A new method for the postsynthetic generation of abasic sites in oligomeric DNA.
A new method for the postsynthetic generation of abasic sites in oligomeric DNA.
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一种在寡聚 DNA 中合成后生成脱碱基位点的新方法。
DOI：
10.1021/tx000108s
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发表时间：
2000
影响因子：
4.1
通讯作者：
Johnson,F
中科院分区：
文献类型：
--
作者：
Shishkina,IG;Johnson,F
无碱基位点是 DNA 中最常见的损伤，被认为在诱变中发挥着关键作用。然而，一直缺乏在寡脱氧核苷酸中位点特异性生成真正脱碱基位点的通用化学方法。我们现在描述了一种程序，该程序允许在单链或双链 DNA 寡聚物中合成后生成脱碱基位点，而不考虑其碱基组成。合成了适当保护的 3-脱氧己糖醇，并将其用作使用自动化 DNA 合成的标准亚磷酰胺方法掺入 DNA 寡聚物中的单体。所得稳定的含二醇寡核苷酸通过HPLC纯化，并通过温和的高碘酸盐氧化定量转化为相应的脱碱基DNA序列。 DNA 中的脱碱基位点在室温下相对稳定，但在 95 °C 下用腐胺处理时完全裂解。主要降解产物的鉴定通过凝胶电泳、HPLC 分离和电喷雾电离质谱表征来完成。研究了含有天然脱碱基位点的双链体寡核苷酸的热稳定性，并将结果与含有 T/dA、F/dA 或同一位点与 dA 相对的前体二醇的寡聚物的结果进行了比较。
The abasic site is the most common lesion in DNA and is thought to play a critical role in mutagenesis. However, a general chemical method for the site-specific generation of true abasic sites in oligodeoxynucleotides has been lacking. We now describe a procedure which permits the postsynthetic generation of abasic sites in single- or double-stranded DNA oligomers without regard to their base composition. An appropriately protected 3-deoxyhexitol was synthesized and used as the monomer that was incorporated into DNA oligomers using the standard phosphoramidite method for automated DNA synthesis. The resulting stable diol-containing oligonucleotides were purified by HPLC and converted quantitatively into the corresponding abasic DNA sequences by mild periodate oxidation. The abasic site in DNA was found to be relatively stable at room temperature, but was completely cleaved when treated with putrescine at 95 °C. Identification of the major degradation products was accomplished by gel electrophoresis, HPLC isolation, and characterization by electrospray ionization mass spectrometry. The thermal stabilities of duplex oligonucleotides containing a natural abasic site were studied, and the results were compared with those from oligomers containing T/dA, F/dA, or the precursor diol opposite dA at the same site.