Multiplex Bioorthogonal Labelling of Nucleic Acids: A Tool for Super-Resolution Imaging

Understanding how mammalian cells function in normal development and disease is central to life science research, and is necessary to discover new drugs and treatments. Many biological systems are extremely complex and comprise many different components. To dissect the individual parts of this puzzle it is necessary to use specific labels that can be used to mark individual components. Over the last 30-years many different approaches have been used for labelling specific parts of the puzzle, often in different colours. Some of the key factors in a cell are the individual components made of proteins and analogous to the parts of a car. These different proteins can be labelled with specific reagents called antibodies that can be tagged in different colours and visualised by microscopy to explore how they relate to each other in 3D space. Similarly this approach can be used on a molecular level to ask if these components interact with each other. There are very good tools for analysing proteins, in contrast it is much more difficult to label DNA and RNA in cells. DNA encodes our genetic information and is carefully folded with proteins to protect it from damage and to regulate how the information is accessed. To make proteins cells copy the information stored in DNA into a temporary code called RNA. In turn RNA is used as a recipe to make proteins. Together RNA and DNA are described as nucleic acids and although these are critical components it is difficult to separately label them, as they look very similar. We have recently shown that in cells, DNA is packaged with RNA and proteins to make a mesh, similar to a fishing net, that is important for regulating how cells function, but we realised that we lacked the tools to investigate this relationship. Understanding these interactions is crucial as one of the proteins for making the mesh is often mutated in cancer, and genetic mutations in this protein cause developmental defects in children. Therefore, to understand how proteins, DNA and RNA work together we will develop a new palette of reagents that will allow us to mark these different components in cells in different colours simultaneously. This multiplex approach will be applicable to many different studies but will critically enable use to understand how individual components work together in regulating DNA and how in disease this can go wrong. To dissect the complex workings of life it is necessary to use innovative methods. To achieve our goals we have assembled a team of chemists and molecular biologists who together can make new tools for understanding how life works.

Molecular labelling reagents are important for marking cellular components to understand how they interact and regulate each other and how this is altered in disease. To date antibodies and protein tags are the reagents of choice for labelling proteins; in contrast fewer methods are available for labelling nucleic acids. This is particularly important for studying interactions within the nucleus using advanced imaging techniques such as super resolution microscopy where multiplex labelling reagents are required. The reaction between an azide and an alkyne, typically coined "click chemistry" is widely used for tagging DNA or RNA, separately. In this approach a modified nucleoside (with an azide or alkyne handle) is metabolically incorporated into the synthesised nucleic acid and this is subsequently labelled with a coupling partner, conjugated to a fluorophore. To complement this method we will develop a novel nucleoside analogue for labelling nucleic acids, within cells, that can be used combinatorially with azide/alkyne "click chemistry" to enable multiplex labelling of DNA and RNA, simultaneously. The approach is based on the inverse electron demand Diels-Alder addition between a strained-alkene and a tetrazine, a recently exploited bioorthogonal reaction that has fast kinetics, excellent orthogonality and biocompatibility. This reaction method will enable simultaneous multiplex labelling of DNA, RNA or other biological molecules. To demonstrate the utility of this approach we will analyse the distribution of an abundant nuclear protein SAF-A (scaffold attachment factor A) with DNA and RNA in mammalian cells, using super resolution microscopy. This proposal brings together chemists and molecular biologists to develop a new palette of tools for labelling nucleic acids and other biological molecules and showcases their use to investigate physiologically relevant molecular interactions in human cells.

In this proposal we will generate a novel tool for labelling nucleosides in a bioorthogonal reaction. This method will work in combination with already available reagents to enable multiplex labelling of nucleotides and biological molecules. To achieve this we will synthesise novel nucleoside analogues with strained-alkene handles that will use an inverse electron demand Diels-Alder reaction to couple strained-alkenes to tetrazines, "tetrazine ligation", in a bioorthogonal reaction. The novel nucleosides and "tetrazine ligation" will work in combination with the already widely used azide/alkyne "click chemistry" reaction and will be useful to academics and research scientists who need to combinatorially tag biological components. This will enable new experiments to be undertaken which in turn will lead to new discoveries. In the short term we also plan to make the new tools available both through disseminating information via publications and presentations but also to interest commercial suppliers to help distribute the reagents. The clear benefits of encouraging industrial companies is their ability to help develop reagents further and to provide a clear avenue for their commercialisation.

Longer term benefits will come from new scientific discoveries. As an example, related to this proposal, we will analyse how SAF-A, a highly abundant nuclear protein, interacts with chromatin associated RNA and DNA. This is relevant as we have shown that the interaction between SAF-A / RNA regulates chromatin structure and is important for genome stability. We have recently found that SAF-A is frequently mutated in cancer and we believe this will change its properties. Furthermore SAF-A has been shown to be mutated in children with developmental neurological disorders; the molecular basis for this is unknown but this is an exciting area of future research. Understanding the molecular basis of this disease will give families better understanding whilst learning about the cancer mutations might open avenues for future treatments. In addition to these examples we anticipate there will be many other projects that will benefit from the new tools that we will develop in this proposal. We also predict that development of these new tools will give rise to future research projects where the tools can either be developed further for variant approaches or can be used directly in new experimental studies. Potential new uses are the bioconjugation of eosin dyes to cellular components for subsequent electron microscopy analysis or the attachment of combinatorial reactive reagents to both proteins and RNA or DNA. Cross-linkers can then be used to join the two together to ask how they relate to each other. We envisage this could be highly beneficial to identify the relationship between proteins and RNAs at centromeres, a critical component of the chromosome important for cell division, where aberrant function leads to aneuploidy and genome instability.