Abstract
Antibody to 2'-5' linked triadenylate, A 2' p 5' A 2' p 5' A, was prepared by injecting bovine serum albumin conjugated with A 2' p 5' A 2' p 5' A into rabbits. The [8-14C]-A 2' p 5' A 2' p 5' A used in the study was synthesized by nonenzymatic template-directed condensation of [8-14C] AMP and 5'-phosphorimidazolide of A 2' p 5' A on poly U. The specificity of the antibody was determined by the inhibition of the binding of the radioisotope probe to the antibody by a competitor using the ammonium sulfate precipitation method. The cross reactivity of a number of modified oligonucleotides has been evaluated in order to gain information on the structural feature involved in the binding to the antibody. The adenine moiety and 2'-5' internucleotide linkage are crucial regions for the binding. The antibody reacted not only on A 2' p 5' A 2' p 5' A and A 2' p 5' A 2' p 5' A 2' p 5' A, but also on a cordycepin analogue of the trimer, (3' dA) 2' p 5' (3' dA) 2' p 5' (3' dA), significantly. Considerable cross reaction was observed with oligoadenylates bearing both 2'-5' and 3'-5' internucleotide linkages, while there was little cross reaction with 3'-5' linked oligoadenylates. The antibody showed very low or no affinity for adenosine, adenine mononucleotides, other nucleotides and other 2'-5' linked trinucleotides. The cross reactivity of the modified oligonucleotides is indicated to have relation to the biological activity of the corresponding 5'-phosphorylated oligonucleotides, so far reported.
The antibody was further purified by affinity chromatography using RNA-Sepharose and AMP-Sepharose giving high specificity.
