Synthesis, characterization, and applications of a fluorescent probe of DNA damage.
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| Abstract |
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.
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| Authors | T J Carnelley, S Barker, H Wang, W G Tan, M Weinfeld, X C Le |
| Journal | Chemical research in toxicology (Chem Res Toxicol) Vol. 14 Issue 11 Pg. 1513-22 (Nov 2001) ISSN: 0893-228X [Print] United States |
| PMID | 11712909 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
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