Extending the Boundaries of Nucleic Acid Chemistry

拓展核酸化学的界限

• 批准号: BB/J001694/2
• 负责人: Tom Brown
• 金额: $ 177.6万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ001694%2F2
• 关键词: Extending; Boundaries; Nucleic; Acid; Chemistry

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.

该项目将一种新的范式引入核酸研究，“点击连接”，这是一种非常有效的纯化学（与生物学相反）方法，用于连接DNA和RNA链以制造大型生物活性结构（DNA和RNA是细胞中储存和传递遗传信息的分子）。尽管新的化学反应产生了一种非自然的连接，但它可以被DNA聚合酶读取，DNA聚合酶是在细胞分裂过程中在生命系统中制造新的DNA拷贝的酶。因此，我们新的人工DNA连接是真正的生物相容性。与生物连接不同，这种化学反应可以在正常和化学修饰的DNA上进行，在广泛的物理条件下以任何规模进行。这使得它可用于生物技术，即大规模生产医学上重要的生物构建体。我们将使用点击连接来制造非常长的DNA链，从而能够组装化学修饰的合成基因，这些基因可用于制造蛋白质。我们的工作将允许插入结构基序，如四链体和化学修饰，如甲基化和羟甲基化的碱基到基因中的基因表达和表观遗传学的研究。这些修饰被认为是通过尚未完全理解的机制来打开和关闭基因。它们目前是密集研究的焦点，因为异常的基因开关与癌症等疾病以及衰老有关。我们将制作荧光标记的DNA和RNA构建体，并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来了解染色质（染色体结构的一部分）中长程相互作用的动态。这将使我们能够理解细胞中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，以研究流感病毒制造蛋白质和复制（复制）自身的机制：这些现象将通过单分子FRET（一种测量两个荧光标记之间距离的非常灵敏的技术）和超分辨率成像进行研究。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构建人工分子机器，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。一个来自南安普顿和牛津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。

项目成果

Pentacyclic adenine: a versatile and exceptionally bright fluorescent DNA base analogue.

• DOI: 10.1039/c7sc05448c
• 发表时间: 2018-04-14
• 期刊: Chemical science
• 影响因子: 8.4
• 作者: Bood M;Füchtbauer AF;Wranne MS;Ro JJ;Sarangamath S;El-Sagheer AH;Rupert DLM;Fisher RS;Magennis SW;Jones AC;Höök F;Brown T;Kim BH;Dahlén A;Wilhelmsson LM;Grøtli M
• 通讯作者: Grøtli M

RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks.

• DOI: 10.15252/embj.201796664
• 发表时间: 2017-07-14
• 期刊: The EMBO journal
• 作者: Abdullah UB;McGouran JF;Brolih S;Ptchelkine D;El-Sagheer AH;Brown T;McHugh PJ
• 通讯作者: McHugh PJ

Transcription of click-linked DNA in human cells.

• DOI: 10.1002/anie.201308691
• 发表时间: 2014-02-24
• 期刊: ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
• 影响因子: 16.6
• 作者: Birts, Charles N.;Sanzone, A. Pia;El-Sagheer, Afaf H.;Blaydes, Jeremy P.;Brown, Tom;Tavassoli, Ali
• 通讯作者: Tavassoli, Ali

Searching for the ideal triazole: Investigating the 1,5-triazole as a charge neutral DNA backbone mimic

• DOI: 10.1016/j.tet.2019.130914
• 发表时间: 2020-02-14
• 期刊: TETRAHEDRON
• 影响因子: 2.1
• 作者: Baker, Ysobel R.;Traore, Diallo;Brown, Tom
• 通讯作者: Brown, Tom

Expanding the chemical functionality of DNA nanomaterials generated by rolling circle amplification.

• DOI: 10.1093/nar/gkab720
• 发表时间: 2021-09-20
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Baker YR;Yuan L;Chen J;Belle R;Carlisle R;El-Sagheer AH;Brown T
• 通讯作者: Brown T