Extending the Boundaries of 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.

Click ligation is an extremely efficient method for joining DNA and RNA strands to produce an unnatural linkage at the site of ligation that can be replicated by DNA polymerases. Unlike biological ligation, it can be carried out on both normal and chemically-modified DNA, on any scale, under a wide range of physical conditions, making it particularly useful for biotechnology. Click ligation will be used for the enzyme-free synthesis of very long DNA strands, enabling the assembly of chemically-modified synthetic genes and plasmids for in vivo applications. The project will open up new areas in synthetic biology, allowing the insertion of structural motifs such as quadruplexes, methylated and hydroxymethylated bases into genes for the studies of the control of gene expression and of epigenetics. We will investigate gene loops and their relationship to non-coding RNA and transcription to understand the dynamics of long range chromatin interactions in yeast by super resolution microscopy using long synthetic click DNA templates containing fluorescent nucleotides at defined sites. We will prepare chemically modified, fluorescently labelled RNA substrates for the study of influenza viral transcription and replication using single-molecule FRET and super-resolution imaging. This will provide unprecedented opportunities to observe these mechanisms and will contribute to the development of models of transcription and gene expression in general, thus benefiting both systems and synthetic biology. We will extend the scope of synthetic biology through combinatorial nanostructure assembly by building molecular machines that are controlled by synthetic genes assembed by click ligation, and by using click ligation to create combinatorial libraries of functional nanostructures. An internationally-leading team from Southampton and Oxford has been assembled and extensive preliminary studies have been carried out to prove feasibility.

We propose to exploit a new technology (click ligation) for joining synthetic nucleic acid fragments by chemical methods. This versatile technique can generate long pieces of DNA or RNA that contain specific modifications, which can be used in synthetic biology, chemical biology, molecular biology and nanotechnology. Site-specific incorporation of modifications such as fluorescent labels and methylated bases cannot be achieved by conventional (enzymic and biological) methods. An internationally-leading team from Southampton and Oxford has been assembled to demonstrate the power of this technology in a number of research areas, including the preparation of synthetic genes; the generation of plasmids that contain permanent unusual DNA structures; the effects of specific methylation patterns in epigenetics; a mechanistic analysis of influenza viral transcription and replication using clicked RNAs; and the generation of combinatorial libraries of functional nanostructures. The potential applications of this technology extend well beyond these examples and the full potential impact of this reseach will be achieved if our technology is widely adopted in other areas of fundamental and applied research that could benefit from the facile preparation of long nucleic acids which contain modifications at specific positions. The main beneficiaries from this research will be those involved in fundamental and applied research in the fields of chemical biology, synthetic biology, biochemistry, molecular biology, genetics, and genomics whose research will benefit from the adoption of our methods. It is therefore important for the results to be rapidly communicated to the scientific community through peer-reviewed scientific journals, general-interest publications, university seminars, conference lectures and posters, webpages. The Universities have dedicated Media Centres to aid with public announcements and production of newsletters, web pages and social networking blogs to further inform the public of the research. This multidisciplinary project will provide training for post-doctoral workers at the interface between Chemistry and Biology. Scientists who can collaborate across wide range of disciplines will have an impact that goes beyond the five years of this project with long-term benefits to the UK research base and wider impacts on society and the economy. PhD students and undergraduate project students in our laboratories will also benefit from interaction with this work. Southampton and Oxford are research intensive Universities and our research activities inform the final year undergraduate teaching. Exploitation of the results will be coordinated through Southampton University's Research and Innovation Services and Oxford University's intellectual property company Isis Innovation. These organizations support world-class research by focusing on its impact on national and global economies and society. They have excellent track records of developing and maintaining relationships with external partners and exploiting research outputs for the benefit of society. The involvement of an SME (atdbio Ltd) will provide a further means for exploiting the technology and will ensure that the project will increase UK competitiveness. The wider public will also benefit from the applicants' engagement with the 'public understanding of science' through public lectures, university Science and Engineering days and other open days. The activities are supported by members of the research team including PhD students and postdoctoral researchers. The general public have considerable interest in DNA and are generally keen to learn more about its properties. The applicants will therefore continue to be involved in visits to local schools to give lectures on a wide variety of topics relating to nucleic acids. We have included communication skills and media training in the budget to ensure that we explain their importance of our work to a wide audience.