Probing the DNA-binding affinity and specificity of designed zinc finger proteins

Abstract

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.

Figure 1

(A) Sequences of the seven probes evaluated with the QNK-QDK-RHR protein, showing the location of fluorescein [F] or Texas Red [T] labels and the protein-binding site (boxed). (B) Amino acid sequence of the QNK-QDK-RHR protein, showing metal-binding (bold) and DNA-contacting (numbered) residues. (C) Plots of anisotropy as a function of QNK-QDK-RHR concentration for the seven probes. (D) Structure of fluorescein-dT.

Figure 2

Schematic structures for three zinc finger proteins aligned with their binding sites. (A) The structure of QNK-QDK-RHR aligned with the binding site used in the probes shown in Fig. 1. The asterisk indicates the position of the fluorescein-dT residue in probe 7. (B) The structures of RHR-QDK-Q(N,D)K aligned with the binding site used in probe 8. The asterisk indicates the position of the fluorescein-dT residue in this probe.

Figure 3

Binding curves for two different proteins. Plots of anisotropy as a function of either (A) QNK-QDK-RHR or (B) RHR-QDK-QNK protein concentration using probe 7 or probe 8 as the labeled DNA. Curves were fit to generate dissociation constants for all four complexes. Not all points are shown for the two lower-affinity complexes. The dissociation constants shown are the mean of three measurements and the uncertainty shown in the standard deviation from the mean derived from these measurements.

Figure 4

Competition experiments to determine binding specificity. (A) Plots of anisotropy as a function of competitor DNA concentration for 12 variants of the 5′-most triplet in the QNK-QDK-RHR binding site. First, 15 nM of QNK-QDK-RHR protein were added to a probe 7 solution to bind the majority of the probe. Unlabeled DNA was then added to compete the protein off of the probe, resulting in a decrease in fluorescence anisotropy. Each of the three charts shows plots corresponding to all four bases substituted in the first, second, or third position in the triplet (not all points are shown for lower-affinity complexes). (B) The curves in A were fit to generate K_a values for each of the 12-point variants in the QNK-QDK-RHR binding site. The resulting binding-site “signature” is a quantitative determination of the preferred base at each position in the triplet. (C) The same analysis as in B for the RHR-QDK-QNK protein. In this case, probe 8 is the fluorescent species being followed, and each of the 12 oligonucleotide sets used as a competitor is a point variant in the 5′-most triplet of the RHR-QDK-QNK binding site.

Figure 5

(A) Association constants for RHR-QDK-QNK binding to probes derived from the sequence -GAA-GCA-GAG- in 50 mM NaCl. The ability of this protein to discriminate between bases at each of the three positions within the first base triplet is shown. (B) Corresponding association constants for RHR-QDK-QDK. (C) Summary of the derived association constants for probes with the sequence -GXA-GCA-GAG- for the proteins RHR-QDK-QNK and RHR-QDK-QDK.

Figure 6

NaCl-concentration dependence of the binding affinity of the RHR-QDK-QNK and RHR-QDK-QDK proteins for probes including the sequence -GXA-GCA-GAG-. The logarithm of the K_a for each interaction is plotted as a function of the logarithm of the NaCl concentration.