Determination of preferential binding sites for anti-dsRNA antibodies on double-stranded RNA by scanning force microscopy

The monoclonal anti-dsRNA antibody J2 binds double-stranded RNAs (dsRNA) in an apparently sequence-nonspecific way. The mAb only recognizes antigens w ith double-stranded regions of at least 40 bp and its affinity to poly(A) p oly(U) and to dsRNAs with mixed base pair composition is about tenfold high er than to poly(I) poly(C). Because no specific binding site could be deter mined, the number, the exact dimensions, and other distinct features of the binding sites on a given antigen are difficult to evaluate by biochemical methods. We therefore employed scanning force microscopy (SFM) as a method to analyze antibody-dsRNA interaction and protein-RNA binding in general. S everal in vitro-synthesized dsRNA substrates, generated from the Dictyostel ium PSV-A gene, were used. In addition to the expected sequence-nonspecific binding, imaging of the complexes indicated preferential binding of antibo dies to the ends of dsRNA molecules as well as to certain internal sites. A nalysis of 2,000 bound antibodies suggested that the consensus sequence of a preferential internal binding site is A(2)N(9)A(3)N(9)A(2) thus presentin g A residues on one face of the helix. The site was verified by site-direct ed mutagenesis, which abolished preferential binding to this region. The da ta demonstrate that SFM can be efficiently used to identify and characteriz e binding sites for proteins with no or incomplete sequence specificity. Th is is especially the case for many proteins involved in RNA metabolism.