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.
Kabakov
A E
AE
Medical Radiology Research Center, Russian Academy of Medical Sciences, Obninsk.
Poverenny
A M
AM
D015082:2,2'-Dipyridyl; D000911:Antibodies, Monoclonal; D004247:DNA / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D004278:DNA, Superhelical / Q000032:analysis; D006801:Humans; D007120:Immunochemistry; D007074:Immunoglobulin G; D009690:Nucleic Acid Conformation; D009993:Osmium Tetroxide / Q000276:immunology; D013936:Thymidine / Q000032:analysis
D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D007074:Immunoglobulin G; D015082:2,2'-Dipyridyl; D004247:DNA; D009993:Osmium Tetroxide; D013936:Thymidine