基于液相CH3-HS疏水结构域表征和PNA侵袭特性为基础的DNA甲基化定量分析关键技术研究

Quantitative analysis of methylation is the key technique for accurate assessment of DNA methylation bias. Currently, the bisulfite-sequencing technology has been used for the detection of genome methylation, but it has some shortcomings such as the requirements of a deep-sequencing, incomplete conversion and big data analysis. The MSRE analysis was limited by the RE recognition sequence, therefore, the number of 5mC sites could not be precisely quantified with this technique. Our previous research showed that the molecular characterization of methylated DNA would change into hydrophobic grouping CH3- in liquidoid, and form a CH3-core hydrophobic sphere. .Therefore, in this study, we intend to establish an electrochemical analytic technique for exact quantitative DNA methylation level based on the liquidoid characteristic of CH3-hydrophobic sphere structure, the electroneutral backbone pcPNA would be used to capture target DNA chain which methylation status is designed at different amount sites. On the electrode surface, the distribution density of the target CH3-hydrophobic sphere is different in the membrane microenvironment and the space resistance of the surface reaction medium is also different. Based on this mechanism, the electrochemical mathematical model was constructed for quantitative detection of DNA methylation level (5mC sites). Then, deeply parse response mechanism of pcPNA electrochemical analysis in liquid phase. With the development, the different amount sites methylation on dsDNA would be studied based on the pcPNA as probe with the characteristic of the double helix invasion. .Through this project, we hope to construct a novel original technical of Quantitative analyzing DNA methylation level, which would support the research of epigenetic and clinical quantitative detection of DNA methylation bias. On the other hand, the related basic theory will be clarified such as the optimum base spacing for electrochemical analysis of DNA methylation.

甲基化定量分析是精确评估DNA甲基化偏倚的关键技术。目前的基因组Bisulfite-Seq分析存在测序深度、转换效率和庞大数据分析等不足；MSRE分析引物设计受RE识别序列限制更不能精确定量5mC位点数量。本项目在对甲基化DNA结构分析和项目组前期研究证实，甲基化DNA液相条件下形成以CH3-为内核的疏水球这一物化特性基础上，利用pcPNA捕获不同位点甲基化的靶序列DNA后，靶序列CH3-疏水球在电极表面膜微环境分布的密度与对膜表面反应介质的空间位阻差异，建立DNA 5mC位点数量与电化学响应数学模型，达到精确定量DNA甲基化水平的目的。并在深入解析pcPNA电化学传感液相响应机理基础上，利用pcPNA双螺旋侵袭特性，进一步探索dsDNA甲基化定量分析的可行性和基础参数。通过研究，拟为表观遗传和临床甲基化定量检测建立一种原创性的新的技术手段，同时阐明甲基化电化学分析碱基最适间距等基础理论。

甲基化定量分析是精确评估DNA甲基化偏倚的关键技术。目前的基因组Bisulfite-Seq分析存在测序深度、转换效率和庞大数据分析等不足；MSRE分析引物设计受RE识别序列限制更不能精确定量5mC位点数量。本项目在对甲基化DNA结构分析和项目组前期研究证实，甲基化DNA液相条件下形成以CH3-为内核的疏水球这一物化特性基础上，利用捕获探针捕获不同位点甲基化的靶序列DNA后，靶序列CH3-疏水球在电极表面膜微环境分布的密度与对膜表面反应介质的空间位阻差异，建立DNA 5mC位点数量与电化学响应数学模型，达到精确定量DNA甲基化水平的目的。我们的研究结果显示，与非甲基化的DNA序列相比，甲基化的DNA序列很容易被PNA探针入侵，从而导致链置换位移和显著的电信号，具有不同甲基化位点数量的目标DNA序列，因tohlod链置换会导致置换效率的显著变化，根据相应的峰值电流响应关系来分析相应的甲基化，目标DNA浓度在10-14M至10-7M范围内时具有良好的线性关系，回归方程为I=0.8671 LogC（μM）+7.775，相关系数R为0.9904，该方法的检测限为0.075 pM；接下来构建AuNPs/g-C3N4@rGO三元纳米复合材料的电化学生物传感器，用于特定序列DNA甲基化定量分析研究，在10-17M到10-8M的范围内靶序列的浓度与电响应信号的大小呈现良好的线性关系，最低检测限达到了8.6 aM；在此基础上，进一步利用三条特殊设计的类回形ssDNA序列合成DNA三棱柱结构（3D-TPN）纳米材料，以实现对甲基化靶序列的捕获，构建DNA纳米材料的光电化学生物传感器用于DNA甲基化位点数量分析，在最优实验条件下可检测到的DNA甲基化位点数为7，甲基化位点数与光电响应值有良好的线性关系，回归方程为∆I=-0.0047+4.01141×N，相关系数R2为0.9997，同时，构建的光电化学传感器也可拓展应用于其他DNA靶序列的检测；另外还拓展了多模态甲基化5mC和6mA光电化学传感定量分析技术，在最适实验条件下，m6A-RNA靶序列浓度与电流强度的关系为I(nA)=282.02+17.495*logC (R2=0.995)，m5C-RNA靶序列浓度与电流强度关系为I(nA)=561.72+48.154*logC (R2=0.994)，最低检测限分别为1.96 fM 和7.37。