Sequence specific antibody-DNA recognition

CERUTTI, M.L; CENTENO, J.M; FERREIRO, D.U; ALONSO, L.G; SCHWARTZ, F; GOLDBAUM, F.A; DE PRAT GAY, G

San Diego, CA, USA

Congreso; The Protein Society 14th Annual Symposium; 2000

Taking advantage of the extreme stability of a protein-DNA complex, we developed a method for obtaining highly specific monoclonal antibodies against a double stranded DNA oligonucleotide, corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus.  Two characterised antibodies bind to the DNA in solution with dissociation constants in the low and sub-nanomolar range, matching the affinity of the natural DNA binding domain.  These antibodies discriminate effectively among a number of double and single stranded synthetic DNAs with factors ranging from 300 to 15,000-fold the dissociation constant of the specific sequence used as the immunogen.  The binding strategy of the antibodies is clearly different from the natural binder, as judged by a positive ∆Cp of binding and tolerance to high ionic strength.  Our calorimetric and circular dichroism results show that while the E2-DNA interaction is enthalpically favoured with retention of the overall classical B conformation on the bound DNA, the antibody-DNA interaction is entropically driven where bound DNA resembles heat denatured or solvent dehydrated DNA.  We propose that, contrary to the natural binder, the antibody-DNA interaction is driven to a large extent by the hydrophobic effect with participation of aromatic base rings in the binding interface that must require substantial base unstacking and DNA distortion.