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."
These differences account for the unique role each cellular protein plays as it interacts with DNA. Cells have proteins to copy DNA, "read" DNA to express genes and repair DNA when it is damaged. There are cellular proteins that unwind DNA, while others bunch DNA tightly together.
Biologists have long recognized that proteins have different structures to perform these roles, but the physical motion of proteins as they work on DNA has been difficult to detect directly.
"When you have the kind of resolution that SPRNT offers, you can start to pick apart the minute steps these proteins take," said Laszlo.
Gundlach and his team show that SPRNT is sensitive enough to differentiate between the mechanisms that two cellular proteins use to pass DNA through the nanopore opening. One protein, which normally copies DNA, moves along the DNA one letter at a time as it guides DNA through the pore. The second protein, which normally unwinds DNA, instead takes two steps along each DNA letter, which they could pick up by tracking minute changes in the current, according to co-author and UW physics doctoral student Jonathan Craig. They even 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 Laszlo.
Gundlach believes this tool may open a new window for understanding how cellular proteins process DNA, which could help genetically engineer proteins to perform novel jobs. These fine details may also help scientists understand how mutations in proteins can lead to disease or find protein properties that would be ideal targets for drug therapies.
"For example, viral genes code for their own proteins that process their DNA," said Gundlach. "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."
Other UW authors on the paper include lead author Ian Derrington, a postdoctoral researcher in physics, and Brian Ross, Henry Brinkerhoff, Ian Nova, Kenji Doering and Benjamin Tickman. Co-authors at Illumina are Kevin Gunderson, Eric Stava, Mostafa Ronaghi and Jeffrey Mandell. Gundlach's laboratory received funding for this project from the National Institutes of Health's $1,000 Genome Project, grant number R01HG005115.
For more information, contact Gundlach at 206-543-8774 or firstname.lastname@example.org
James Urton | EurekAlert!
Funding of Collaborative Research Center developing nanomaterials for cancer immunotherapy extended
28.06.2017 | Johannes Gutenberg-Universität Mainz
Zeolite catalysts pave the road to decentral chemical processes Confined space increases reactivity
28.06.2017 | Technische Universität München
An international team of scientists has proposed a new multi-disciplinary approach in which an array of new technologies will allow us to map biodiversity and the risks that wildlife is facing at the scale of whole landscapes. The findings are published in Nature Ecology and Evolution. This international research is led by the Kunming Institute of Zoology from China, University of East Anglia, University of Leicester and the Leibniz Institute for Zoo and Wildlife Research.
Using a combination of satellite and ground data, the team proposes that it is now possible to map biodiversity with an accuracy that has not been previously...
Heatwaves in the Arctic, longer periods of vegetation in Europe, severe floods in West Africa – starting in 2021, scientists want to explore the emissions of the greenhouse gas methane with the German-French satellite MERLIN. This is made possible by a new robust laser system of the Fraunhofer Institute for Laser Technology ILT in Aachen, which achieves unprecedented measurement accuracy.
Methane is primarily the result of the decomposition of organic matter. The gas has a 25 times greater warming potential than carbon dioxide, but is not as...
Hydrogen is regarded as the energy source of the future: It is produced with solar power and can be used to generate heat and electricity in fuel cells. Empa researchers have now succeeded in decoding the movement of hydrogen ions in crystals – a key step towards more efficient energy conversion in the hydrogen industry of tomorrow.
As charge carriers, electrons and ions play the leading role in electrochemical energy storage devices and converters such as batteries and fuel cells. Proton...
Scientists from the Excellence Cluster Universe at the Ludwig-Maximilians-Universität Munich have establised "Cosmowebportal", a unique data centre for cosmological simulations located at the Leibniz Supercomputing Centre (LRZ) of the Bavarian Academy of Sciences. The complete results of a series of large hydrodynamical cosmological simulations are available, with data volumes typically exceeding several hundred terabytes. Scientists worldwide can interactively explore these complex simulations via a web interface and directly access the results.
With current telescopes, scientists can observe our Universe’s galaxies and galaxy clusters and their distribution along an invisible cosmic web. From the...
Temperature measurements possible even on the smallest scale / Molecular ruby for use in material sciences, biology, and medicine
Chemists at Johannes Gutenberg University Mainz (JGU) in cooperation with researchers of the German Federal Institute for Materials Research and Testing (BAM)...
19.06.2017 | Event News
13.06.2017 | Event News
13.06.2017 | Event News
28.06.2017 | Physics and Astronomy
28.06.2017 | Physics and Astronomy
28.06.2017 | Health and Medicine