Nanopore DNA sequencing inches closer to commercial debut Free

Inspired by the transport of ions and molecules through protein channels in cell membranes, scientists began research in the early 1990s on what was projected to be a simple and fast technique to sequence DNA. Conventional methods require that DNA be fragmented, replicated, and fluorescently labeled before the order of its bases can be optically determined. But one can, in principle, electrokinetically drive an intact, unlabeled strand of DNA through a pore slightly wider than the diameter of the DNA molecule in an ionic solution. As each successive base enters the pore, it disrupts the flow of ions through the pore, resulting in a measurable “blockade current” that’s different for each of the four bases. Some nanopore technology startups are developing systems that combine nanopores and optical detection or that analyze bases that have been cleaved from the DNA strand. An ideal nanopore sequencer, such as the one being created by ONT, would analyze long strands of DNA and would eliminate the need for costly optics and multiple chemical reagents.

The fundamental challenge with strand sequencing, says Muthukumar, “is that entropy dominates the dynamics, and the longer the polymer chain, the harder it is to control the entropy of the wagging tail.” Researchers have long struggled to control DNA’s pore-transport dynamics, which directly impacts the quality of the measurement of the blockade current, says NIST biophysicist John Kasianowicz, who has conducted groundbreaking nanopore-sensing experiments using α-hemolysin, a tube-shaped protein pore. “Our results show that the more time a base spends in or near a pore,” says Kasianowicz, “the stronger and more resolved the electrical signal.”

To that end, Jens Gundlach, a physicist at the University of Washington, has developed a nanopore-sequencing approach that both strengthens the electrical signal and slows DNA’s movement through the pore. He uses the funnel-shaped bacterial protein MspA as the nanopore and a ratchet-like enzyme that delivers the DNA strand into the pore at a controllable rate. When a DNA base enters the funnel’s constricted region, it generates a more distinguishable electrical signal than it does in α-hemolysin. And with the ratchet-like enzyme, the MspA sequencer slows DNA’s passage through the pore from microseconds to a few milliseconds per base.

For its part, ONT says it will use a biological pore in its initial commercial offering. Protein pores are ideal, says Gundlach, because one can tailor their surface charge by inducing mutations in their genes. “You can essentially engineer them at the angstrom level, and nature [through self-assembly] reproduces them at zero cost.” But ONT, other nanopore-sequencing companies, and noncommercial researchers are also pursuing systems that use more durable inorganic materials.

Leading alternatives to biological pores include silicon nitride, graphene, and other solid-state materials that can be fabricated to any specified pore size, need no mechanical support, and can be integrated into a semiconductor chip. With solid-state materials, DNA bases could be detected by measuring the tunneling current across the pore entrance. Graphene is promising because, like MspA, it has a narrow and short constriction region that fits only one base. But “DNA tends to stick to [graphene’s] exterior surface before it can enter the pores,” says NIST biophysicist Joseph Robertson. Another problem, he says, is that designing a tunneling junction for solid-pore systems has “proven to be a great fabrication challenge.”

Researchers from both the biological and solid-state camps are turning to simulations to accelerate the transition to technology development, says Aleksei Aksimentiev, a computational physicist at the University of Illinois at Urbana-Champaign. Using molecular dynamics simulations, Aksimentiev models DNA translocation and its response to pore shape and chemistry in biological and solid-state nanopores. Because “it’s very difficult to make a precise [solid-state] pore” with existing fabrication techniques, the MspA pore offers a clear advantage, he says.

Whenever it enters the market, nanopore-sequencing technology “will be a disruptive force,” in large part because of its projected high sequencing speeds and low costs, says John Quackenbush, a computational biologist at the Dana-Farber Cancer Institute in Boston and CEO of GenoSpace, a cloud-computing resource for personal and clinical genomic data. “Everyone is interested in low-cost sequencing,” he says. “A few years ago, I would have had to mortgage my house to get my personal genome. Today I can charge it to my credit card.”