Extending the Boundaries of Nucleic Acid Chemistry

这个项目为核酸研究引入了一种新的范式，“点击连接”，这是一种非常有效的纯化学（而不是生物）方法，用于连接DNA和RNA链，以制造大型生物活性结构（DNA和RNA是细胞中存储和传递遗传信息的分子）。尽管这种新的化学反应产生了一种非自然的连接，但它可以被DNA聚合酶解读，这种酶在细胞分裂过程中在生命系统中产生新的DNA拷贝。因此，我们新的人工DNA链是真正的生物相容性。与生物结扎不同的是，这种化学反应既可以在正常DNA上进行，也可以在化学修饰的DNA上进行，而且可以在各种物理条件下以任何规模进行。这使得它对生物技术非常有用，即大规模生产具有重要医学意义的生物结构。我们将使用点击连接来制造非常长的DNA链，使化学修饰的合成基因能够组装，这些基因可以用来制造蛋白质。我们的工作将允许在基因中插入结构基序，如四联体和化学修饰，如甲基化和羟甲基化碱基，用于基因表达和表观遗传学的研究。这些修饰被认为通过尚未完全了解的机制来开关基因。由于异常的基因开关与癌症和衰老等疾病有关，它们目前是激烈研究的焦点。我们将制作荧光标记的DNA和RNA结构，我们将使用它们来研究基因的物理结构（包括基因环），并通过超分辨率显微镜了解染色质（染色体结构的一部分）中远程相互作用的动力学。这将使我们了解细胞中DNA的紧密包装与其调节蛋白质合成的能力之间的关系。我们将制备荧光标记的RNA底物来研究流感病毒制造蛋白质和自我复制的机制：这些现象将通过单分子FRET（一种非常敏感的技术，用于测量两个荧光标记之间的距离）和超分辨率成像来研究。这将有助于我们了解RNA病毒的生物学，这是开发改进疗法的重要一步。我们将使用点击连接来建造人工分子机器，这些机器将被设计成以精确控制的方式进行独特的化学反应。这项技术可能会导致开发包括药物在内的生物活性化合物的新方法。来自南安普顿和牛津大学的国际领先团队已经组建，并进行了广泛的初步研究以证明可行性。

This project introduces a new paradigm into nucleic acids research, 'click ligation', which is an extremely efficient purely chemical (as opposed to biological) method for joining DNA and RNA strands to make large biologically active constructs (DNA and RNA are the molecules in cells that store and transmit genetic information). Although the new chemistry produces an unnatural linkage, it can be read through by DNA polymerases, the enzymes that make new copies of DNA in living systems during cell division. Thus our new artificial DNA linkage is truly biocompatible. Unlike biological ligation, this chemical reaction can be carried out on both normal and chemically-modified DNA, on any scale under a wide range of physical conditions. This makes it useful for biotechnology, i.e. the large scale production of medicinally important biological constructs. We will use click ligation to make very long DNA strands, enabling the assembly of chemically-modified synthetic genes which can be used to make proteins. Our work will allow the insertion of structural motifs such as quadruplexes and chemical modifications such as methylated and hydroxymethylated bases into genes for the study of gene expression and epigenetics. These modifications are thought to switch genes on and off by mechanisms that are not yet fully understood. They are currently the focus of intense research as aberrant genetic switches are implicated in diseases such as cancer and also in ageing. We will make fluorescently labelled DNA and RNA constructs and we will use them to investigate the physical structures of genes (including gene loops) and to understand the dynamics of long-range interactions in chromatin, part of the structure of a chromosome, by super resolution microscopy. This will allow us to understand the relationship between the tight packaging of DNA in cells and its ability to regulate the synthesis of proteins. We will prepare fluorescently labelled RNA substrates to investigate mechanisms used by the influenza virus to make proteins and to replicate (copy) itself: theses phenomena will be studied by single-molecule FRET (a very sensitive technique for measuring distances between two fluorescent labels) and super-resolution imaging. This will help us to understand the biology of RNA viruses, an important step towards developing improved therapies. We will use click ligation to build artificial molecular machines that will be designed to carry out unique sets of chemical reactions in a precisely controlled manner. This technology may lead to new ways to develop biologically active compounds including drugs. An internationally-leading team from Southampton and Oxford has been assembled and extensive preliminary studies have been carried out to prove feasibility.