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PacBio-Northeastern Team Demonstrates Increased Loading Efficiency with Combo of Nanopore, Zero-Mode Waveguide

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NEW YORK (GenomeWeb News) — Researchers at Northeastern University and Pacific Biosciences have developed a method for reversibly drawing molecules into the nanowells of PacBio's chips, thus increasing the efficiency for loading proteins and DNA.

Potentially, the approach could enable PacBio's real-time single-molecule sequencing technology to analyze samples with limiting amounts of DNA, such as epigenetically modified mammalian DNA, and to sequence long DNA fragments more efficiently. It could also allow the company's chips to be reused more easily.

Essentially, the researchers drilled a narrow solid-state nanopore into the bottom of a PacBio zero-mode waveguide — a small well — and used the pore to draw protein-DNA complexes as well as free DNA into the well under a voltage. By doing so, they were able to improve the capture of DNA into the well by two to three orders of magnitude compared to the current diffusion-based approach. They described their method in a paper in Nano Letters this month.

The publication is the first result from a three-year research grant that PacBio and Meni Wanunu's group at Northeastern University won from the National Human Genome Research Institute in 2012. While it provides proof-of-concept for the nanopore-ZMW combo and its ability to draw in and release molecules, it does not yet demonstrate that the device can be used for sequencing, or manufactured in large numbers.

"The immediate next step is to demonstrate that the devices are active, meaning that we can read a sequence, and that's what we are currently working on," Wanunu told In Sequence.

In parallel, he and his colleagues are working on ways to produce high-density arrays of nanopore zero-mode waveguides, or NZMWs, using electron or ion beams.

It is unclear whether PacBio plans to implement the new method once it succeeds in improving sequencing efficiency. The published work "does not in any way reflect any projections about future commercial implementations or features of the current or future SMRT sequencing platforms," Jonas Korlach, the company's chief scientific officer, told In Sequence. However, PacBio and Northeastern University patented the approach prior to applying for the NHGRI grant, Wanunu said.

To make the NZMWs, the researchers first fabricated ZMWs in a silicon nitride membrane instead of a glass slide, which is normally used. They then drilled nanopores of 2.5 to 4 nanometer diameter into the bottom of the ZMWs using transmission electron microscopy.

Next, they added biotinylated DNA with a fluorescently labeled streptavidin protein attached to it to a single NZMW and drew the DNA into the pore under voltage. At high voltage, the streptavidin dissociated from the DNA, and when the voltage was reversed, the protein-DNA complex was ejected from the NZMW.

They also floated DNA-streptavidin complexes into eight NZMWs at once and found that they could measure fluorescent signals from five of them, indicating that 60 percent of the wells had a complex bound to the nanopore. This is higher than the occupation rate of PacBio's sequencing chips with single polymerase molecules, which is limited to 37 percent by Poisson statistics.

The researchers also tested the loading of free DNA into the device by adding 6-kilobase labeled DNA fragments to an array of ZMWs with a single NZMW and applying a voltage. Compared to the loading rate by diffusion or magnetic bead loading, which PacBio currently employs to insert the DNA, they found the loading rate into the NZMW to be "orders of magnitude more efficient."

Also, instead of incubating the ZMWs with DNA for an hour or so, which PacBio's current method requires, "what we observed is that as soon as we apply voltage, within a second or two DNA just goes into the waveguides," Wanunu said.

Especially for long fragments of DNA, which do not readily diffuse into a ZMW, the NZMW approach could have advantages, he said, and would require lower concentrations of DNA.

The increased DNA loading efficiency could be particularly useful for analyzing samples with small amounts of DNA that cannot be amplified, for example mammalian DNA with epigenetic modifications that would be lost by amplification. That, Wanunu said, is currently not feasible with PacBio's sequencing system. "Because the concentration is so low, [the DNA] will stick to other places before it gets into the waveguides and you will not get a lot of reads from it," he said.

Being able to release DNA and polymerase from the NZMW by reversing the voltage could also make it easier to reuse PacBio's chips, which currently require lengthy washing steps. "We think that in our device, you can just reverse the bias, trap a new complex, and continue to read with the same device," Wanunu said, which would increase the lifetime of the chip and provide more data for the same cost.

There are two ways in which NZMWs could be used for sequencing, Wanunu said. One is to immobilize the polymerase first through a DNA tag that inserts into the pore, similar to the DNA-streptavidin complex in their experiment, and then to add template DNA. "Even if your pore has a DNA-protein complex in it, there is still some current flowing through the pore, and that additional current can be used to also flow in [additional] DNA," he explained.

The other is to skip the polymerase immobilization, which takes about half an hour, and to load template DNA with polymerase bound to it directly. This approach could save both time and reagents, he said.

Other researchers have worked on alternative approaches that could help increase the sequencing efficiency of PacBio's device. Earlier this year, for example, a group in Germany published a method that uses DNA origami adaptors to position single molecules at the bottom of ZMWs.

But while that approach may increase the loading efficiency for DNA polymerase, it would not improve the loading of DNA, Wanunu said, and the polymerase may interact with the DNA origami.

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