Immunogenicity of Z-DNA depends on the size of polynucleotide presented in complexes with methylated BSA.

The importance of polynucleotide size for immunogenicity was tested with size-fractionated Z-DNA. High molecular weight Z-DNA, larger than 1000 bp, was fragmented by digestion with micrococcal nuclease. Fractions corresponding to less than 60, 60-120, 100-200, 200-400 and 400-900 bp were isolated by gel filtration on Sepharose 4B. These fractions and the greater than 1000 bp Z-DNA were mixed with methylated BSA and the complexes were injected into C57BL/6 mice with RIBI adjuvant. Only one of four mice responded to the less than 60 bp immunogen. All the fractions larger than 60 bp induced specific anti-Z-DNA antibodies, mostly of IgG isotype, in all animals injected. Fractions larger than 200 bp induced antisera of higher titer than did 60-120 or 100-200 bp fractions. All positive sera reacted with Z-DNA but not with B-DNA and only very weakly with denatured DNA. In competitive assays, similar concentrations of fragments larger than 60 bp inhibited binding to immobilized Z-DNA. A higher concentration of less than 60 bp fragments was required for competitive binding. Even for a highly immunogenic nucleic acid that differs from the B-DNA conformation, a polynucleotide larger than 100 bp is much more immunogenic than smaller fragments.