Building zinc fingers by selection: toward a therapeutic application.
A phage display approach was utilized to modify the specificity of each of the three fingers of the murine transcription factor Zif268. Selections were performed by using the consensus binding sequence of the natural protein and a conserved sequence in the genome of the type 1 human immunodeficiency virus. By using an extensive randomization strategy, the entire 3-bp specificity of a finger has been changed. Rapid analysis of selected zinc fingers was facilitated by the development of an immunoscreening assay for DNA binding and specificity. To investigate the mechanism of binding and specificity, the binding kinetics of Zif268 and 10 selected variants were determined in real time with an assay based on surface plasmon resonance. Differential mechanisms for sequence-specific recognition were observed. No evidence in support of a single general coding relationship between zinc finger and target DNA sequence was observed. The prospects for the development of this class of proteins in human therapy are considered.
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037.
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D051766:Early Growth Response Protein 1; D015723:Gene Library; D005815:Genetic Code; D015497:HIV-1 / Q000235:genetics; D017874:Immediate-Early Proteins; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D012641:Selection, Genetic; D017421:Sequence Analysis; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D016335:Zinc Fingers / Q000235:genetics*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D010446:Peptide Fragments; D014157:Transcription Factors