The investigators have designed and synthesized a 21-base long DNA hairpin to serve as a model test antigen to probe the specificity of lupus anti-DNA autoantibodies. The investigators propose that the single-stranded and double-stranded determinants on DNA hairpins can model the ssDNA and dsDNA antigens that are recognized by lupus anti-ssDNA and anti-dsDNA autoantibodies.Unlike many previous anti-DNA test antigens such as synthetic polymers and plasmids, the investigators construct is defined with respect to sequence, size and conformation. A simple chemical method based on disulfide bond crosslinking between modified thymidine bases has been developed to stabilize the conformation of DNA hairpins. The solution- phase geometry of the hairpin test antigen is also being determined by NMR spectroscopy so that binding specificity can be correlated with antigenic structure. This type of comparison has not been possible in many previous studies of anti-DNA specificity because of the size and heterogeneity of the test antigens used (e.g., calf thymus DNA). The investigators synthetic hairpin is recognized by 37 percent (9 of 24) of anti-DNA hybridomas generated from an 8-week old lupus prone MRL-1pr mouse that did not show signs of lupus. More significantly, this hairpin was recognized by all of the anti-DNA monoclonal autoantibodies (4 of 4) produced in a second fusion with a 20-week old MRL-1pr mouse that showed signs of advanced lupus. In binding studies, the investigators propose to measure equilibrium dissociation constants of antibody-hairpin complexes and examine the sequence and conformational specificity of the antibodies. In addition, antibody complexes with a plasmid that contains this hairpin sequence will be studies as a model for anti-DNA autoantibody recognition of """"""""native"""""""" DNA. Antibodies against the investigator's hairpin have also been raised in healthy mice to enable a direct comparison of lupus anti-DNA antibodies. The investigators propose that a comparative study like this will help to uncover the basis for these differences. This research should lead to a better understanding of immune-complex mediated tissue injury and will hopefully identify the molecule(s) that triggers and/or drives the lupus anti-DNA response.

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National Institute of General Medical Sciences (NIGMS)
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University of Michigan Ann Arbor
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Bobeck, Melissa J; Cleary, Joanne; Beckingham, Jenny A et al. (2007) Effect of somatic mutation on DNA binding properties of anti-DNA autoantibodies. Biopolymers 85:471-80
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