Antisera specific for the oligoribonucleotide sequences AAU and A2U2 and their recognition of oligonucleotide conformation

Abstract

Antisera specific for two synthetic oligoribonucleotide sequences, AAU and A2U2, were elicited in rabbits. The oligonucleotides were synthesized using polynucleotide phosphorylase under high salt conditions. Each oligomer was isolated by ion exchange chromatography, and was conjugated to bovine serum albumin, and injected into rabbits as an emulsion with complete Freund's adjuvant. The specificities of the resulting sera were analyzed using a modified Farr-type radioimmunoassay employing homologous oligonucleotide-protein conjugates radiolabeled with [3H]acetic anhydride and unlabeled free oligonucleotides as inhibitors. The antiserum elicited by AAU-BSA reacted well with AAU-RSA but a major fraction of the antibodies was directed to determinants of the conjugate that were not present on the free hapten. With respect to the haptenic determinants, AAU was a better inhibitor than any of the constituent mono- or dinucleotides, implying that features of the entire trinucleotide were being recognized. The other members of the A2Un family reacted to about the same extent as AAU, while other trinucleotides required an up to 21-fold higher concn in order to achieve similar inhibition. The most striking aspect of this antiserum was its failure to bind free ApA, although it could bind the ApA-containing oligonucleotides A3, AAG, AAC and A2Un. It seems likely that the ApA sequence in solution does not contain a significant proportion of a conformation present to a great extent in the ApA-containing oligomers. The antiserum elicited by A2U2-BSA was like anti-AAU-BSA in that some of the antibodies were directed against determinants not present on the free hapten. The most striking result of the inhibition experiments was the specificity of the antiserum for members of the A2Un series. When the A2Un series was compared with AA, AMP or any member of the Un series, approximately four orders of magnitude separated the inhibition curves. The poor binding of component mono- and dinucleotides implies that the conformation recognized by the antibody is present only to a significant extent in the trimeric sequence; the equality of binding of AAU with A2U2, A2U3 and A2U4 suggests that this conformation of the triplet is preserved in the longer sequences. These studies demonstrate the utility of immunochemical procedures for the study of oligonucleotide conformation in solution.