Extending the Boundaries of Nucleic Acid Chemistry

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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 research 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.

Publications

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Related Projects

Project Reference Relationship Related To Start End Award Value
BB/J001694/1 01/05/2012 30/09/2013 £1,829,817
BB/J001694/2 Transfer BB/J001694/1 01/10/2013 30/04/2017 £1,393,815
 
Description BBSRC sLoLa grant Extending the Boundaries of Nucleic Acid Chemistry
BB/J001694/1, BB/J001694/1

What were the most significant achievements from the award?

Click ligation- gene synthesis
Objective:
To explore the use of click chemistry for the synthesis of genes and genomes by developing one-pot gene synthesis by click ligation, to probe the biocompatibility of click-DNA in eukaryotes, specifically yeast and mammalian cells, and to determine the basis for the observed biocompatibility of click-DNA in E.coli (replication or repair) and (if observed) in eukaryotes.

The above objective has been entirely fulfilled, details are below:
Collaborative research in the Brown group at Oxford University and the Tavassoli group at Southampton University was recently published in Nature Chemistry.1 It describes the first synthesis of a chemically modified gene by entirely chemical methods (CuAAC reaction).1 Fluorescent and epigenetically-modified versions of the iLOV gene were assembled, replicated and encoded into functional iLOV protein in E.coli. This study is significant because high density site-specific incorporation of epigenetic (or other chemical) information into genomic DNA is not currently feasible by conventional gene synthesis methods based on PCR. The "click ligation" methodology was originally developed in the Brown group2,3 for use in various applications4 including the synthesis of crosslinked and cyclic DNA duplexes5 and biologically active RNA constructs.6 The remarkable biocompatibility of the triazole linkage in DNA3 stems from its detailed molecular structure and hydrogen bonding capacity.7-9 One potential limitation of the triazole linkage is that DNA polymerases read through it more slowly than natural DNA.1, 8 However, it functions well in vitro, and in bacterial7 and human cells10, and has established the important principle that unnatural DNA backbones can be read through by polymerase enzymes. TB's group recently showed that other designed modified backbones can be biocompatible;8 one of these is an amide linkage which is read-through by DNA polymerases almost as efficiently as a natural phosphodiester backbone. Another related approach involves the chemical ligation to create a phosphoramidate linkage. This chemistry was used recently to synthesis the 760 base pair GFP gene.11

A molecular assembler machine - nanotechnology
Objective
To expand the scope of synthetic biology through combinatorial nanostructure assembly. To demonstrate combinatorial synthesis using natural and unnatural molecular machinery with products controlled or recorded by synthetic 'genes' assembled by click ligation. To use click ligation to create combinatorial libraries of functional nanostructures.

The Meng et al. Nature Chemistry volume 8, pages 542-548 (2016)
• doi:10.1038/nchem.2495

We developed an autonomous molecular assembler for combinatorial oligomer synthesis, based on the principle of DNA-templated synthesis and a DNA hybridization chain reaction. Oligomers were assembled according to a molecularly-encoded program in a one-pot reaction using both biomimetic (peptide) and completely synthetic (Wittig) backbone assembly reactions. Non-deterministic assembly programs were used to produce combinatorial libraries of oligomers. Each product molecule was individually tagged with a DNA 'gene', assembled using click ligation, to record its sequence, opening the way to the use of such assemblers to discover and optimise new functional oligomers by library synthesis, selection and evolution.12 Other have used our click ligation methodology to construct and encode DNA libraries for drug discovery.13, 14 Key publication: Meng et al. Nature Chemistry volume 8, pages 542-548 (2016) doi:10.1038/nchem.2495


To investigate gene loops
Objective
To investigate gene loops and their relationship to non-coding RNA and transcription by the use of specifically modified long synthetic DNA molecules. To understand how installation of precise patterns of DNA methylation in a CGI impact surrounding DNA methylation and CGI function in vivo, to understand the effect of 5 hydroxymethycytosine on transcription in mammals, to understand the dynamics of long range chromatin interactions in yeast using super resolution microscopy and long synthetic DNA templates containing fluorescent nucleotides at defined sites.

