NEW YORK (GenomeWeb) – Researchers at the University of Washington and Illumina are turning nanopore sequencing on its head, using the method to study not nucleic acids but, rather, the motor proteins that process these molecules.
In a study published last month in Nature Biotechnology, a team led by UW researcher Jens Gundlach used a mutated Mycobacterium smegmatic porin A nanopore to collect information on the helicase Hel308, an enzyme thought to be involved in the repair of stalled DNA replication forks.
Using a method they have termed single-molecule picometer-resolution nanopore tweezer (SPRNT), the researchers were able to determine that Hel308 moves along DNA in half-nucleotide steps, one of which is ATP-dependent and one of which is ATP-independent.
The work presents proof of concept for a method that could have broad applicability to the study of proteins such as helicases, polymerases, recombinases, and repair enzymes that translocate on nucleic acids, Gundlach told GenomeWeb. The SPRNT technique, he said, could prove a more sensitive approach to the study of these enzymes than currently used techniques like optical tweezers and fluorescence resonance energy transfer.
Gundlach is one of the pioneers of nanopore-based DNA sequencing, and Illumina has licensed technology developed by his lab and the lab of University of Alabama, Birmingham researcher Michael Niederweis. Nanopore-based sequencing involves passing a nucleic acid strand through a nanopore and observing the ion current changes that take place as it moves through the pore. Different changes can be associated with different nucleic acid bases, and in this way a sequence can be put together.
Typically, some sort of enzyme is used to move the target nucleic acid strand through the pore. Gundlach and his team realized that in addition to collecting information on the nucleic acids, the nanopore also delivered data that could be used to better understand the workings of the enzymes used to pass them through the pore.
"We realized that we could really see the tiniest movements – much, much smaller than a single nucleotide when the enzyme draws the DNA through the nanopore," he said. "So we were really analyzing the physics behind it all and really understanding the innermost parts of that system. And from that observation we figured we could turn this whole method around and look at the enzymes."
Currently, optical tweezers, which use lasers to manipulate analytes at the single-molecule level, are the highest-resolution tool for protein analyses similar to that presented in the Nature Biotechnology study, Gundlach said. According to the authors, optical tweezers provide spatial resolution of around 300 picometers and temporal resolution of around 1 millisecond. By way of comparison, the SPRNT method offers spatial resolution of around 40 picometers and sub-1 millisecond temporal resolution, they wrote.
Additionally, because the nanopore is simultaneously collecting nucleic acid sequencing information, SPRNT allows researchers to observe the relationship between an enzyme's activity and the sequence it is located on.
"So the tool is really very powerful and can be applied to a whole host of enzymes," Gundlach said.
In the Nature Biotechnology paper the researchers applied it to Hel308, finding, among other things, that the enzyme moves in pairs of half-nucleotide steps along DNA, one of which is ATP-dependent and one of which is ATP-independent.
"There are models of superfamily II helicases, such as Hel308, that people have developed from crystal structures in which they posited that there were multiple steps, but they couldn't see it directly," Gundlach said. SPRNT offered "just a whole host of information that was unavailable with any other tool so far."
He said that he and his colleagues have filed a patent application covering the technique. They have also established collaborations with a number of outside researchers to look at enzymes currently being studied using techniques like optical tweezers.
Gundlach highlighted the pharmaceutical industry and infectious disease research as examples of fields where the approach might find uptake in the future. For instance, he said, it could enable deeper research into helicases and polymerases encoded by viruses that pharma firms are working to combat.
"Many viruses code their own helicases and polymerases, and if you really understand those then you can figure out how to interfere with them and therefore interfere with the virus," he said, adding that researchers could, for example, use the technique in screening for drugs that disrupt the normal function of a particular viral enzyme.
"So in the long run, that is a really interesting application," he said.