A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites.
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 DNA. This strategy may provide important information about protein-DNA recognition as well as powerful tools for biomedical research.
Greisman
H A
HA
Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Pabo
C O
CO
D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D016158:Genes, p53; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019151:Peptide Library; D011487:Protein Conformation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics; D016385:TATA Box; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D019151:Peptide Library; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D004247:DNA