基于脱氧核酶催化苯胺聚合的人类甲基化转移酶活性传感器的构建

Human DNA methyltransferase 1 (Dnmt1) overexpression is found in various types of cancer，the accumulated Dnmt1 induces tumor suppressor gene silencing and inactivation. The detection of Dnmt1 activity is of great importance for both early cancer diagnosis and treatment. Most of the reported DNA methyltransferase activity biosensors are focused on those methyltransferases from prokaryote, which differ a lot with Dnmt1 in both recognition sequence of DNA substrate and size. Thus, the development of human Dnmt1 methyltransferase activity biosensors still needs further exploration. In this project, DNAzyme catalyzed polymerization of aniline is proposed as the signal producer and amplifier for the development of a novel biosensing platform with two different detection modes for the monitoring of human Dnmt1 activity. Firstly, by using a hairpin-structured substrate DNA, Dnmt1 and the methylation-sensitive restriction enzyme BssHII catalyzed methylation and digestion are carried out in a homogenous solution. Then, the released DNAzyme functional segments after the methylation/digestion are introduced to a microplate or a gold electrode that pre-immobilized with capturing DNA probes. In the presence of hemin, the released segments and the capture probes form the G-quadruplex DNAzyme structure, which catalyzes the polymerization of aniline. Finally, the produced polyaniline can be detected by either UV-vis spectrometry or differential pulse voltammetry. Since the amount of the released DNAzyme functional segments is related to the methylation level of the substrate DNA, the final signal readout of polyaniline reflects the activity of Dnmt1. The separation of the Dnmt1/BssHII catalyzed enzymatic reaction from the detection platform makes the proposed biosensor avoid several drawbacks commonly encountered in conventional DNA methyltransferase activity biosensors, such as the non-uniformity caused by heterogenous reaction, the steric hindrance for the enzyme recognition, and the interference from the complex real samples. The development of this biosensor may provide a potentially effective tool for the early cancer diagnosis.

人类DNA甲基化转移酶1（Dnmt1）在多种癌症中过表达，检测Dnmt1活性对于癌症早期诊断及治疗具有重要意义。目前所报道的传感器多针对原核生物甲基化转移酶，其对底物的识别以及尺寸等都与Dnmt1有较大差异，开发新型的针对于Dnmt1活性的检测方法仍是需要深入研究的。本项目拟以脱氧核酶（DNAzyme）催化苯胺聚合的反应作为信号输出，发展一种灵活的双检测模式的Dnmt1传感器。首先以Hairpin DNA作底物，经Dnmt1甲基化和甲基化敏感的限制性内切酶剪切后，释放出能构成DNAzyme的功能片段，在血红素存在的情况下共同构成DNAzyme结构，利用其催化苯胺聚合生成聚苯胺，可实现灵活的紫外可见分光光度法或者示差脉冲伏安法的双检测模式。因酶催化反应过程与信号输出过程分立，可避免异相反应非均一性、酶识别位阻和复杂基质干扰。项目实施可为癌症早期诊断提供潜在有效手段。

人类甲基化转移酶（DNMT1）是一种与癌症等疾病相关的生物标志物，检测DNMT1活性对于癌症的早期诊断及治疗具有重要意义。G-四链体是一种富含鸟嘌呤的功能化核酸片段，可作为灵活的信号源被广泛应用于传感器的构建。基于此，本项目首先建立了一种基于G-四链体的双通道检测模式的DNMT1活性传感器，即将G-四链体序列设计在DNMT1的发卡底物DNA中，在对甲基化敏感的限制性内切酶BssHII的辅助下，通过对底物DNA甲基化反应响应释放G-四链体的策略，从而将DNMT1活性与所释放的G-四链体关联起来，最终一方面可通过G-四链体与血红素共同构成的脱氧核酶（DNAzyme）催化2,2'-联氮双(3-乙基苯并噻唑啉-6-磺酸)二铵盐（ABTS）氧化生成蓝色产物（ABTS•⁻），以紫外可见分光光度法进行检测；另一方面可通过N-甲基卟啉二丙酸IX（NMM）与G-四链体复合产生增强的荧光信号，以荧光分光进行检测。两种检测模式对DNMT1在0-20 U/mL均获得了较好的线性结果，并以人血清样品进行DNMT1的回收率实验，两种检测模式的回收率均在98.0%-105.0%之间，验证了所构建的传感器的实际样品检测能力。本项目所建立的DNMT1活性传感器在均相反应体系中进行，易于操作，且实现了紫外分光光度法检测和荧光分光光度法检测的双检测模式，可提供一种简易、灵活的DNMT1检测方法，在癌症早期诊断中具有潜在应用价值。此外，基于项目申报书中的DNAzyme和苯胺聚合的扩充内容，还分别建立了基于Zn2+特异性DNAzyme和苯胺聚合为信号源的两种金属离子传感器，分别应用于婴儿奶粉中Zn2+的测定和水样中Cu2+和Fe3+的检测，均取得了较为满意的结果，所构建的金属离子传感器对于食品分析以及环境监测等具有潜在应用价值。