NEW YORK (GenomeWeb) − Researchers at the Technische Universität Braunschweig in Germany have developed a method to precisely place single molecules inside of nanowells that could help Pacific Biosciences increase the throughput of its single-molecule sequencing platform.
The method, which uses DNA origami nanoadapters to position single dye molecules on the bottom of PacBio's zero-mode waveguides, was published online in Nano Letters this month. However, PacBio and others have been working independently on their own approaches, and it is currently unclear which one the company will pick for its commercial single-molecule real-time sequencer, the PacBio RS II.
PacBio's sequencing system uses chips called SMRT cells that are patterned with 150,000 zero-mode waveguides, or ZMWs – tiny wells with metal sides and a glass bottom. To sequence DNA, a single active polymerase needs to be immobilized at the bottom of each ZMW, where light from below creates a small observation volume. Individual fluorescently labeled nucleotides can then be monitored when the polymerase incorporates them into a nascent strand of DNA.
Up until now, PacBio has been using diffusion to fill the ZMWs with polymerase, meaning some will carry one, some two, and others no enzyme, following a Poisson distribution. Using diffusion, the theoretical limit for obtaining ZMWs with exactly one polymerase is 37 percent, so the company makes no use of almost two-thirds of its ZMWs.
Since it started presenting its sequencing technology publicly six year ago, PacBio has been talking about wanting to increase the loading efficiency of the ZMWs with the help of nanotechnology self-assembly methods but has not done so yet.
During the firm's first-quarter earnings call last month, CEO Mike Hunkapiller said the company is working on improving the efficiency of the SMRT cells "so that more reads can be obtained from the same cell" and plans to increase the throughput per chip fourfold this year, but he did not provide details about the method.
According to the company's website, each SMRT cell currently typically generates about 50,000 reads and between about 275 megabases, using the P4-C2 chemistry, and 375 megabases, using the P5-C3 chemistry, of sequence data per three-hour run.
The method developed by the German researchers, which has been patented but not yet licensed, represents one way PacBio might be able to increase the loading efficiency of its ZMWs, and hence its throughput. "PacBio's R&D team continues to evaluate various strategies for this and other performance enhancements to the PacBio RS II," Chief Technology Officer Steve Turner told In Sequence via e-mail, but did not offer specifics.
Earlier this month, for example, PacBio was issued US Patent No. 8,715,930 that describes methods for loading polymerase-DNA complexes into ZMWs using magnetic or other types of beads that have a diameter larger than the ZMWs.
For their published approach, the German researchers, led by Philip Tinnefeld, a professor of biophysical chemistry at the Institute for Physical and Theoretical Chemistry at TU Braunschweig, created two types of DNA origami nanoadapter structures.
One type is a DNA disk, which consists of an array of six-helix bundles that is held together by so-called staple strands and has a diameter of 62 nanometers. The disks fit into all ZMWs, which range in diameter from 85 to 200 nanometers. The other type is a rectangular DNA origami that measures 122 nanometers diagonally and does not fit into the smaller ZMWs.
Each DNA origami carries a single fluorescent dye on top − which could in the future be replaced by a polymerase enzyme – and several biotin molecules near the center on the bottom. Once a DNA origami enters the ZMW and floats parallel to the bottom, the biotins bind to neutravidin molecules on the bottom, thus fixing the structure inside the ZMW.
Because the DNA origami structures block most of the ZMW's bottom surface, usually only one fits inside, allowing the researchers to use relatively high concentrations to try and saturate the ZMWs.
Using the rectangular DNA origami adapters, they were able to fill about 60 percent of 200-nanometer ZMWs, while about 13 percent of the ZMWs were occupied by two DNA adapters and the remainder had none. The reason why they could not load all ZMWs with DNA adapters, even with longer incubation times, might be that the density of neutravidin anchors on the bottom of the ZMWs was too low, the authors speculated.
Increasing the occupancy of ZMWs with single DNA origami structures beyond 60 percent is now "a matter of optimization," Tinnefeld said, noting that the study served as a proof of principle.
He and his colleagues have not yet looked at DNA origami adapters with polymerases attached to them, but they have previously shown that attaching DNA origami to DNA-binding proteins does not affect the binding strength or kinetics of the proteins, "so we have good indications that this is absolutely compatible with such an assay," he said.
In addition to placing DNA origami adapters inside ZMWs, he and his colleagues also studied how the position of the single dye on the adapter affects its fluorescence, which is influenced by the walls of the ZMWs. "Interestingly, we found that the intensity does not change so much depending on the localization [of the dye]," he said. "It seems like the molecules at the edges experience more light, but they are also more quenched. And these two effects seem to cancel [each other] out a bit, which is good news for Pacific Biosciences because this explains why their technique works," even when the polymerase is randomly placed anywhere on the bottom of the ZMW.
Tinnefeld and his colleagues did not collaborate with PacBio for their study, and used their own custom microscope, though they did purchase SMRT cells from the company.
Going forward, the researchers plan to use their method in single-molecule Förster resonance energy transfer, or FRET, experiments, in particular to study transcription. "There are many weaker interactions involved which we couldn't study so far, and with the help of this technology we hope that we can get better data on this," Tinnefeld said.
In the meantime, PacBio appears to have been working on its own approaches for positioning single polymerase enzymes inside of ZMWs. At a scientific conference last year, for example, the company presented work from a collaboration with Meni Wanunu at Northeastern University. According to the abstract, the method, for which PacBio filed a patent in 2012, achieved "full polymerase occupancy of ZMWs" by using ZMWs with a silicon nitride bottom, through which they drilled nanopores with an ion beam. They then threaded a DNA fragment with biotin at each end through the pore, anchored it with streptavidin from below, and had a polymerase-streptavidin complex bind to it from the inside of the ZMW.
"From my point of view this seems quite complex, and just flowing DNA origami appears much simpler, but certainly time will tell what is more practicable and efficient," Tinnefeld said.