New fluorescent probes for labelling nucleic acids

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

Abstract

The human body is composed of billions of cells of different types. Each cell type (e.g. skin cells, liver cells, red blood cells) has its own specific roles. Many diseases arise from a breakdown or defect in the complex biochemistry within specific types of cells. A clinically important example of this is thalassaemia. The alpha and beta thalassaemias are common severe inherited forms of anaemia caused by imbalance of alpha and beta globin gene expression which affects the production of haemoglobin, the protein that carries oxygen through the body. Current therapy involves lifelong blood transfusion and iron chelation therapy, but these are invasive and only moderately effective. Consequently new therapies to correct globin chain imbalance are urgently needed. A crucial requirement in the development of new treatments is a detailed understanding of the regulation of alpha and beta globin gene expression, i.e. why these genes do not make their products in the correct amounts in patients suffering from thalassaemia. It is now recognized that the ability of many genes to make proteins is regulated by enhancer elements in their DNA. These are binding sites for proteins that switch genes on or off, and mutations at these sites underlie many human diseases. It has been shown by special fluorescence cell imaging techniques that enhancers and promoters can be located far apart on DNA, but come together when they are controlling protein synthesis. However it is not clear how this physical interaction exerts its effect. To analyse this we must obtain much clearer physical pictures of the process to determine how dynamic this enhancer-promoter association is, when it occurs and how long it persists. This will require new methods for high-resolution imaging of genomic DNA using small and intensely bright pieces of DNA (fluorescent probes). These probes will be much brighter than those currently available. They will be synthesised chemically by high-throughput automated methods using a modular approach which will greatly reduce the cost of their manufacture. This new approach to DNA and RNA imaging is generic and will be used in a wide variety of clinical and research applications by scientists working in academia, research institutes and the pharmaceutical industry.

Technical Summary

We aim to transform the resolution and sensitivity of imaging of cellular nucleic acids using a new generation of probes. Densely labelled and intensely fluorescent probes will be prepared by modular approaches that combine large scale with high throughput automated oligonucleotide synthesis and post-synthetic DNA crosslinking and labelling. This new strategy will make it possible to synthesise large numbers of probes at low cost, and will allow the synthesis of probes with unique fluorescent signatures to image various regions of genomic DNA in different colours. The generic approach is applicable to a wide range of cell-imaging research projects including super-resolution studies. It will be evaluated by investigating the regulation of expression of globin genes implicated in the alpha and beta thalassaemias. These are common severe inherited forms of anaemia caused by imbalance of alpha and beta globin gene expression. Current therapy involves lifelong blood transfusion and iron chelation therapy, but these are invasive and only moderately effective. Consequently new therapies are urgently needed. The expression of many genes is regulated by enhancer elements which are binding sites for proteins that switch genes on or off. Mutations at these sites can grossly alter levels of transcription of the gene, and hence can underlie many human diseases. Fluorescence imaging suggests that enhancers and promoters come into physical proximity when transcriptionally active but it is not clear how this increases transcription. We will visualise the spatial organisation of the alpha globin gene regulatory region at high resolution on a cell by cell basis and for the first time we will visualise short meRNAs that run off intragenic enhancers. These RNAs are also implicated in the regulation of alpha-globin gene expression, but their precise biological role is unknown. We aim to relate their appearance to that of the alpha globin nascent transcript.

