Preparation and Characterization of Polyclonal and Monoclonal Antibodies Specific for Covalently Linked DNA/RNA Cross Sections

ABSTRACT

Covalently linked cross sections refer to structures that mimic hydrogen-bonded purine–pyrimidine, purine– purine, and pyrimidine–pyrimidine duplexes. Cross sections dA [unk] U and A [unk] dT, which have been synthesized chemically, have molecular dimensions similar to purine–pyrimidine base pairs in a double helix. We propose that antibodies to such covalent cross sections might facilitate the study of the pathogenesis of specific diseases or of biochemical processes in which base pair involvement is suspected and/or demonstrated. We have made polyclonal antibodies against "A:U" and "A:T" cross sections by immunizing rabbits with dA [unk] U and A [unk] dT, each conjugated to keyhole limpet hemocyanin (KLH). The antibodies were found to be highly specific for the cross sections and to cross react minimally to single nucleosides. Hybridomas secreting monoclonal antibodies to "A:T" were then generated from spleen cells of mice immunized with A [unk] dT conjugated to KLH. The MAbs produced were also found to be highly specific for "A:T" among various nucleosides. In fact, the binding of most of the monoclonal antibodies to "A:T" was only partially inhibited by high concentrations of adenosine or thymidine. All monoclonal antibodies to "A:T" cross react, but with lower affinity, to "A:U." Selected MAbs showed greater inhibition of binding to "A:T"–BSA by A + T than by A or T alone.