Scientists at Brown University have explored a new approach for controlling the passage of DNA through a nanopore that involves magnetic force pulling the DNA against an electric field.
The researchers, who published their results last week in Nanotechnology, say their "reverse DNA translocation" method could be useful for solid-state nanopore as well as protein nanopore sequencing and is suitable for multiplexing.
Methods for sequencing DNA by feeding it through a nanopore require some way to slow down the DNA molecule, and so-called magnetic tweezers, which use a magnetic field gradient to exert and measure forces on magnetic particles, might provide a way for doing so, according to the authors. The basic idea is to pull one end of the DNA through a nanopore using an electric field, and then to apply a magnetic force to a magnetic bead attached to the other end of the DNA, so it is "in a tug-of-war between the magnetic bead and the nanopore," the authors write.
By increasing the magnetic force slightly, the DNA can then be pulled out again slowly against the electric force. "That's why it is a reverse translocation — it's moving in the reverse direction of the force of the electrical field," explained Xinshen Sean Ling, a professor of physics at Brown and a senior author of the paper.
In their study, the researchers put lambda-phage DNA that was attached to a magnetic bead through fairly large solid-state nanopores — 10 to 20 nanometers in diameter — and found that the average speed of the reverse DNA translocation was more than 2,000-fold slower than usual.
Ling said he and his colleagues are currently working on combining magnetic tweezers with hybridization-assisted nanopore sequencing, which aims to detect short probes hybridized to the DNA and to reconstruct the sequence from the patterns of DNA probes. Detecting short probes is difficult, he said, "meaning you need to slow down the DNA in a way that our current work offers."
Hybridization-assisted nanopore sequencing has been developed commercially by NABsys, a Brown University spin-off that Ling co-founded (see In Sequence 1/9/2007).
Ling said the method is also suitable for multiplexing. "There is nothing to prevent us from doing two, 20, or 2 million pores," he said, though scaling up would require "non-trivial electrical engineering."
In addition, he suggested that magnetic tweezers could be useful to researchers pursuing nanopore sequencing with protein pores (see other feature in this issue).
"I think it's a rather appropriate technique for controlling the dynamics of DNA going through a nanopore," said Jens Gundlach, a professor at the University of Washington who is working on nanopore sequencing using the MspA protein. His lab has also explored using magnetic tweezers to slow down the DNA. "What remains to be shown is how well this works with a nanometer-sized aperture," he said. "Then it becomes interesting in terms of sequencing."
According to Yann Astier, a researcher at the New University of Lisbon in Portugal, reverse threading "can introduce more stability to the process" of nanopore translocation, and stretching the DNA by applying opposing forces can help iron out secondary structures.
Regarding the Nanotechnology paper, "though this is a step in the right direction, it is probably [too] early to link it to sequencing, because the quality of the nanopores used in this study is not characterized nor controlled at a small enough scale to reveal any qualitative information about the DNA that is threaded through it," he said.
However, he added that "it would be extremely interesting to apply the magnetic reverse threading method to single-stranded DNA threaded through alpha-hemolysin" — a protein pore that several research groups are exploring for nanopore sequencing. While the solid-state nanopores used in the Nanotechnology paper were on the order of 10 to 20 nanometers, the alpha-hemolysin pore ranges between 1.5 and about 4 nanometers in diameter.
Astier and his colleagues at the Massachusetts Institute of Technology recently published a paper in the journal Small in which they trapped single gold nanoparticles in the entrance of an alpha-hemolysin pore in order to slow down the DNA inside.
Magnetic tweezers and gold nanoparticles are not the only approaches researchers are pursuing to control the speed of DNA. Scientists have also tried changing environmental factors such as the viscosity of the solution, or by using DNA-binding proteins. Also, a team at the IBM Watson Research Center that includes a former graduate student from Ling's lab is working on a DNA transistor to slow down DNA in solid-state nanopores.