A new single-molecule tool to observe enzymes at work
A team of scientists at the University of Washington and the biotechnology company Illumina have created an innovative tool to directly detect the delicate, single-molecule interactions between DNA and enzymatic proteins. Their approach provides a new platform to view and record these nanoscale interactions in real time. As they report Sept. 28 in Nature Biotechnology, this tool should provide fast and reliable characterization of the different mechanisms cellular proteins use to bind to DNA strands—information that 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,” said senior author and UW physics professor Jens Gundlach. “We can really pick up atomic-scale movements that a protein imparts onto DNA.” As can happen in the scientific process, they developed this tool—the single-molecule picometer-resolution nanopore tweezers, or SPRNT—while working on a related project.
The UW team has been exploring nanopore technology to read DNA sequences quickly. Our genes are long stretches of DNA molecules, which are made up of combinations of four chemical DNA “letters.” In their approach, Gundlach and his team measure an electrical current through a biological pore called MspA, which is embedded within a modified cell membrane. As DNA passes through a tiny opening in the pore—an opening that is just 0.00000012 centimeters wide, or 1/10,000th the width of a human hair—the current shifts based on the sequence of DNA letters. They use these changes in current to infer DNA sequences.
Gundlach and his team, in the process of investigating nanopore sequencing, tried out a variety of molecular motors to move DNA through the pore. They discovered that their experimental setup was sensitive enough to observe motions much smaller than the distance between adjacent letters on the DNA. As they report in their paper, SPRNT is more than 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 Gundlach. “With SPRNT, we can have 40 picometer resolution.” For reference, 40 picometers are 0.000000004 centimeters, or about 0.0000000016 inches.
“We realized we can detect minute differences in the position of the DNA in the pore,” said UW physics postdoctoral researcher Andrew Laszlo, a co-author on the paper. “We could pick up differences in how the proteins were binding to DNA and moving it through the pore.”
Sourced through Scoop.it from: phys.org
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