Department of Chemistry, Wake Forest University Wake Downtown Campus, 455 Vine St., Winston-Salem, NC, 27101, USA.
Chemistry. 2021 Oct 21;27(59):14681-14689. doi: 10.1002/chem.202102263. Epub 2021 Sep 23.
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.
为了研究一种有效的铂吖啶类抗癌药物（PA）在癌细胞中引起的 DNA 损伤，开发了一种基于生物正交后标记的测定方法，使用点击化学使叠氮化物修饰的衍生物（APA）。该方法涉及生物素化、亲和捕获和基于珠子的 APA 修饰基因组 DNA 的富集。该测定法的关键步骤在模型双链体中进行了验证和优化，包括全长质粒、限制片段和 DNA 梯。用 APA 处理的天然 DNA 随后用生物素亲和标签进行后标记，并用酶消化，并通过在线 LC-MS 和 MS/MS 进行分析。APA 在双链 DNA 中 5'-嘧啶/鸟嘌呤序列中形成的单功能嵌入加合物通过应变促进的 1,3-偶极环加成化学被定量生物素化。当应用于从 A549 肺癌细胞中提取的 DNA 时，该测定法与 qPCR 扩增相结合，证明铂吖啶在编码核糖体前 RNA 的基因序列中形成加合物，这是这些药物的潜在药理学靶标。
