Fluorescence Detection of Protein/Z-DNA Interactions

Abstract

A left-handed helical conformer of double-stranded DNA (dsDNA), Z-DNA, is observed in crystals of a synthetic hexamer (dC-dG)3 (dC: deoxycytosine, dG: deoxyguanosine) 1 and is formed best in vitro when DNAs consisting of alternating dG and dC, poly(dG-dC), are placed in high salt conditions. 2 The Z-DNA conformation is stable at physiological salt concentrations when poly(dG-dC) is either methylated or brominated. 3 The in vivo relevance of Z-DNA, i.e., the role of Z-DNA inside a cell, is still unclear, but the discovery that negative supercoiling would stabilize Z-DNA indicated biological involvement of Z-DNA in vivo. 4 The first two questions to be answered in order to elucidate the in vivo role of Z-DNA would be (1) the presence of a class of nuclear proteins that recognize the Z-DNA conformer and (2) the role(s) of the proteins in a cell. Rich and collaborators reported that an RNA-editing enzyme, human dsRNA adenosine deaminase (ADAR1), contains a domain (Zα ) that binds specifically to the Z-DNA conformation with high affinity. 5 Subsequently, Rich and Jacobs reported that the ability of a viral protein to bind to the Z-DNA conformation was essential for pathogenesis of vaccinia virus - a poxvirus that is used in smallpox vaccines - in mice: the N-terminal domain of viral E3L protein of vaccinia virus has a sequence similarity to the Zα family, and mutational studies clearly demonstrated that the Z-DNA-binding capability was necessary for pathogenicity in vaccinia virus. 6 These recent reports imply that blocking of the binding of viral proteins to Z-DNA would prevent the lethality associated with vaccinia infection and it would be possible to design a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L protein. 7 The binding of Zα family to Z-DNA has been assessed by electrophoretic mobility shift assay (EMSA), analytical ultracentrifuge, and surface plasmon resonance (SPR) spectroscopy. 5,8 However, to identify possible candidates for antiviral reagents ( e.g., against smallpox) by targeting ZDNA-binding proteins, it is needed to find more general and robust high-throughput techniques for detecting protein/ Z-DNA interactions. One of the promising approaches for the high-throughput screening is the microarray technology, where biospecific interactions (DNA/DNA, protein/protein, protein/DNA, small molecule/protein, etc) can be screened and evaluated at a time. Another approach would be the microplate-based detection, the principle of which is intrinsically the same as the microarray technology and could be incorporated into the microarray technology. Herein, we investigated a possibility of fluorescence detection of protein/Z-DNA interactions in the microplates, onto which Z-DNA was attached, with the ultimate goal of establishing a high-throughput detection of protein/Z-DNA interactions. Our approach to detecting the protein/Z-DNA interactions is based on glutathione S-transferase (GST)-mediated dimerization of proteins. Multiple and simultaneous interactions, polyvalent interactions, have unique collective properties that are qualitatively different from properties displayed by their constituents, 9 and bidentate ligands have an affinity that can approach the product of the individual binding constants. 10 We used a dimerized Zα protein (Zα GST ), formed by the dimerization of GSTs, as a model of Z-DNA-binding proteins and GST as a background control to access the specific interaction between Z-DNA-binding proteins and Z-DNA, and compared the results with the monomeric counterpart. The procedure of fluorescence detection is depicted in