Generation of sequence-specific, high affinity anti-DNA antibodies

By taking advantage of the extreme stability of a protein-DNA complex, we h ave obtained two highly specific monoclonal antibodies against a predetermi ned palindromic DNA sequence corresponding to the binding site of the E2 tr anscriptional regulator of the human papillomavirus (HPV-16), The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constant s in the low and subnanomolar range. This affinity matches that of the natu ral DNA binding domain and is severalfold higher than the affinity of a hom ologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibod ies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125- to 20,000-fold the dissociati on constant of the specific DNA sequence used in the immunogenic protein-DN A complex. Moreover, they are capable of fine specificity tuning, since the y both bind less tightly to another HPV-16 E2 binding site, differing in on ly 1 base pair in a noncontact flexible region. Beyond the relevance of obt aining a specific anti-DNA response, these results provide a first glance a t how DNA as an antigen is recognized specifically by an antibody. The accu racy of the spectroscopic method used for the binding analysis suggests tha t a detailed mechanistic analysis is attainable.