The long term goal is to understand immune responses to nucleic acids, particularly the origin and nature of autoantibodies associated with autoimmune diseases such as systemic lupus erythematosus. This goal is approached through knowledge of the structural correlates of nucleic acid antigenicity, the principles of protein-nucleic acid interaction, and the gene pool available for anti-nucleic acid antibody formation. The next phase of this research will determine whether autoantibodies to DNA share structural motifs and gene origins with antibodies induced by immunization. These experiments will test the proposal that anti-DNA autoantibody formation is a response to specific immunization with nucleic acid-containing antigens. This research also increases the repertoire of antibodies that serve as useful biochemical reagents. The structural basis for recognition of Z-DNA, denatured DNA and native DNA will be analyzed with bacterially expressed antigen-binding (Fv) domains of monoclonal antibodies. A plasmid vector has been prepared for expression of anti-Z-DNA Fv by E. coli. Replacement of Heavy or Light Chain segments and directed mutagenesis, guided by molecular modeling, will be used to replace amino acids in the antigen-binding sites of the Fv protein. These experiments will identify amino acids that are important, directly or indirectly, for antigen binding. Original and modified Fv proteins by will be characterized by electrophoresis, antigen-binding selectivity, affinity, idiotype expression, and stability. When mutation studies identify amino acids that are important for DNA binding, NMR spectroscopy will be used to test whether the amino acid makes direct contact with DNA. Experiments will test whether variable region genes and rearrangements for immunization-induced antibodies to nucleic acids are the same as those used for disease-associated or natural anti-DNA autoantibodies in the same animal. Studies of immune responses to DNA will evaluate the role of foreign protein carriers. Complexes containing DNA and a bacterial DNA- binding protein will be tested for their ability to induce formation of antibodies to DNA.
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