The proposed research will extend studies of antibodies as examples of proteins that recognize specific helical nucleic acid structures. The goals are to determine the structural basis for these specific interactions. At the first level, recognition for immune responsiveness will be studied. The minimal size and difference from B-DNA that is required for immunogenicity of nucleic acids will be determined. Immunogens to be tested will include ligated oligomers of immobile junctions, bent DNA, oligonucleotides with RNA-DNA hybrid and DNA-DNA duplex sections in one molecule, oligonucleotides with backbone modifications and with mismatched or unpaired bases, and poly(A).poly(U) of varying size. Murine hybridomas will be prepared with selected immunogens, and detailed studies of specificity and interaction will be carried out with purified monoclonal antibodies. Current studies of monoclonal anti-Z-DNA antibodies will be extended. Primary sequences of the heavy and light chain variable regions will be determined, and Fab fragments will be prepared for attempts to grow crystals and for nuclear magnetic resonance (NMR) spectroscopy. NMR will be used to study oligonucleotide conformation. Emphasis will be placed on short helical molecules with non-uniform structures and junctions between regions of divergent conformation. NMR spectrometry will also be used to examine the oligonucleotide- Fab interaction, reflected in changes in either oligonucleotide or protein (or both) resonances upon binding. As tests of the role of specific amino acids in the antibody binding sites, variants of hybridomas producing antibodies with reduced anti-Z-DNA activity will be selected and their variable region sequences determined. In addition, expressed immunoglobulin genes of the hybridomas will be isolated, mutated and transferred to mutated myeloma cells for expression and growth of modified antibodies.
The aims will be to test hypotheses about the oligonucleotide-protein interacting sites, and to modify the specificity and affinity of the antibodies.
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