By Mark Dwortzan
Low-cost, ultra-fast DNA sequencing would revolutionize healthcare and biomedical research, sparking major advances in drug development, preventative medicine and personalized medicine. By gaining access to the entire sequence of your genome, a physician could determine the probability that you’ll develop a specific genetic disease or tolerate selected medications.
In pursuit of that goal, Associate Professor Amit Meller (BME, MSE) has spent much of the past decade spearheading a method that uses solid state nanopores—two-to-five-nanometer-wide holes in silicon chips that read DNA strands as they pass through—to optically sequence the four nucleotides (A, C, G, T) encoding each DNA molecule.
Now Meller and a team of researchers at Boston University—Professor Theodore Moustakas (ECE, MSE) and research assistants Nicolas Di Fiori (Physics, PhD’13) and Allison Squires (BME, PhD’14)—and Technion-Israel Institute of Technology—have discovered a simple way to improve the sensitivity, accuracy and speed of the method, making it an even more viable option for DNA sequencing or characterization of small proteins.
In the November 3 online edition of Nature Nanotechnology, the team demonstrated that focusing a low-power, commercially available green laser on a nanopore increases current near walls of the pore, which is immersed in salt water. As the current increases, it sweeps the salt water along with it in the opposite direction of incoming samples. The onrushing water, in turn, acts as a brake, slowing down the passage of DNA through the pore. As a result, nanoscale sensors in the pore can get a higher-resolution read of each nucleotide as it crosses the pore, and identify small proteins in their native state that could not previously be detected.
“The light-induced phenomenon that we describe in this paper can be used to switch on and off the ‘brakes’ acting on individual biopolymers, such as DNA or proteins sliding through the nanopores, in real time,” Meller explained. “This critically enhances the sensing resolution of solid-state nanopores, and can be easily integrated in future nanopore-based DNA sequencing and protein detection technologies.”
Slowing down DNA is essential to DNA or RNA sequencing with nanopores, so that nanoscale sensors, like sports referees, can make the right call on what’s passing through.
“The goal is to hold a base pair of DNA nucleotides in the nanopore’s sensing volume long enough to ‘call the base’ (i.e, determine if it’s an A, C, G or T),” said Squires, who fabricated nanopores and ran experiments in the study. “The signal needs to be sufficiently different for each base for sensors in the nanopore to make the call. If the sample proceeds through the sensing volume too quickly, it’s hard for the sensors to interpret the signal and make the right call.”
Other methods designed to slow down DNA in nanopores change the sensing properties of the pore, making it more difficult to ensure accuracy of detected base pairs. Shining laser light on the nanopore alters only the local surface charge, an effect that’s completely reversible within milliseconds by switching the laser off.
As an added bonus, the researchers found that the sudden increase in surface charge and resulting flow of water reliably unblocks clogged nanopores, which can take a long time to clean, significantly extending their lifetime.
Meller and his team characterized the amount of increase in current under varying illumination in many different-sized nanopores. They next aim to explore in greater detail the mechanism underlying the increase in surface current when the green laser is applied to a nanopore, information that could lead to even more sensitivity and accuracy in DNA sequencing.
The research is funded by a $4.2 million grant from the National Institute of Health’s National Human Genome Research Institute under its “Revolutionary Sequencing Technology Development—$1,000 Genome” program, which seeks to reduce the cost of sequencing a human genome to $1,000.