A novel computational approach used to redesign an enzyme's selectivity for its DNA target could have important genomics and therapeutic uses if it proves applicable to a wide range of proteins and sequences.
The approach was developed by grad student Justin Ashworth and biochemistry professor David Baker of the University of Washington, Seattle, and coworkers (Nature 2006, 441, 656). They used Rosetta, a molecular modeling program developed earlier by Baker's group, to calculate how to change the DNA-cleavage specificity of a homing endonuclease, an enzyme that cuts phosphodiester bonds.
When the Rosetta-predicted modifications were made, the enzyme cleaved a new DNA sequence, which the researchers had selected because it "looked like it was going to be 'designable,' " Baker says. However, "the method should be generalizable to any protein-DNA interface redesign problem," the researchers note. "Our long-term goal [is] designing novel proteins able to recognize and cleave any desired DNA site with high specificity for targeted genomics applications."
"The approach the authors take is exciting," but until they prove that their method can be extended to more demanding redesign targets, "the general utility remains to be determined," says assistant professor of biochemistry Matthew H. Porteus of the University of Texas Southwestern Medical Center in Dallas. They also don't show that the redesigned enzyme can cut genomic DNA and that it fails to cut other off-target sequences. However, "the ability to redesign using computational approaches is novel and with further development might end up being the long-sought-after method to broadly reprogram such enzymes to a wide variety of sites."
"This is a tour de force in protein design," says biochemistry professor Dana Carroll of the University of Utah. "Zinc-finger nucleases still have multiple advantages over homing endonucleases as DNA-targeting agents, including ease of design and range of accessible target sequences." But if computational methods "allow essentially arbitrary changes in specificity without relaxation in selectivity, endonucleases could pull even or possibly ahead as candidates for targeted gene therapy."
Professor of biochemistry and chemistry Gregory A. Petsko of Brandeis University notes that a number of techniques have been used to reengineer enzyme specificity and function and says he would be more impressed if Ashworth, Baker, and coworkers "had taken a case known to be difficult to reengineer from first principles and shown that their computational strategy would get you there much faster." But historically, "this sort of switch has required a lot of fits and starts to achieve, and here it seems to have been done in something much more like a single smooth step. The interplay of computation and experiment in this paper is also nice and undoubtedly represents the way such studies will be done in the future."