Chemists have invented a purely chemical method—called click ligation—for stitching together DNA and RNA strands into large, biologically active molecules, bypassing the enzymes cells normally use. This matters because existing biological ligation is fussy: it only works on natural DNA, at small scales, under narrow conditions. The new chemical approach works on both natural and chemically modified DNA, at any scale, under a wide range of conditions. That opens the door to making synthetic genes with precise chemical modifications—such as methylated bases—that are thought to switch genes on and off. These epigenetic switches are poorly understood but implicated in cancer and ageing. The team will also use click ligation to build fluorescently labelled DNA and RNA constructs to study how DNA is packaged in chromosomes, and to investigate how the influenza virus replicates itself using single-molecule FRET and super-resolution microscopy. If successful, the project could enable large-scale production of medicinally important DNA constructs, provide tools to decode epigenetic regulation, and support development of improved antiviral therapies. The researchers will also build artificial molecular machines that carry out controlled chemical reactions, potentially leading to new ways to synthesise drugs. This is fundamental chemistry with clear biotechnological and medical promise.
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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.
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