Yuan Fang, Bi Ying, Zhang Jia-Yuan, Zhou Ying-Lin, Zhang Xin-Xiang, Song Chun-Xiao
Ludwig Institute for Cancer Research, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford OX3 7FZ UK
Beijing National Laboratory for Molecular Sciences (BNLMS), MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry, Peking University Beijing 100871 China
RSC Adv. 2019 Sep 16;9(50):29010-29014. doi: 10.1039/c9ra04375f. eCollection 2019 Sep 13.
DNA cytosine modifications are important epigenetic modifications in gene regulation and pathogenesis. DNA hydrolysis followed by HPLC-MS/MS is the gold standard in DNA modification quantification. In particular, it is the only sensitive and accurate method for low abundance modifications, such as 5-carboxylcytosine (5caC). Here, we report the discovery of the nuclease resistance property of 5caC to snake venom phosphodiesterase I (PDE1), a 3' to 5' exonuclease commonly used in several DNA hydrolysis protocols. We conducted a systematic evaluation of six commonly used hydrolysis protocols and found that all protocols that use PDE1 underestimate the level of 5caC. Finally, we identified the best method for cytosine modification quantification of biological samples, which leads to an over 10-fold higher amount of 5caC being detected compared with other methods. Our results highlight that caution should be taken when choosing a DNA hydrolysis protocol to quantify certain DNA modifications.
DNA胞嘧啶修饰是基因调控和发病机制中重要的表观遗传修饰。DNA水解后通过高效液相色谱-串联质谱（HPLC-MS/MS）是DNA修饰定量的金标准。特别是，它是用于低丰度修饰（如5-羧基胞嘧啶（5caC））的唯一灵敏且准确的方法。在此，我们报告发现5caC对蛇毒磷酸二酯酶I（PDE1）具有核酸酶抗性，PDE1是一种常用于多种DNA水解方案的3'至5'核酸外切酶。我们对六种常用的水解方案进行了系统评估，发现所有使用PDE1的方案都低估了5caC的水平。最后，我们确定了生物样品胞嘧啶修饰定量的最佳方法，与其他方法相比，该方法检测到的5caC量高出10倍以上。我们的结果强调，在选择DNA水解方案以定量某些DNA修饰时应谨慎。
