Extending the Boundaries of Nucleic Acid Chemistry

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

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 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.

Publications

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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 group[2,3] for use in various applications[4] including the synthesis of crosslinked and cyclic DNA duplexes[5] and biologically active RNA constructs.[6] The remarkable biocompatibility of the triazole linkage in DNA[3] 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 bacterial[7] and human cells[10], 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.

'Click ligation' has a very broad range of possible applications, including the development of new methods for the discovery of new functional molecules. Techniques that we have developed for the creation of libraries of molecules, from which those with particular properties can be selected, will be developed with the aim of creating a new technology that can be applied to, e.g., the discovery of new antimicrobial compounds. 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] The importance of click ligation in this process is that it is effective in reaction conditions that would be incompatible with enzymatic ligation, allowing us to explore the full potential of synthetic, non-ribosomal systems for programmed synthesis of abiotic compounds. We have also explored the construction of DNA-based molecular motors as potential components in the next generations of molecular assembly lines. Other have used our click ligation methodology to construct and encode DNA libraries for drug discovery.[13, 14]


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.

Guanine-rich DNA sequences can fold into four-stranded structures that consist of stacks of G-quartets. Sequences with the potential to adopt this structure are common throughout the human genome and are frequently found in the proximal promoter region of genes, particularly those involved in growth-control and in several oncogenes. Our key finding is that DNA supercoiling alone is not sufficient to drive quadruplex formation.[16]

Objective: To carry out a mechanistic analysis of influenza viral transcription and replication using clicked RNAs. 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. We used our expertise in structural analysis of the influenza virus polymerase 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, providing novel insights into the replication mechanism of the influenza virus.[17, 18] 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.
16. D. Sekibo and K. R. Fox, Nucleic Acids Research, 2017, 45, 12069-12079.
17. A. I. Tomescu et al., PNAS, 2016, 111, E3335-E3342.
18 N. C. Robb et al., Nucleic Acids Research, 2016, 44, 10304-10315.
Exploitation Route With our collaborators in Southampton University, we are demonstrating and publishing a wide range of applications of click ligation, providing technical and scientific data and application examples to underpin the wider uptake and development of this new technology. Specific findings will form the basis of protected intellectual property, and may be developed through, e.g., licensing agreements through Oxford University Innovation and Southampton University's Research and Innovation Services.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
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.
 
Description 14-ERASynBio BioOrigami
Amount £415,854 (GBP)
Funding ID BB/M005739/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 08/2017
 
Description Artificial synthesis of the bacterial flagellar motor with DNA nanostructures
Amount $1,200,000 (USD)
Funding ID RGP0030/2013 
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 09/2013 
End 08/2016
 
Description BBSRC Responsive mode grant
Amount £393,000 (GBP)
Funding ID BB/N018656/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2016 
End 06/2019
 
Description Bio-Inspired Quantum Technologies
Amount £1,500,000 (GBP)
Organisation University of Oxford 
Department Oxford Martin School
Sector Academic/University
Country United Kingdom
Start 03/2013 
 
Description Critical Mass Award
Amount £2,340,288 (GBP)
Funding ID EP/P000479/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 10/2020
 
Description DNA Strand Displacement driven Molecular Additive Manufacturing (DSD-MAM)
Amount $787,627 (USD)
Funding ID DE-EE0008310 
Organisation U.S. Department of Energy 
Sector Public
Country United States
Start 07/2018 
End 12/2021
 
Description Dorothy Hogkin Fellowship for Nicole Robb
Amount £516,625 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2022
 
Description EPSRC & BBSRC Centre for Doctoral Training in Synthetic Biology
Amount £8,261,498 (GBP)
Funding ID EP/L016494/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 04/2014 
End 09/2022
 
Description EScoDNA Marie Curie Initial Training Network
Amount € 4,070,204 (EUR)
Funding ID 317110 
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 02/2013 
End 01/2017
 
Description International Exchanges Scheme
Amount £11,750 (GBP)
Funding ID IE150554 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2015 
End 01/2019
 
