Cells are packed with DNA and RNA, but existing methods to map which proteins and molecules interact with these nucleic acids are too imprecise or inefficient to catch the most fleeting or fragile connections. This project aims to build a new tool—a photocatalytic system that, when activated by blue light, generates short-lived chemical intermediates capable of labelling DNA and RNA within a few nanometres of a target. Current proximity labelling techniques either label over hundreds of nanometres, blurring true interactions, or fail to tag nucleotides efficiently. The researchers will design and test precursor molecules and a photocatalyst, first on synthetic DNA and RNA in the lab, then inside living cells. If successful, the technology could offer 100-fold greater precision than existing nucleotide labelling methods, enabling super-resolved interaction maps and high-resolution imaging in both fixed and live cells. This is fundamental science: it does not directly treat a disease or improve a product. But better maps of how nucleic acids interact with other molecules could, in the long run, reveal new drug targets or illuminate basic mechanisms of gene regulation and cellular function.
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Interactions between biomolecules sustain life as we know it. Accordingly, the spatiotemporal coordination of biomolecular interactions is critical to all biological processes. Methods to precisely measure biomolecular interactions are thus critical to the entirety of all biological research. Historically, biomolecular interactions have been studied by isolating molecules of interest and looking at what else was present. In the ‘omics-era’ of modern biology, this could be scaled up and combined with unbiased screening platforms to profile the interaction networks of isolated biomolecules at high throughput. These approaches, however, struggle to identify interactions that are either transient or labile (i.e. destroyed upon preparation of the sample). Proximity labelling technologies offer a solution by labelling nearby ‘interactors’ that can be detected in downstream analyses. Most rely on enzymes that convert inert small molecule probes into free radical intermediates that, via diffusion, bind promiscuously to nearby biomolecules. The probes contain a conjugation handle that can be used for identification, for example, biotin, which enables enrichment with streptavidin. These technologies have facilitated discoveries in various aspects of cell biology but suffer from poor resolution, labelling molecules over hundreds of nanometres. This prevents such technologies identifying bona fide interactors. The nature of the reactive intermediate dictates both the spatial resolution of proximity labelling and the types of biomolecules that are labelled. Recent advances employ photocatalytic approaches to generate localised carbene intermediates with extremely short half-lives, transforming labelling by bringing it within nanoscale proximities (~4 nm) enabling the precise identification of interactors. This ‘super-resolution’ approach, termed µMap, has defined targets of immunotherapies and small molecule-protein interactions with unparalleled precision. Although these technologies have transformed proximity proteomics, they do not efficiently label other biomolecules, such as nucleotides. Moreover, current nucleotide proximity labelling technologies also suffer from poor labelling efficiency, requiring large amounts of starting material. Nucleic acid (DNA and RNA) interactions are as ubiquitous in biology as protein-protein interactions and improvements to technologies that enable efficient and precise nucleotide proximity labelling would be of great importance, value, and interest. Here, we will develop a novel nucleotide labelling technology based on the photocatalytic generation of new nucleotide-targeting reactive intermediates that can label DNA and RNA at nanoscale distances. We will design and examine a range of precursor molecules that can be activated by a photocatalyst, adapting µMap technology for nucleotide labelling. We will undertake the development of both the photocatalyst and nucleotide-targeting probes, optimising their nucleotide proximity labelling potential. We will test our technology in vitro on synthetic DNA and RNA constructs, allowing us to establish both the efficiency and resolution of our labelling technology. We will then showcase its nanoscopic labelling potential in cells by demonstrating DNA/RNA labelling compared to the current state-of-the-art approaches. Our technology could provide 100-fold greater precision than current nucleotide proximity labelling approaches. Our technology will also be modular, so that it can be used for numerous downstream applications. Consequently, our technology could i) enable precise, efficient and sensitive nucleotide labelling to generate super-resolved nucleotide interaction maps, ii) enable precise nucleotide labelling for high-resolution imaging (electron or light microscopy; in both fixed or live cells), iii) enable the first spatially restricted labelling of nucleotides (due to reactive intermediate generation being dependent on targeted blue light irradiation), and iv) enable simultaneous super-resolution protein and nucleotide proximity labelling, in combination with µMap.
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