The interaction of the zinc finger
protein WT1 with
RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the
DNA binding properties of this
protein. A recombinant
peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of
RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to
DNA. An
isoform that contains an insertion of three
amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to
DNA. The equilibrium binding of WT1-ZFP to the highest-affinity
RNA molecule was compared to the equilibrium binding to a consensus
DNA molecule as a function of temperature, pH, monovalent
salt concentration, and divalent
salt concentration. The interaction of WT1-ZFP with both
nucleic acids is an entropy-driven process. Binding of WT1-ZFP to
RNA has a pH optimum that is narrower than that observed for binding to
DNA. Binding of WT1-ZFP to
DNA is optimal at 5 mM MgCl(2), while the highest affinity for
RNA was observed in the absence of MgCl(2). Binding of WT1 to both
nucleic acid ligands is sensitive to increasing monovalent
salt concentration, with a greater effect observed for
DNA than for
RNA. Point mutations in the zinc fingers associated with
Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with
DNA, but a consistent and modest effect on the interaction with
RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus
nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.