配体靶向DNA酶化修饰以研究并调控其对转录响应的分子机制

The genomic DNA is constantly being modified by both environmental and endogenous factors, among which there are some groups of DNA modifications that are catalyzed by enzymes and potentially have regulatory roles in gene expression. However, except the well-studied methylation and hydroxymethylation in the fifth position of cytosine, functional roles of these enzyme-catalyzed endogenous DNA modifications in transcriptional regulation remain elusive. This project will focus on understanding molecular mechanisms of transcriptional response to these DNA modifications via the approach of chemical biology, as well as developing novel small ligands that can specifically target DNA modifications to unveil and further manipulate their transcriptional effects. In this project, defined in vitro transcriptional system by RNA polymerase II will be utilized to investigate the influence of DNA modifications before and after the ligand targeting during transcriptional elongation. Detailed kinetic response of RNA polymerase II to enzyme-catalyzed DNA modifications and their ligand adducts will be characterized to reveal the quantitative change of efficiency and fidelity during transcription elongation. Furthermore, rationally designed small ligands will be used to interfere and regulate gene transcription related to these DNA modifications in living cells. An EGFP plasmid will be constructed with the modified DNA site located in the 5’-untranslated region. Expression level of GFP will be monitored to investigate the effects of enzyme-catalyzed DNA modifications before and after the ligand targeting. These studies are expected to reveal the relationship between enzyme-catalyzed DNA modifications and the transcriptional response of RNA polymerase II in the molecular level. This project will also try to develop new small molecules that can manipulate gene expression via targeting enzyme-catalyzed DNA modifications. Since the enzyme-catalyzed DNA modifications are highly related to epigenetics and cancer development, these studies will pave the road for the development of new chemical approaches to treat transcription-related diseases.

基因组DNA上广泛存在着由细胞自主酶促反应产生的修饰，而且这种类型的修饰被认为很有可能与基因的转录调控密切相关。然而，除去胞嘧啶的甲基化和羟甲基化外，人们对其他DNA酶化修饰的认知却极其有限。本项目将从转录响应的分子机制出发，利用化学生物学手段靶向这些依然知之甚少的DNA酶化修饰，以研究并调控其在基因转录中的影响。本项目拟构筑单一而定点的体外转录体系，研究DNA酶化修饰本身和其被配体靶向之后，对RNA聚合酶II转录行为的改变；并以此为指导，设计发展配体分子干扰并调控与DNA酶化修饰相关的基因表达。通过此项研究，我们期望揭示DNA酶化修饰与RNA聚合酶II的转录响应在分子水平上的关联作用；并以DNA酶化修饰作为靶标，发展出干扰并调控基因表达的新型分子手段。鉴于DNA的酶化修饰可能与细胞癌变和表观遗传密切相关，此研究将为以后发展新型化学手段以抗击基因转录失调相关的疾病奠定基础。

核酸作为生命的遗传物质，一直处于动态变化中。这种变化既因为其结构的多样可变，也表现为各种化学修饰的动态可逆。核酸的酶化修饰即是一种细胞自我主动发生的，并在基因表达调控中具有重要作用的化学行为。在这个项目中，我们重点围绕核酸酶化修饰及其化学靶向，核酸修饰相关酶的作用机制，以及发展基于核酸的新型分子工具展开研究。在核酸修饰及其化学靶向上，我们探究了DNA糖基化修饰和甲酰基修饰这两个酶化修饰产物。我们化学构筑了DNA糖基化修饰，探究了其对DNA结构的影响，并进一步发现其能够在聚合酶转录延伸中产生定点阻滞作用。在甲酰基修饰中，我们一方面采用苯基羟胺类配体靶向并分析其对聚合酶转录延伸的影响，另一方面我们采用钌配合物的光学性质靶向染色体中的甲酰基修饰并探究其细胞内分布行为。在核酸修饰相关酶的作用机制方面，我们重点针对TET和TDG这两个酶，并采用核苷酸类似物作为分子工具，揭示了一些通常生物学化学手段不易发现的分子机制。通过对这些核酸修饰的靶向及其相关修饰酶的分析，我们不仅揭示了一些的核酸动态修饰中重要分子机制，对阐明其在基因表达中的可能行为提供了化学基础，而且还发展了一些具有潜在调节和探测功能的靶向分子，为相应化学修饰的功能调控提供了新途径。此外，我们还利用核酸动态结构和人为构筑的化学修饰发展新型分子工具，以调控细胞功能和基因表达。细胞内存在各种复杂的基因关联网络，针对这个方面的研究在于能够创造新型化学手段，以实现对细胞基因表达的目的性调控。综合而言，我们基于核酸结构和修饰的动态可变，一方面阐释核酸修饰及其相关酶的分子行为，另一方面发展可化学调控核酸分子功能的新工具。相关研究成果将为发展基于动态核酸行为以调节细胞功能的分子工具，并进而为实现细胞基因表达网络的人工调控提供了可能途径。鉴于核酸的动态修饰可能与细胞癌变和表观遗传密切相关，此项目研究将为以后发展新型化学手段以抗击基因转录失调相关的疾病提供指导。