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
Meng W
(2016)
An autonomous molecular assembler for programmable chemical synthesis.
in Nature chemistry
Brown T
(2018)
Antisense transcription-dependent chromatin signature modulates sense transcript dynamics.
in Molecular systems biology
Benn F
(2018)
Chiral DNA Origami Nanotubes with Well-Defined and Addressable Inside and Outside Surfaces
in Angewandte Chemie
Benn F
(2018)
Chiral DNA Origami Nanotubes with Well-Defined and Addressable Inside and Outside Surfaces.
in Angewandte Chemie (International ed. in English)
Mazumder A
(2019)
Closing and opening of the RNA polymerase trigger loop
Mazumder A
(2020)
Closing and opening of the RNA polymerase trigger loop.
in Proceedings of the National Academy of Sciences of the United States of America
Gilboa B
(2019)
Confinement-Free Wide-Field Ratiometric Tracking of Single Fluorescent Molecules.
in Biophysical journal
Duchi D
(2018)
Conformational heterogeneity and bubble dynamics in single bacterial transcription initiation complexes.
in Nucleic acids research
Howe FS
(2017)
CRISPRi is not strand-specific at all loci and redefines the transcriptional landscape.
in eLife
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 | 08/2014 |
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 | 08/2013 |
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 | 05/2016 |
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 | 06/2018 |
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 | 09/2017 |
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 | 03/2014 |
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 | 07/2017 |
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 | 11/2021 |
Description | Programme Grant |
Amount | £1,800,000 (GBP) |
Funding ID | MR/R009945/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2018 |
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 | 03/2016 |
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 | 03/2012 |
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 | 03/2016 |
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 | 09/2013 |
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 | 09/2017 |
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 |
Title | SINGLE-MOLECULE PHENOTYPING AND SEQUENCING OF NUCLEIC ACID MOLECULES |
Description | This invention relates to a method of sequencing a nucleic acid molecule, in particular the method comprising: (a) providing the nucleic acid immobilised on a surface; (b) forming a single-stranded gap section by partially duplexing the nucleic acid such that the single-stranded gap section is flanked by duplex sections, wherein the sequence to be sequenced is a sequence of the single-stranded gap section; (c) providing a set of at least four fluorescently labelled oligonucleotide probes, (d) detecting binding, or absence thereof, of the fluorescently labelled oligonucleotide probes with the gap section of the immobilised nucleic acid, wherein the identity of the interrogated nucleotide of the gap section of the immobilised nucleic acid is identified as the complementary base of the nucleotide X of the fluorescently labelled oligonucleotide probe that has the highest incidence of binding; (e) repeating steps (c) and (d) for interrogating subsequent nucleotide positions of the gap section of the immobilised nucleic acid until sufficient nucleotides of the gap section have been identified to be able to determine a sequence. Also provided are methods of single-molecule phenotyping and sequencing; methods of single-molecule phenotyping and identification of a molecule tagged with nucleic acid; an immobilised nucleic acid molecule; and a composition comprising oligonucleotide probes. |
IP Reference | US2021180126 |
Protection | Patent / Patent application |
Year Protection Granted | 2021 |
Licensed | No |
Impact | Impact generation is still in progress |
Company Name | PicturaBio |
Description | PicturaBio develops rapid pathogen diagnostics tools, utilising machine learning alongside single-molecule fluorescent microscopy. |
Year Established | 2021 |
Impact | Rapid Diagnostics on Respiratory Viruses. Raised $3.55M in seed funding in late 2021. |
Website | https://pictura.bio/ |
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 |