Separation and identification of DNA-carcinogen adduct conformers by polyacrylamide gel electrophoresis with laser-induced fluorescence detection.
We have developed a separation protocol utilizing high-resolution polyacrylamide gel electrophoresis (PAGE) to isolate stable anti-benzo[a]pyrene diol epoxide adducts of oligodeoxynucleotides. Both enantiomers produced multiple adduct species. The distribution of adduct types could be quantitated by densitometry of autoradiograms or Cerenkov counting of eluted oligomers modified by anti-BPDE isomers. Laser-induced fluorescence (LIF) spectra of eluted adducts at 4.2 K (fluorescence line-narrowing spectroscopy) and 77 K revealed that bands corresponded to pure conformers of pyrene chromophore. Carcinogen-modified oligodeoxynucleotides were single-stranded, but there were often considerable stacking interactions between the pyrenyl residues and the oligonucleotide bases, indicating that electrophoresed oligomers were single-stranded but in a native, versus random coil, conformation. The ability to identify and quantitate adducts by PAGE-LIF, coupled with the high resolution and sensitivity of both techniques, makes PAGE and LIF in tandem a potentially powerful tool in the study of chemical carcinogenesis or other ligand-DNA interactions.
Marsch
G A
GA
Ames Laboratory-USDOE, Iowa State University 50011.
Jankowiak
R
R
Farhat
J H
JH
Small
G J
GJ
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D001345:Autoradiography; D001483:Base Sequence; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification*; D004249:DNA Damage; D004591:Electrophoresis, Polyacrylamide Gel; D007834:Lasers; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D013050:Spectrometry, Fluorescence / Q000379:methods*
D009838:Oligodeoxyribonucleotides; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA