DNA Adduct Detection after Post-Labeling Technique with PCR Amplification (DNA-ADAPT–qPCR) Identifies the Pre-ribosomal RNA Gene as a Direct Target of Platinum–Acridine Anticancer Agents |
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Authors: | Dr Xiyuan Yao Prof Ulrich Bierbach |
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Institution: | Department of Chemistry, Wake Forest University Wake Downtown Campus, 455 Vine St., Winston-Salem, NC, 27101 USA |
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Abstract: | To study the DNA damage caused by a potent platinum–acridine anticancer agent (PA) in cancer cells, an assay based on biorthogonal post-labeling using a click chemistry-enabled, azide-modified derivative (APA) was developed. The method involves biotinylation, affinity capture, and bead-based enrichment of APA-modified genomic DNA. The key steps of the assay were validated and optimized in model duplexes, including full-length plasmids, restriction fragments, and a DNA ladder. Native DNA treated with APA and subsequently subjected to post-labeling with a biotin affinity tag was enzymatically digested and fragments were analyzed by in-line LC–MS and MS/MS. The monofunctional–intercalative adducts formed by APA in 5′-pyrimidine/guanine sequences in double-stranded DNA were quantitatively biotinylated by strain-promoted 1,3-dipolar cycloaddition chemistry. When applied to DNA extracted from A549 lung cancer cells, the assay in combination with qPCR amplification demonstrates that platinum–acridines form adducts in the gene sequences encoding pre-ribosomal RNA, a potential pharmacological target of these agents. |
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Keywords: | antitumor agents click chemistry DNA damage platinum pre-rRNA gene |
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