PREFERENTIAL RECOGNITION OF SPECIFIC DNA MOTIFS BY ANTI-DOUBLE-STRANDED DNA AUTOANTIBODIES

Although antibodies (Ab) specific for double-stranded (ds) DNA are tho ught to be involved in the etiopathogenesis of systemic lupus erythema tosus (SLE), the fine structure of their DNA targets remains elusive. We have adapted a polymerase chain reaction (PCR)-assisted immunopreci pitation method to define the binding sites in DNA sequences recognize d by high affinity anti-dsDNA Ab of SLE patients. SLE sera were used t o bind templates from a pool of double-stranded oligonucleotides (ON). A central part of 20 base-pair random sequence was flanked by restric tion endonuclease recognition sites and sequences complementary to pre defined PCR primers. Immunoselected ON were precipitated, isolated fro m the immune complexes and then subjected to a further immunoprecipita tion step after amplification by PCR. After five cycles of immunopreci pitation and PCR, the resulting ON were cloned. Sequence analysis reve aled that sera from SLE patients and two human monoclonal anti-dsDNA A b obtained from SLE patients preferentially select sequences expected to form non-B-DNA structures. Inhibition studies of the Farr assay con firmed the increased affinity of the selected epitopes for anti-DNA Ab as compared to random B-DNA.