Synthesis, characterization, and applications of a fluorescent probe of DNA damage

We have designed and generated a 90-mer oligonucleotide that contains a sin gle 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 thi s probe a useful standard for DNA damage assay. The BPDE-90-mer was fluores cently labeled with tetramethylrhodamine to allow for high sensitivity dete ction with laser-induced fluorescence (LIF). The binding of both double-str anded and single-stranded BPDE-90-mer with three anti-BPDE antibodies was s tudied 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 8E1 1 has high-affinity suitable for immunoassay of BPDE-DNA adducts. A competi tive immunoassay using the fluorescent probe and CE/LIF is demonstrated for the analysis of BPDE-DNA adducts in A549 human lung carcinoma cells incuba ted with 2.5, 5, and 10 muM 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 sequenc e of the two center oligonucleotides may be changed depending on the desire d DNA lesion measurement. By inserting different damaged oligonucleotides, a variety of DNA damage systems can be investigated using the same CE/LIF a pproach.