Synthesis, characterization, and applications of a fluorescent probe of DNA damage.
We have designed and generated a 90-mer oligonucleotide that contains a single adduct of benzo[a]pyrene diol epoxide (BPDE) and that is fluorescently labeled. The known amount of BPDE adduct in a given length of DNA makes this probe a useful standard for DNA damage assay. The BPDE-90-mer was fluorescently labeled with tetramethylrhodamine to allow for high sensitivity detection with laser-induced fluorescence (LIF). The binding of both double-stranded and single-stranded BPDE-90-mer with three anti-BPDE antibodies was studied using affinity capillary electrophoresis (CE). Formation of antibody complex with BPDE-90-mer results in a shift in migration time from that of the unbound BPDE-90-mer. Affinity CE/LIF studies suggest that antibody 8E11 has high-affinity suitable for immunoassay of BPDE-DNA adducts. A competitive immunoassay using the fluorescent probe and CE/LIF is demonstrated for the analysis of BPDE-DNA adducts in A549 human lung carcinoma cells incubated with 2.5, 5, and 10 microM BPDE for 2 h. The design of the 90-mer probe is flexible to substitute different DNA damage types with relative ease. The fluorescent 90-mer is composed of six shorter oligonucleotides. The sequence of the two center oligonucleotides may be changed depending on the desired DNA lesion measurement. By inserting different damaged oligonucleotides, a variety of DNA damage systems can be investigated using the same CE/LIF approach.
Carnelley
T J
TJ
Department of Public Health Sciences, University of Alberta, Edmonton, AB T6G 2G3, Canada.
Barker
S
S
Wang
H
H
Tan
W G
WG
Weinfeld
M
M
Le
X C
XC
D000906:Antibodies; D001580:Benzopyrenes / Q000737:chemistry; D004249:DNA Damage; D004586:Electrophoresis; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008175:Lung Neoplasms / Q000473:pathology; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000906:Antibodies; D001580:Benzopyrenes; D005456:Fluorescent Dyes; D009841:Oligonucleotides; C072019:benzo(a)pyrene diolepoxide I