The 20th-century author and biochemist Issac Asimov famously wrote that “the most exciting phrase to hear in science, the one that heralds new discoveries, is not ‘Eureka!’ but ‘That’s funny'…” Often during the scientific process, researchers set out on one project only to make a discovery along the way that changes the entire scope of their work—which is exactly what happened to a research collaboration with scientists from the University of Washington (UW) and the biotechnology company Illumina.
The researchers created an innovative tool to directly detect single-molecule interactions between DNA and enzymatic proteins, viewing and recording these nanoscale interactions in real time. The research team is optimistic that this tool will provide fast and reliable characterization of various mechanisms cellular proteins use to bind to DNA strands—information they believe could shed new light on the atomic-scale interactions within our cells and help design new drug therapies against pathogens by targeting enzymes that interact with DNA.
“There are other single-molecule tools around, but our new tool is far more sensitive,” explained senior author Jens Gundlach, Ph.D., professor of physics at UW. “We can really pick up atomic-scale movements that a protein imparts onto DNA.”
The findings from this study were published recently in Nature Biotechnology through an article entitled “Subangstrom single-molecule measurements of motor proteins using a nanopore.”
The scientists were originally investigating different ways of improving nanopore sequencing—a relatively new method that sequences native DNA, allowing a single strand to pass through a tiny pore one nucleotide at a time—when they discovered that their experimental setup was sensitive enough to observe motions much smaller than the distance between adjacent letters on the DNA.
Their newly developed tool, dubbed single-molecule picometer-resolution nanopore tweezers, or SPRNT, is seven times more sensitive than existing techniques to measure interactions between DNA and proteins.
“Generally, most existing techniques to look at single-molecule movements—such as optical tweezers— have a resolution, at best, of about 300 picometers,” said Dr. Gundlach. “With SPRNT, we can have 40 picometer resolution.”
For comparison, the pore in which the DNA strand passes through for the sequencer is approximately 1.2 nm wide (roughly 10,000 times smaller than the width of human hair), whereas the 40 picometer resolution for the SPRNT is about 30 times even smaller.
“We realized we can detect minute differences in the position of the DNA in the pore,” noted co-author Andrew Laszlo, Ph.D., a postdoctoral researcher in Dr. Gundlach’s laboratory. “We could pick up differences in how the proteins were binding to DNA and moving it through the pore.”
Those differences describe the distinctive role each cellular protein plays as it interacts with DNA. Cells have unique proteins to copy and repair DNA, as well as express genes. Moreover, researchers have been searching for better techniques for many years to study the physical motion of proteins as they work on DNA molecules.
“When you have the kind of resolution that SPRNT offers, you can start to pick apart the minute steps these proteins take,” stated Dr. Laszlo.
The researchers showed that SPRNT was able to differentiate between the mechanisms that two cellular proteins use to pass DNA through the nanopore opening. One protein to guide the DNA through the pore, one base at a time, and a second protein, which normally unwinds DNA, instead takes two steps along each DNA letter—which the team could pick up by tracking minute changes in the current. Furthermore, the investigators discovered that these two steps involve sequential chemical processes that the protein uses to walk along DNA.
“You can really see the underlying mechanisms, and that has a ton of implications—from understanding how life works to drug design,” said Dr. Laszlo.
The researchers believe that this new tool may open a new window for understanding how cellular proteins process DNA, which could help genetically engineer proteins to perform novel jobs.
“Viral genes code for their own proteins that process their DNA. If we can use SPRNT to screen for drugs that specifically disrupt the functioning of these proteins, it may be possible to interfere with viruses,” concluded Dr. Gundlach.