EQUILIBRIUM BINDING-STUDIES OF RECOMBINANT ANTI-SINGLE-STRANDED DNA FAB - ROLE OF HEAVY-CHAIN COMPLEMENTARITY-DETERMINING REGIONS

We previously isolated nucleic acid-binding antibody fragments (Fab) f rom bacteriophage display libraries representing the immunoglobulin re pertoire of autoimmune mice to expedite the analysis of antibody-DNA r ecognition, In the present study, the binding properties of one such a nti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1 ), were defined using equilibrium gel filtration and fluorescence titr ation, Results demonstrated that DNA-1 had a marked preference for oli go(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleo tides in length, A detailed analysis of the involvement of the individ ual heavy chain (H) complementarity determining regions (CDR) ensued u sing previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding Fab (D5), a Fab that binds poorly to D NA (Calcutt, M. J. Komissarov, A. A., Marchbank, M. T., and Deutscher, S. L. (1996) Gene (Amst.) 168, 9-14), Circular dichroism studies indi cated that the wild type and mutant Fab studied were of similar overal l secondary structure and may contain similar combining site shapes. T he conversion of D5 to a high affinity oligo(dT)-binding Fab occurred only in the presence of DNA-1 HCDR3. Results with site-specific mutant s in HCDR1 further suggested a role of residue 33 in interaction with nucleic acid, The results of these studies are compared with previousl y published data on DNA-antibody recognition.