A General Strategy for Selecting High-Affinity Zinc Finger Proteins for Diverse DNA Target Sites

  title={A General Strategy for Selecting High-Affinity Zinc Finger Proteins for Diverse DNA Target Sites},
  author={Harvey A. Greisman and Carl O. Pabo},
  pages={657 - 661}
A method is described for selecting DNA-binding proteins that recognize desired sequences. The protocol involves gradually extending a new zinc finger protein across the desired 9- or 10-base pair target site, adding and optimizing one finger at a time. This procedure was tested with a TATA box, a p53 binding site, and a nuclear receptor element, and proteins were obtained that bind with nanomolar dissociation constants and discriminate effectively (greater than 20,000-fold) against nonspecific… 
A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter
A rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range is presented.
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Design of novel sequence-specific DNA-binding proteins.
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The novel functionality obtained by engineered zinc Finger proteins and the computational approaches for prediction of recognition helices of zinc finger proteins that can raise the ability to re-program zinc finger protein with desired novel DNA-binding specificities are explored.
DNA-binding specificity is a major determinant of the activity and toxicity of zinc-finger nucleases.
The results of these cell-based assays reveal that the DNA-binding specificity--in addition to the affinity--is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity, and provide the first evidence that engineering strategies, which account for context-dependent DNA- binding effects, yield ZFs that function as highly efficient ZFNs in human cells.