Designer Zinc Finger Proteins

By Jason Socrates Bardi

For the past several years, Carlos Barbas, who, at 36, holds the Janet and Keith Kellogg II Chair in Molecular Biology, has been pursuing one line of research aimed at answering a simple—but important—question: can one design proteins to regulate the expression of any human gene?

With zinc fingers, a common structural element found in proteins, says Barbas, the answer is “yes.”

Barbas has found a set of these small zinc finger protein "motifs" that each specifically bind to a particular three base pair sequence of DNA—a codon. By stringing several of these zinc fingers together, he can create a multiple zinc finger protein that can bind any sequence of interest, including unique regulatory regions, and to which he can fuse repressor and activator proteins to specifically down- and up-regulate those genes.

Finding Zinc Fingers that Bind

Barbas has accomplished this feat using the same technology that he developed for his antibody research—phage display.

In his antibody research, which is a separate, thriving area within his laboratory, Barbas isolates catalytic antibodies—human immunoglobins that have useful chemical activity in addition to their antigen specificity.

For instance, Barbas and his colleagues designed the first commercially available catalytic antibody, 38C2, which can be made to bind certain markers on a cancer cell and catalyzes reactions there. This is useful, because patients can be injected with the antibody and then treated with a highly powerful anti-cancer agent they otherwise could not tolerate. Many such agents are too toxic to administer, but they can be rendered nontoxic by slightly modifying their chemical structure. But the catalytic antibodies, hinged to surface of the cancerous cells, can modify them back to their potent form once they reach the cells.

Barbas and his colleagues find these antibodies through phage display.

Phage display is a method of generating billions of protein variants and selecting for those that bind to a particular target. In the technique, a protein is fused to a viral coat protien of the phage—a filamentous virus that infects bacteria. Then the virus is allowed to reproduce in culture, where it copiously makes new copies of itself. Billions of variants of a single protein can be studied in this way.

Since the phage virus displays these proteins on the surface of the virions, it makes them easy to select for in vitro by simply passing the viral stew over a stationary phase containing the target substrate. Those that can bind will, and the ones that bind the best will bind the tightest.

"It became obvious to me that we should use this approach to evolve proteins that bind specific DNA sequences," says Barbas. "If we could do that, then we could do almost anything in the genome that we wanted to—turn genes on or off."

Zinc fingers are a common protein motif in nature because they bind to DNA. They come in various shapes and sizes, but they all chelate a zinc ion in their binding domain, and they all have a long alpha helix that inserts into the major groove of DNA, making contact with the bases.

Using phage display and oligonucleotide hairpins (short, single-strand pieces of DNA that twist into tiny helices to which the zinc fingers can bind), the Barbas laboratory selected for zinc fingers that bind their target codons with at least 100-fold greater affinity than they bind other sequences that are a single base change different.

Since three base pairs is the length of a codon and there are 43=64 possible ways to combine the four types of bases into a codon, Barbas needed to develop 64 such zinc fingers to cover all possibilities—a goal which is now in sight.

"We have just a few zinc fingers [left] to find," he says.

Regulating the Regulators

Finding the fingers was only the first step, however. The next was to combine several of these zinc finger proteins into a "hand," so that they have a highly selective specificity for a longer, more unique sequence of DNA. Barbas knew he had to combine a minimum of six or seven fingers together in order to recognize 18 to 21 base pairs.

"That DNA address is long enough that it is potentially found only once in a genome," says Barbas.

Then he had to fuse on the activator or repressor domains that would do the work of turning the gene on or off. A particular six zinc finger combination fused with a repressor should silence the gene’s expression, and the same six zinc fingers fused to an activator should increase expression.

And he had to design switches that would regulate the activation or repression of the action of small molecules which, when bound to the protein, would prevent the zinc fingers from binding to the DNA. And he had to design reporter systems—green fluorescent protein and other markers—so that the binding and to and regulation of the genes could be studied in the laboratory.

The ability to regulate the regulators is particularly compelling, because it increases the possibilities for clinical control. For instance, if one wanted to treat growth hormone deficiency, one could presumably deliver a transcription factor that would enhance transcription of growth hormone. But more importantly, one could couple the transcription factor with a switch that would activate or inactivate the gene whenever a pill was swallowed.

"It can be done either way," says Barbas.

Another possibility is to use a tissue-specific switch or a switch that would be sensitive to endogenous signals. Glucose levels, for instance, could conceivably be a signal that turns on or off a gene that encodes for a protein that increases the production of insulin.

These possibilities are all down the road, but Barbas has already designed several successful zinc finger transcription factors to regulate several different genes.

"What we’re doing now," he says, "is applying this approach to many types of diseases. We have encoded in our own genes the solutions to many diseases."

"What awaits is just a way to switch on those critical genes."


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Principal investigator Carlos Barbas’ interest in designing proteins grew out of his work on antibodies.
















“We have encoded in our own genes the solutions to many diseases. What awaits is just a way to switch on those critical genes.”

—Carlos Barbas III