Nucleotide-specific antibodies as potential blocking agents in the structural analysis of nucleic acids

Abstract

Anti-nucleotide antibodies might be useful blocking agents in directing the nuclease catalyzed hydrolysis of RNA for sequence analysis, but only if the antibodies recognized a nucleotide or sequence with sufficient specificity and affinity to compete with a nuclease at only specific sites in an RNA. As a test, antibodies directed against mononucleotide haptens were induced with 5′-nucleotide (oxidized with periodate) coupled to bovine serum albumin. Nucleotide-specific antibodies, isolated by affinity chromatography on nucleotide-agarose columns, were at least 95 % pure 7-S γ-globulin. The antibodies precipitated RNA, homologous nucleotide-rabbit albumin conjugates, and to a lesser extent, heterologous conjugates. Antibodies further purified by passage through an affinity column of heterologous nucleotides showed specificity for the single homologous nucleotide. Equilibrium dialysis measurements gave dissociation constants for the antibody-homologous nucleotide complex of about 10 −5 M, while binding of a heterologous nucleotide was insufficient to be measured. The 5′-phosphate group, the base, and the ribose derivative in the conjugate were all elements of structure recognized by the antibodies. Hydrolysis of antibodies with insolubilized papain produced monovalent Fab fragments with dissociation constants essentially as determined for intact antibodies. Antibodies were found to compete with pancreatic ribonuclease for dinucleoside phosphate substrates in a manner consistent with the binding results. Anti-nucleotide Fab fragments inhibited the nuclease hydrolysis of f2 RNA, but no directed cleavage to give altered RNA fragmentation patterns was observed.