Description MRC Confidence in Concept
Amount £22,506 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 04/2018
 
Description Marie Sklodowska Curie Innovative Training Network
Amount € 3,979,633 (EUR)
Funding ID 765703 
Organisation European Commission H2020 
Sector Public
Country Belgium
Start 01/2018 
End 12/2021
 
Description Medical and Life Sciences Translational Fund
Amount £72,890 (GBP)
Funding ID 0005941 
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 11/2018 
End 11/2019
 
Description Molecular arrows: DNA markers for electron cryotomography
Amount £652,124 (GBP)
Funding ID MR/R017875/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 12/2018 
End 11/2020
 
Description Programme Grant
Amount £1,800,000 (GBP)
Funding ID MR/R009945/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2018 
End 03/2023
 
Description Responsive mode
Amount £420,000 (GBP)
Funding ID MR/N010744/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2019
 
Description Royal Society Wolfson Research Merit Award
Amount £100,000 (GBP)
Funding ID WM110130 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2012 
End 03/2017
 
Description Single-molecule analysis of influenza virus transcription and replication
Amount £420,000 (GBP)
Funding ID MR/N010744/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 03/2019
 
Description SynbiCITE - an Imperial College led Innovation and Knowledge Centre (IKC) in Synthetic Biology
Amount £5,074,190 (GBP)
Funding ID EP/L011573/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2018
 
Description University of Oxford John Fell Fund
Amount £72,096 (GBP)
Organisation University of Oxford 
Sector Academic/University
Country United Kingdom
Start 10/2017 
End 09/2018
 
Description Wellcome Trust Investigator award
Amount £1,587,000 (GBP)
Funding ID 110164/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2016 
End 02/2021
 
Description 3D structural studies of the influenza virus RNA polymerase 
Organisation University of Oxford
Department Wellcome Trust Centre for Human Genetics
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provided expertise in functional studies of the influenza virus RNA polymerase.
Collaborator Contribution Provided expertise in large scale purification of proteins for structural studies.
Impact PMID: 24145413; PMID: 25071209; PMID: 26503046; PMID: 27396566; PMID: 27694620
Start Year 2007
 
Description Dunn School of Pathology (Prof Ervin Fodor) 
Organisation University of Oxford
Department Sir William Dunn School of Pathology
Country United Kingdom 
Sector Academic/University 
PI Contribution Single-molecule fluorescence methods, ways to study protein-RNA interactions, use of fluorescence-based distance constraints to generate structural models
Collaborator Contribution Influenza RNA polymerase, promoter RNA design, in vitro transcription assays
Impact 5 academic research papers. This is a multidisciplinary commemoration between a biophysics and a virology group
Start Year 2009
 
Description EM structural studies 
Organisation University of Oxford
Department Wellcome Trust Centre for Human Genetics
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We provided material for structural studies using electron microscopy.
Collaborator Contribution The partner provided expertise, data analysis and access to equipment to perform structural analysis by electron microscopy.
Impact 24155385
Start Year 2012
 
Description Single molecule studies 
Organisation University of Oxford
Department Department of Physics
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided materials for investigating influenza virus RNA polymerase - promoter interactions at the single molecule level.
Collaborator Contribution The partner contributed technology to analyse protein-RNA complexes at the single molecule level.
Impact PMID: 25071209; PMID: 27694620; PMID: 27572643
Start Year 2010
 
Description "Back from the Dead" series of public events 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact 'Back to the Dead' temporary exhibition on antibiotics in the Museum of the History of Science, Oxford. An exhibition describing Oxford past and present role in cutting edge science, using Penicillin as the pull but allowing us to describe our current science to the general public and schools.
Year(s) Of Engagement Activity 2017
URL https://www.mhs.ox.ac.uk/backfromthedead/events/
 
Description Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Presentation at open day in the Department on research in the Department. Sparked lots of interest and questions from the participants which included the general public and some prospective undergraduates. General outcomes include engaging the public with research in the Department including gene expression and its regulation and its importance in disease and development.
Year(s) Of Engagement Activity 2014,2015,2016,2017