Planned Impact

Beneficiaries of this research will be:

Research scientists in academia who are developing new techniques for imaging DNA and RNA

Research scientists in academia who are working to develop a better understanding of biology in healthy cells and organisms

Biomedical scientists in academia and industry who are endeavoring to understand the biological processes that occur in the development and progression of diseases

Scientists in the pharmaceutical and biotech industries who are working in discovery biology to determine new targets for therapy in a wide range of diseases

Companies that manufacture and supply fluorescent probes to academia and industry for use in the above applications

The UK economy through companies who will be given access to IP generated in this project

The general public who will benefit from new and improved medicines and therapies

Schools and colleges through outreach activities


How they will benefit from this research:

Scientists in academia and Industry will be able to progress their research more efficiently and cost-effectively and carry out projects that are not possible using existing imaging techniques. This will result in new knowledge and scientific advancement through utilization of new and innovative methodologies and cross-disciplinary approaches. It will contribute towards the health of academic disciplines by developing expertise and knowledge

In particular the project will train a highly skilled postdoctoral researcher who will develop new inter-disciplinary skills

The results of this research project will lead to improved treatments for diseases and a better understanding of the healthy organism, enhancing cultural enrichment, quality of life, health and well-being, thus benefitting the general public

The UK economy will benefit through wealth creation and economic prosperity by the growth of companies and jobs, enhancing business revenue and innovative capacity
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1161
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1152
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1158
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1160
 
Title Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin 
Description Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ~200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization. 
Type Of Art Image 
Year Produced 2020 
URL https://idr.openmicroscopy.org/webclient/?show=project-1159
 
Description The work initiated by this grant has been extremely successful in providing insight into the four-dimensional organisation of the genome. Sara was able to synthesise various pools of oligonucleotides mapping to the alpha globin locus and establish a reliable rolling circle method of amplification and direct labelling. We were able to use these probe pools to visualise the locus at super-resolution and establish that the predicted formation of a decondensed domain structure in actively transcribing nuclei could indeed be visualised. Looking more broadly across the region we used this approach to examine the organisation of sequential domains at super-resolution and how that integrated with other approaches to establish chromatin structure in the nucleus.

The ability to synthesise large numbers of oligonucleotides was further capitalised on by tiling oligos right cross an entire 3Mb section of the genome. This led to the development of an entirely new Chromosome Conformation Capture (3C) technology 'Tiled-C', which allows for the generation of high-resolution contact matrices of loci of interest at unprecedented depth. We have used this approach to study the chromatin architecture of the mouse alpha-globin locus through in vivo erythroid differentiation. Our findings demonstrate that chromatin architecture and gene activation are tightly linked during development and provide insights into the distinct mechanisms contributing to the establishment of tissue-specific chromatin structures.

The success of these studies has led to numerous enquiries about the techniques used. We are in the process of preparing a methods paper and have been invited to submit a methods chapter to 'Methods in Molecular Biology' that will encompass the use of oligonucleotide probe pools in fluorescent imaging.
Exploitation Route An improved understanding of the factors that control and influence gene expression aids in the development of novel therapeutic and diagnostic applications.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology,Other

 
Description The precise relationship between nuclear architecture and gene regulation is still not completely understood, yet this is a critical facet of molecular medicine for our understanding of human disease that will impinge widely on developments in gene therapy and personalised medicine. Our work has contributed to an understanding of the specific changes that need to occur in chromatin organisation during development and differentiation as transcriptional programs are activated. The technologies we have developed have a great visual impact that have been used in many public engagement opportunities involving the Weatherall Institute of Molecular Medicine, in particular New Scientist Live (http://www.doc.gold.ac.uk/csynth/wp/new-scientist-live-22nd-25th-september-2016-the-excel-london/) and the Royal Society Summer Exhibition (https://royalsociety.org/science-events-and-lectures/2017/summer-science-exhibition/exhibits/dna-origami-how-do-you-fold-a-genome/). Images from this work were also used in the development of an educational dance 'The Cell' by Impelo (https://www.impelo.org.uk/cell)(https://www.imm.ox.ac.uk/about/news/arts-meets-science-in-dance-exploration-of-cell-biology) which is now touring to UK primary and secondary schools, science festivals and other festivals eg Hay. A comic and now even an animation featuring Prof Buckle have been developed as teaching resources to support the performances.
First Year Of Impact 2019
Sector Healthcare,Pharmaceuticals and Medical Biotechnology