Monoclonal antibody against DNA adducts with osmium structural probes.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

Immunochemical probing of DNA structure with monoclonal antibody to OsO4/2,2'-bipyridine adduct.

Chemical modification with osmium tetroxide (OsO4) in the presence of 2,2'-bipyridine was employed for selective labeling of certain non-B DNA structures. A murine monoclonal antibody (IgG1) specific for deoxythymidine adduct of the modification was produced. It was demonstrated that the level of single-stranded DNA can be evaluated by enzyme-linked immunosorbent assay using the modification and monoclonal antibody. Site-specific OsO4-bipyridine modification followed by dot-blotting was used for probing of some unusual conformations occurring in supercoiled plasmids. The cruciform structures, B-Z junctions, and intramolecular triplex and long (A-T)n inserts were targets for the modification and small quantities of the labeled plasmids can be immunodetected. The method enabled to catch certain structural transitions within the same plasmid in vitro and to detect the left-handed Z DNA labeled inside bacteria. The antibody was also shown to recognize specifically the in situ modified DNA in cell nuclei and polytene chromosomes isolated from Drosophila melanogaster. Preferentially S1-nuclease-sensitive (single-stranded) regions of the nuclear and chromosomal DNA were stained by indirect immunofluorescence after the modification.