pore 51 times more slowly than a 10.3-nanometer pore; it slowed down from 0.043 microseconds per base to 2.2 microseconds per base.

According to the authors, the fact that double-stranded DNA travels through the same size nanopore much more slowly than single-stranded DNA "could not be predicted from the findings obtained in the previous studies on DNA translocation using [double-stranded] DNA" and means that it interacts more strongly with the pore than single-stranded DNA.

Anazawa told In Sequence that molecular dynamics simulations show the ability to control DNA speed "is dominated by the force to drag DNA molecules away from associated solvent molecules when DNA molecules enter the nanopore," a finding that also explains the experimental result that translocation speed does not depend on the thickness of the nanopore.

The scientists also found that their measured translocation speeds agreed well with those predicted by simulations. Based on such simulations, they estimated that single-stranded DNA would travel through a 1.4-nanometer pore – approximately the same diameter as single-stranded DNA itself – at a rate of 1.4 microseconds per base.

That, they said, would still be too fast for DNA sequencing, which would require a speed of 1 millisecond per base.

But, they wrote, they could in the future use a high-speed amplifier with 1 megahertz resolution, which would improve the time resolution 100 times over standard amplifiers, so a DNA speed of 10 microseconds per base would be sufficient for sequencing.

In addition, they wrote, they could change the chemical properties of the nanopore to increase its interactions with DNA, allowing them to slow single-stranded DNA to 10 microseconds per base.

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