Preparation and analysis of anti-DNA antigen binding fragments
Three DNA-binding antibodies, Jel 72, Jel 274 and Jel 241, were studied to understand their binding characteristics. Binding constant measurements of Jel 274 and Jel 241 IgG, as a function of salt concentration, by fluorescence polarimetry revealed that in both cases the interaction with duplexes was very dependent on ionic strength and 4 to 6 ion pairs were involved in complex formation. A van't Hoff analysis revealed that for both antibodies, the standard heat capacity change was dependent on ionic strength and unlike the case with DNA-binding proteins, a large negative value did not correlate with sequence specific interactions. Jel 72 and Jel 274 single-chain Fv fragments (scFv) were expressed in Escherichia coli (E. coli) and subsequently analyzed. Both scFv, in particular Jel 72, showed a considerable change of specificity compared to the parent IgG examined previously at high ionic strength (Lee et al., 1984b). Detailed analysis of Jel 72 Fab and IgG by fluorescence polarimetry at low ionic strength revealed that similar to its scFv derivative, Jel 72 IgG was not specific for dG•dC sequence. These results demonstrates that the specificity may be derived from salt dependent DNA conformational changes. In separate experiments, the effect of three parameters on Jel 72 scFv specificity was tested: linker length, chain polarity, and two non-CDR (CDR = complementary determining region) positively charged amino acids in the vicinity of the antibody combining site. Thus, six different scFv were constructed and analyzed. Binding to (dG)20•(dC)20 and (dA)20•(dT)20 showed that none of the three parameters had any effect on antibody affinity or specificity. The role of CDR H3 (H = heavy chain) arginine residues at positions 98, 100, 100a in Jel 72 affinity and specificity was also examined. Mutation to asparagine or glutamine did not result in any significant specificity for (dA)20•(dT)20 as suggested on the basis of structural and biochemical studies (Seeman et al., 1976; Mol et al., 1994b; Choo and Klug, 1994a). As well, substitution of all three arginine with asparagine, glutamine, serine, and lysine, surprisingly, did not change the affinity and specificity of Jel 72, demonstrating that these arginine are not important in antigen binding. Further studies were performed with Jel 274 scFv. Experiments in which the light chain of Jel 274 scFv was replaced by those from Jel 72 and Jel 318 (a triplex-binding antibody) revealed that, in both cases, the specificity and, in the case of latter, the affinity was altered, demonstrating the importance of the light chain in antibody binding. To search for DNA-binding antibodies, a phage display library was panned against various duplexes and many positive clones were selected against poly (d(GC)), poly(dA)•poly(dT), poly(rA)•poly(dT), and a few against calf thymus DNA and the triplex DNA. Poly(dA)•poly(dT)-, and poly(rA)•poly(dT)-binders, in general, did not bind to calf thymus DNA, suggesting that structure and sequence specific antibodies are present in phage display libraries.