DNA and RNA contain diverse chemical modifications that exert important influences in a variety of cellular processes. In addition to enzyme-mediated modifications of DNA and RNA, previous in vitro studies showed that pre-modified nucleoside triphosphates (NTPs) can be incorporated into DNA and RNA during replication and transcription. Herein, we established a chemical labeling method in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis for the determination of endogenous NTPs in the mammalian cells and tissues. We synthesized 8-(diazomethyl)quinoline (8-DMQ) that could efficiently react with the phosphate group under mild condition to label NTPs. The developed method allowed sensitive detection of NTPs, with the detection limits improved by 56–137 folds. The results showed that 12 types of endogenous modified NTPs were distinctly determined in the mammalian cells and tissues. In addition, the majority of these modified NTPs exhibited significantly decreased contents in human hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues. Taken together, our study revealed the widespread existence of various modified NTPs in eukaryotes.

中文翻译：

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哺乳动物中存在修饰的三磷酸核苷†

DNA和RNA包含多种化学修饰，这些修饰在各种细胞过程中均具有重要影响。除了酶介导的DNA和RNA修饰外，以前的体外方法研究表明，在复制和转录过程中，可以将预修饰的三磷酸核苷（NTPs）掺入DNA和RNA中。在这里，我们建立了一种结合液相色谱-电喷雾电离质谱（LC-ESI-MS）分析的化学标记方法，用于确定哺乳动物细胞和组织中的内源性NTPs。我们合成了8-（重氮甲基）喹啉（8-DMQ），可以在温和条件下与磷酸基团有效反应以标记NTP。所开发的方法可以灵敏地检测NTP，检测限提高了56–137倍。结果表明，在哺乳动物细胞和组织中可清楚地确定出12种内源性修饰NTP。此外，与邻近肿瘤的正常组织相比，这些修饰的NTP中的大多数在人类肝细胞癌（HCC）组织中表现出明显降低的含量。两者合计，我们的研究表明真核生物中广泛存在各种修饰的NTP。