New fluorescent probes for labelling nucleic acids

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.

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.

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