Autoantibodies to nuclear components of cells (antinuclear antibodies, ANA), including DNA (a-DNA), are widely used in the diagnosis and subtyping of certain autoimmune diseases, including systemic lupus erythematosus (SLE). Despite clinical use over decades, precise, reproducible measurement of a-DNA titers remains difficult, likely due to the substantial sequence and length heterogeneity of DNA purified from natural sources. We designed and tested a panel of synthetic nucleic acid molecules composed of native deoxyribonucleotide units to measure a-DNA. ELISA assays using these antigens show specificity and reproducibility. Applying the ELISA tests to serological studies of pediatric and adult SLE, we identified novel clinical correlations. We also observed preferential recognition of a specific synthetic antigen by antibodies in SLE sera. We determined the probable basis for this finding using computational analyses, providing valuable structural information for future development of DNA antigens. Synthetic nucleic acid molecules offer the opportunity to standardize assays and to dissect antibody-Antigen interactions.