This objective was hard to complete due to difficulties with transforming the fluorescently-labelled CLICK assembled double stranded DNA into yeast and cultured cells at a sufficiently high frequency to facilitate the high resolution microscopy. In consultation with the Kapanidis group, who had successfully used similar methodology in E.coli, we trialed a number of different methods, but without obtaining high enough transformation frequency for microscopy. In consultation with the project team, we devised a new biological application for CLICK chemistry which proved very successful. We used this technology to build a library of guide RNAs to target a catalytically dead Cas9 protein to block transcription. The application was to test whether tethering dCas9 can block antisense transcription and to ask how this influences sense transcription on the same gene. At one locus studied in detail, we discovered that targeting dCAS to block antisense transcription successfully blocks the targeted transcription event but leads to a new transcription event initiating upstream and extending into the promoter for the sense transcript. In addition, we observe truncation of the sense transcript suggesting that targeting dCas9 to one strand of DNA blocks transcription on both strands of DNA. This work was published in eLIFE.15

Objective
To investigate the biological effects of non-canonical DNA structures. To use click ligation to prepare plasmids that contain permanent unusual DNA structures, in particular quadruplexes, within gene promoters and to determine their biological effects.

One publication concerning supercoiling and quadruplex formation:
Sekibo, D., & Fox, K. R. (2017). The effects of DNA supercoiling on G-quadruplex formation. Nucleic Acids Research 45, 12069-12079. DOI: 10.1093/nar/gkx856

An important finding would be that DNA supercoiling alone is not sufficient to drive quadruplex formation.

Objective

To carry out a mechanistic analysis of influenza viral transcription and replication using clicked RNAs.

We used our expertise in structural analysis of the influenza virus polymerase (Hengrung et al. Nature 527:114, 2015) and single-molecule techniques to study the interactions and dynamics of the influenza polymerase and viral RNA. This work allowed us to propose a model of the dynamic processes that occur during initial viral replication (Tomescu, Robb et al. PNAS 111:E3335, 2014, Robb et al. NAR 44(21):10304, 2016).

To use single molecule fluorescence techniques on large chemically modified click RNA constructs to elucidate these mechanisms.

The influenza polymerase wasn't able to read through long clicked RNAs efficiently but we were able to obtain good results using short synthetic RNA fragments.

Key findings / achievements:

1. This work has provided novel insights into the replication mechanism of the influenza virus.
2. Summarised in two papers (Tomescu, Robb et al. PNAS 111:E3335, 2014, Robb et al. NAR 44(21):10304, 2016).
3. On the basis of the work carried out in this grant we were able to secure additional funding (an MRC grant to A.N.K. and E.F. and a Royal Society Fellowship to N.C.R.).



1. M. Kukwikila, N. Gale, A. H. El-Sagheer, T. Brown and A. Tavassoli, Nat. Chem., 2017, 9, 1089-1098.
2. R. Kumar, et al., J. Amer. Chem. Soc., 2007, 129, 6859-6864.
3. A. H. El-Sagheer and T. Brown, J. Amer. Chem. Soc., 2009, 131, 3958-3964.
4. A. H. El-Sagheer and T. Brown, Accounts Chem. Res., 2012, 45, 1258-1267.
5. P. Kocalka, A. H. El-Sagheer and T. Brown, Chembiochem, 2008, 9, 1280-1285.
6. A. H. El-Sagheer and T. Brown, Proc. Natl. Acad. Sci. USA, 2010, 107, 15329-15334.
7. A. H. El-Sagheer, A. P. Sanzone, R. Gao, A. Tavassoli and T. Brown, Proc. Natl. Acad. Sci. USA, 2011, 108, 11338-11343.
8. A. Shivalingam, A. E. S. Tyburn, A. H. El-Sagheer and T. Brown, J. Amer. Chem. Soc., 2017, 139, 1575-1583.
9. A. Dallmann, et al., Chemistry - A European Journal, 2011, 17, 14714-14717.
10. C. N. Birts, et al., Angew. Chem. Int. Ed., 2014, 53, 2362-2365.
11. A. H. El-Sagheer and T. Brown, ChemComm, 2017, 53, 10700 - 10702.
12. W. J. Meng, et al., Nat. Chem., 2016, 8, 542-548.
13. A. D. Keefe, M. A. Clark, C. D. Hupp, A. Litovchick and Y. Zhang, Curr. Opin. Chem. Biol., 2015, 26, 80-88.
14. A. Litovchick, et al., Nat. Sci. Rep., 2015, 5, 1-8.
15. F. S. Howe, et al., eLife, 2017, 6, e29878.
Exploitation Route in commercial synthesis of chemically modified genes, possibly for synthesis of chemically modified mRNA.
Sectors Manufacturing, including Industrial Biotechology

 
Description We (Oxford and Southampton University) have filed several patents on the technology that we developed and are seeking industrial partners to take these further. A number of companies have used the nucliec acid click ligation methodology after seeing our publications
First Year Of Impact 2019
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Title DNA click ligation 
Description 3'-propargyl-5-methyl deoxycytidine on solid support for oligonucleotide synthesis, commercially available through Glen Research 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact Wider use of DNA click ligation in the research community 
URL http://www.glenresearch.com/GlenReports/GR24-21.html