Small Molecule Modulation of Zinc‐Finger Sequence Specificity

The high resolution x‐ray crystal structure of Aart, a synthetic six finger zinc‐finger protein, bound to DNA revealed the presence of glycerol molecules at the protein‐DNA interface. Analysis of the hydrogen bonding network suggests that the glycerol may enhance the affinity of Aart for particular sequences of DNA. To determine the role that glycerol molecules may play in the DNA sequence specificity of Aart, we have designed and carried out Fluorescence Polarization Anisotropy (FPA) measurements of Aart for a series of DNA sequences in the presence and absence of 10% glycerol. The hypothesis we tested was that the presence of 10% glycerol would enhance binding behavior for sequences containing either adenine or guanine at the 5′ position of two of the six triplet base pairs (F3 and F4) recognized by Aart, with greater enhancement in the case of 5′ adenine nucleotides. The FPA measurements showed first that a sequence containing no 5′ purine nucleotide in F3, but a 5′G in F4 exhibited no difference in binding affinity to Aart in the presence and absence of 10% glycerol (141± 15 nM in the absence of glycerol, 140± 12 nM in 10% glycerol). Second, a sequence containing a 5′A in F3 and a 5′G in F4 was bound slightly tighter in the presence of glycerol (283± 4 nM in the absence of glycerol, 218± 12 nM in 10% glycerol). Finally, a sequence with 5′A in both F3 and F4 showed no difference in binding affinity with and without 10% glycerol (68± 15 nM in the absence of glycerol, 97± 33 nM in 10% glycerol). We therefore conclude that glycerol at a concentration of 10% has little to no effect on the binding affinity of Aart for these sequences. We next plan to test the effect of other small molecules also hypothesized to alter the specificity of this designed zinc finger. Research sponsored by NIH‐ GM008718