"Zinc fingers" are an exciting protein engineering strategy for activating gene expression. These small proteins have a simple fold—a β-hairpin that nestles against one α-helix—and can recognize a sequence of three DNA bases with excellent specificity. They are called zinc fingers because they require a coordinated zinc ion to fold properly.
DNA base-pair recognition by these proteins occurs by means of six amino acids that span the beginning of the α-helix. A nearly complete palate of zinc-finger proteins that can specifically recognize every possible series of three base-pair sequences (64 in all) has been discovered in nature or engineered in the laboratory. Because any number of zinc finger proteins can be linked in tandem, researchers can build agents that target extremely long tracks of DNA. At the end of the agents' DNA-binding domains goes the payload, such as a transcription activator that can recruit the transcription machinery to a specific gene.
Other payload possibilities include DNA-modifying enzymes that splice out disease genes and replace them with healthy sequences or remodeling enzymes that render accessible to transcription machinery portions of DNA that are otherwise inaccessible due to their being wound tightly around histone proteins, says Carlos F. Barbas III, a chemist at Scripps Research Institute. Yet another application is to use the zinc fingers as sensors of DNA methylation, which silences the gene to which the methyl groups are attached.
The problem is getting these proteins to cross both the cell and nuclear membranes. "The limitation to protein-based transcription factors is that gene therapy is required for delivery," says Barbas. Gene therapy relies on viruses to transport a genetic blueprint for the protein drug of interest into cells. Once inside, the virus inserts the DNA for the artificial transcription factor into the patient's genome. Then the patient's own protein machinery produces the artificial transcription factor. That artificial transcription factor then turns on the genes of interest.
This indirect delivery has its challenges—namely, controlling where exactly in the genome the genetic freight is delivered. Inserting the gene into the wrong part of the DNA can disrupt a healthy gene and lead to the onset of cancer. That's why researchers are looking to engineer smart viruses to direct precise delivery, so that gene therapy shipments arrive at specific addresses in the genome.
This has become far more than an academic exercise. The biotech company Sangamo has moved protein-based artificial transcription factors into Phase II clinical trials. The Richmond, Calif. business is evaluating whether zinc-finger-based transcription factors can activate genes that protect sensory and motor nerves from diabetes-induced damage. The company's investigators, in collaboration with Dow AgroSciences, also are trying to activate plant genes with zinc fingers. The benefit of using zinc fingers in agricultural applications is that plant genes important for crop yield, flavor, or resistance to plant pathogens could be activated, whereas genes involved in producing allergens could be silenced.