Fabs specific for 8-oxoguanine: Control of DNA binding

Free radicals produce a broad spectrum of DNA base modifications including 7,8-dihydro-8-oxoguanine (8-oxoG). Since free radicals have been implicated in many pathologies and in aging, 8-oxoG has become a benchmark for factor s that influence free radical production. Fab g37 is a monoclonal antibody that was isolated by phage display in an effort to create a reagent for det ecting 8-oxoG in DNA. Although this antibody exhibited a high degree of spe cificity for the 8-oxoG base, it did not appear to recognize 8-oxoG when pr esent in DNA. Fab g37 was modified using HCDR1 and HCDR2 segment shuffling and light chain shuffling. Fab 166 and Fab 366 which bound to 8-oxoG in sin gle-stranded DNA were isolated. Fab 166 binds more selectively to single-st randed oligonucleotides containing 8-oxoG versus control oligonucleotides t han does Fab 366 which binds DNA with reduced dependency on 8-oxoG. Numerou s other clones were also isolated and characterized that contained a spectr um of specificities for 8-oxoG and for DNA. Analysis of the primary sequenc es of these clones and comparison with their binding properties suggested t he importance of different complementarity determining regions and residues in determining the observed binding phenotypes. Subsequent chain shuffling experiments demonstrated that mutation of SerH53 to ArgH53 in the Fab g37 heavy chain slightly decreased the Fab's affinity for 8-oxoG but significan tly improved its binding to DNA in an 8-oxoG-dependent manner. The Light ch ain shuffling experiments also demonstrated that numerous promiscuous light chains could enhance DNA binding when paired with either the Fab g37 or Fa b 166 heavy chains; however, only the Fab 166 Light chain did so in an addi tive manner when combined with the Fab 166 heavy chain that contains ArgH53 . A three-point model for Fab 166 binding to oligonucleotides containing 8- oxoG is proposed. We describe a successful attempt to generate a desired an tibody specificity, which was not present in the animal's original immune r esponse. (C) 1999 Academic Press.