Transcription activator-like effector nucleases (TALENs) are proteins that can be designed to lock on to a specific region of the genome and make a double-stranded break. Though scientists have been using them for about five years, nobody was sure exactly how the protein complexes homed in on particular genomic regions.
"TALENs do not possess the common canonical elements such as helix-turn-helix or leucine zipper motifs found in other DNA-binding proteins capable of sequence-specific recognition," the authors wrote in the paper, so how the proteins found their target sites was somewhat of a mystery. "They are designed to bind to a particular site, but there's this big genome with billions of bases," Charles Schroeder, a professor of chemical and biomolecular engineering and a senior author of the paper said in a statement.
To find out, the researchers used total internal reflection fluorescence microscopy to watch individual TALENs interacting with strings of DNA in a single-molecule assay based on dual-tethered DNA. They found that the proteins slide along and jump around the genome as they search the genome for their target.
"The combination of sliding and hopping means they can cover more ground and potentially move past obstacles that might be in their way," said Luke Cuculis, a co-lead author of the paper. It also allows the protein to access both strands of DNA to find its target site.
The scientists also found that there are two important modes of activity: search and sequence recognition. Within the protein's structure, the scientists found a domain responsible for each mode, an N-Terminal Region domain that is responsible for searching the genome and a central repeat domain that binds to the specified target sequence. Understanding these domains could help address one of the most problematic side effects of genome editing, off-target activity.
"The major goal would be to engineer improved proteins that have lower off-target binding. You don't want them to bind to the wrong place," Schroeder said. "If we engineer a protein in such a way where we don't just naively change the specific binding domain, but design a new protein with distinct parts of the protein in different places, we might be able to increase the efficiency without increasing mistakes."
The experiments were performed in microfluidic flow cells containing a field of DNA templates, so the researchers said their next step is to look at TALEN activity in a live cell nucleus.