Probing the DNA-binding affinity and specificity of designed zinc finger proteins.
Engineered transcription factors and endonucleases based on designed Cys(2)His(2) zinc finger domains have proven to be effective tools for the directed regulation and modification of genes. The introduction of this technology into both research and clinical settings necessitates the development of rapid and accurate means of evaluating both the binding affinity and binding specificity of designed zinc finger domains. Using a fluorescence anisotropy-based DNA-binding assay, we examined the DNA-binding properties of two engineered zinc finger proteins that differ by a single amino acid. We demonstrate that the protein with the highest affinity for a particular DNA site need not be the protein that binds that site with the highest degree of specificity. Moreover, by comparing the binding characteristics of the two proteins at varying salt concentrations, we show that the ionic strength makes significant and variable contributions to both affinity and specificity. These results have significant implications for zinc finger design as they highlight the importance of considering affinity, specificity, and environmental requirements in designing a DNA-binding domain for a particular application.
Jantz
Derek
D
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA.
Berg
Jeremy M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D015202:Protein Engineering; D012965:Sodium Chloride / Q000494:pharmacology; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D012965:Sodium Chloride; D004247:DNA