For gene-editing proteins to be useful in clinical applications, they need to be able to find the specific site they’re supposed to edit among billions of DNA sequences. Using advanced imaging techniques, University of Illinois researchers have found that one class of genome-editing proteins rapidly travels along a strand of DNA like a rider on a zip line – a unique behavior among documented DNA-binding proteins.
TALE proteins can be programmed to recognize and bind to specific regions of DNA for applications in synthetic biology or clinical gene therapy. The zipline behavior that the new study uncovered is different from what researchers have seen in any other proteins that bind to and travel along DNA.
“Among the classes of known DNA-binding proteins, TALE proteins appear to behave in a unique way in term of target site search,” Schroeder said. “Understanding the search mechanism is helpful in moving toward clinical applications. A major goal is to design these proteins to find a specific target site with minimal off-target binding.”
Using single-molecule imaging techniques, the Illinois team watched how TALE proteins moved along a DNA template that didn’t contain the proteins’ target sequence, a process known as nonspecific search. They expected that TALEs would bind to the DNA backbone and rotate around the double helix, like a nut on a bolt – as do nearly all documented proteins that move along DNA searching for a target site. Instead, they found that the protein wrapped around the helix and slid back and forth, like a rider on a zip line.
“We uncovered a new search process for DNA-binding proteins that doesn’t fit into previous binding classifications,” said Cuculis, now a consultant at the Boston Consulting Group. “We performed several experiments to test these theories, and the results were unexpected based on current thinking about how proteins move along DNA in search of a target site or gene.”
The unusual mechanism could hold advantages for researchers looking to develop TALE proteins for new biomedical applications, Cuculis said. For example, it could be used as a delivery mechanism, like a passenger riding a zip line to a destination, with the TALE protein acting as the hook.
“It opens up more avenues for this protein to be used,” he said. “Because of how well TALEs hook onto the DNA, there’s a potential to attach larger payloads to the proteins. Right now it’s mainly used for gene editing, but based on this work, there may be potential to attach something much larger to deliver to a target site.”
Now that they have detailed how TALE proteins work in vitro, the researchers are beginning to study them at work in live cells.
“It’s providing more fundamental insight into the mechanism behind gene editing,” Schroeder said. “Research is always more interesting when nature behaves in ways we don’t expect, because it enables us to look at things from a new perspective. The hope for this work is ultimately to develop new and optimized systems for genome engineering.”
The Carl R. Woese Institute for Genomic Biology and the David and Lucile Packard Foundation supported this work. Schroeder and Zhao also are affiliated with the department of chemistry and the Center for Biophysics and Quantitative Biology.
Editor’s notes: To reach Charles Schroeder, call (217) 333-3906 ; email: email@example.com. To reach Huimin Zhao, call (217) 333-2631; email: firstname.lastname@example.org. The paper “TALE proteins search DNA using a rotationally decoupled mechanism” is available online. DOI: 10.1038/nchembio.2152