DNA动态甲基化过程中5-醛基胞嘧啶选择性反应型小分子荧光探针研究

This application is intended to provide a novel method for the quantitative and qualitative determination of 5-formylcytosine (5fC) and 5-hydroxymethyl cytosine (5hmC), which contained in active DNA demethylation. By improving the conventional Wittig reagent, 5-formylcytosine was transformed into a novel base with fluorescence after the Wittig reaction and cyclization reaction. Due to the fluorescent of the new formed nucleotide, the content of 5fC could be easily determined by high performance liquid chromatography (HPLC) with a fluorescence detector. The detection method is simple and convenient which could be processed in mild reaction conditions without complicated instruments. As to detection of 5hmC, we first converted 5hmC to 5fC and then conducted the same procedure for 5fC. The content of 5hmC could be obtained by calculating the difference between before and after conversion. On the other hand, after the cyclization reaction, leaving groups will be activated into small molecules with fluorescent properties. Thus the content thereof can be measured by fluorescence spectroscopy. The combination of two-step reaction (Wittig reaction and cyclization), greatly improve the precision and reliability of the detection method. This project is not only to fill the epigenetic quantitative detection methods for 5fC, but also provide a novel method for the detection of 5hmC, which is important for the study of disease mechanisms and regulation of cell function.

本申请旨在提供一种新的检测DNA主动甲基化过程中5-醛基胞嘧啶（5fC）和5-羟甲基胞嘧啶（5hmC）含量的方法。主要通过改良传统的Wittig试剂，经过Wittig反应和环化反应后，将胞嘧啶转变为具有荧光特性的新型碱基。通过带有荧光检测器的高效液相色谱（HPLC）来检测，进而确定5fC的含量。本检测方法反应条件温和，无需复杂的仪器, 可以广泛地应用于基础实验中。而对于5hmC的检测，则先将其转化为5fC再检测，通过对比前后的含量差值，进而确定5hmC的含量。另外一方面，环化反应后，自探针分子离去基团会被激活成为具有荧光特性的小分子，因此可以通过荧光光谱测定其含量。由于Wittig反应和环化反应两步反应的联合，大大提高了该HPLC和荧光双模式检测方法的抗干扰性。本项目的研究为目前表观遗传学中定量检测5fC和5hmC提供一种新的思路和方法，对研究疾病机理和细胞功能调控具有重要意义。

DNA甲基化是表观遗传调控机制之一，相关修饰碱基的定性和定量检测具有重要的研究价值。传统的5-醛基胞嘧啶（5fC）的检测主要基于胺基衍生物的席夫碱反应为主。然而，席夫碱的不稳定性给检测结果容易造成检测的不确定性，而且醛基尿嘧啶（5fU）上的醛基反应活性高于胞嘧啶，会对特异性识别5fC的造成较大的干扰。因此，开发新的稳定性好5fC的定性定量检测方法具有重要的研究意义。. 本项目主要开发一种新的荧光检测DNA甲基化过程中5-醛基胞嘧啶（5fC）和5-醛基尿嘧啶（5fU）含量的方法。通过构建了一系列荧光Wittig探针分子库，首次将Wittig反应应用于5-醛基尿嘧啶的选择性识别和定量检测中，可以实现选择性标记5fU修饰的DNA和成像。此外，我们也建立了一种光辅助和Wittig反应联合技术区分5fC和5fU，是迄今为止5fC选择性标记的最快方法。在此基础上，还进行了细胞内活性小分子的检测及其应用研究，获得了系列原创性成果。. 本项目的研究成果对于核酸化学中修饰碱基的检测具有重要的研究意义，将推动该领域内其它分析方法的构建；同时，该荧光Wittig试剂对进一步应用到蛋白的标记及功能检测提供了丰富的数据支撑。