Molecular imprinting involves making a binding pocket in a polymer which is chemically and shape specific for the target compound. These "smart plastics" offer robustness compared to biological molecular recognition elements such as antibodies and enzymes. They also have the ability to work in extreme environmental conditions. However, they can sometimes lack the necessary specificity/affinity. Aptamers are small pieces of DNA/RNA that have the ability to target proteins and small molecules and bind to them with high specificity and affinity. They are not toxic and are attractive alternatives to antibodies. They have been used primarily in research due to their susceptibility to enzymatic and chemical degradation, though this is slowly changing and they are becoming commercially relevant. The global aptamers market is projected to reach $2.4 billion by 2020, up from $1.1 billion in 2015. A 12-month proof-of-concept study, supported by the EPSRC and led by the PI (a molecular imprinting specialist), created novel hybrid materials made by incorporating aptamers into molecularly imprinted polymers (MIPs). In simple terms, the aptamer structure is modified to allow it to be directly incorporated into a polymer, so it will hold its shape while being protected from environmental conditions. Novel, high affinity and stable materials were created. These "aptaMIPs" demonstrated exceptional molecular recognition and offer significant improvements on both MIPs and aptamers in terms of stability, and specific target recognition, effectively maintaining the best properties of both classes of materials. This proposal seeks to explore the potential of aptaMIPs through a two year study into the core chemistry used to create these novel materials. We will build on the results of the pilot study and create useful, effective materials with high commercial potential. The research in this proposal will focus on: (i) Identifying the right linker chemistry; (ii) Developing polymerisable modifications for all four bases; (iii) Identifying how many linkers are needed; (iv) Identifying the best position for these linkers. An in-depth study on these four points will enable a full understanding of the key chemistry of how the aptamer incorporates itself into the polymer and, through this, allow us to understand what makes a good aptaMIP and why. Alongside these the synthetic strategies used will be analysed to ensure the creation of these hybrids is simple and effective. Two targets have been selected to study these chemistries. These differ in size and application: a protein and a bioactive drug, but both targets have significant commercial potential. Through these model systems we aim to demonstrate the validity and potential of aptaMIP materials. Alongside the PI, two project partners form the research team: The Watts group were collaborators on the pilot study and are based at the University of Massachusetts RNA Therapeutics Institute (a world leading school in novel aptamer synthesis). They will support the proposal through access to state-of-the-art synthesis equipment, combined with know-how in oligomer synthesis and application. Aptamer Group are a commercial aptamer development company based in York. Their expertise will benefit the project by providing the known oligomer sequences which will act as the basis for our studies and access to specialised instrumentation. The impact of the project will be supported by their detailed knowledge of the aptamer field and commercial outlook. The experience of the whole team will allow this interdisciplinary proposal, covering the fields of polymer, nucleic acid, protein and analytical chemistries to succeed. We will take aptaMIPs from the existing proof-of-concept stage and develop them, and their synthetic process, into viable competitors in artificial molecular recognition, ready for application in systems where their functionality can be exploited.

The static structure of biomacromolecules (proteins, DNA, RNA) defines their function but, under physiological conditions, changes in structure (structural dynamics) are equally important in biological mechanisms. This means that, in order to design molecules that bind to large, flexible biomolecules or which influence the conformations that they adopt in solution, we must have access to accurate structural and dynamic information about the target molecule. Current analytical methods fall into two categories, those that provide detailed structures (X-ray crystallography, cryo-EM, protein-observed NMR), but are time consuming to apply (low throughput) and those that report rapidly on intermolecular interactions but provide little structural insight such as ligand-observed NMR, native mass spectrometry or surface plasmon resonance. A step change in our use of structural and dynamic information is offered by two-dimensional infrared (2D-IR) spectroscopy, which uses a sequence of mid-IR laser pulses to excite molecular vibrations and generate a unique 2D 'map' of the 3D structure, structural dynamics and intermolecular interactions of biological molecules. Crucially, modern laser technology has dramatically shortened the amount of time needed to acquire a 2D-IR spectrum, opening up exciting possibilities for 2D-IR to be used as a high-throughput structure-based screening tool or to probe complex and evolving molecular mixtures in real time. Recently, world-leading research led by York has developed 2D-IR measurements of the structure and dynamics of biological molecules in water (H2O) and biofluids. This invention removes the traditional need for replacement of water with 'heavy water' (D2O) before IR measurements, which is both time consuming and expensive. Moreover, this new ability paves the way to label-free molecular analysis of biofluids without sample drying (Chem Sci, 10, 6448-6456, 2019, Editors' Choice) and 2D-IR protein-drug screening experiments in H2O. We believe that rapid structure/dynamics-based 2D-IR analysis of molecules under physiologically relevant conditions will fill an important gap in our analytical capability, transforming biological chemistry research and providing a new tool for healthcare diagnostics. To exploit this enormous potential, we propose to build a globally unique high throughput 2D-IR instrument at York that can measure microlitre volume samples in under a minute. This new capability will: 1) Advance biomedical diagnostics by quantifying the biomolecular content of biofluids for disease diagnosis without labelling, drying or use of antibodies. 2) Enhance next-generation photonic biosensors by enabling structure-based optimisation of sensor-analyte interactions in biofluids. 3) Deliver enabling technology for chemical biology and drug design via real-time mechanistic insight into molecular synthesis and structure-based screening of candidate molecules binding to proteins and nucleic acids without expensive, laborious replacement of H2O with D2O. 4) Measure structural dynamics of biomolecules and ligands in their native solvent for the first time.

Viral infections pose one of the biggest global threats to human populations and agriculture. Successful prevention, monitoring and treatment of viral infections requires the availability of fast and reliable diagnostic methods which can not only sensitively, but rapidly detect a viral infection of interest and differentiate between viral infections. This is particularly important in the winter months where rapid diagnosis of viral infections emerging from SARS-Cov-2 relative to influenza strains is essential in order to assist medical practitioners to suggest the most appropriate interventions and treatment. At present, methods do not exist which can rapidly detect viral infections in a low-cost, point-of-care device. We propose to develop a biosensing technology which can not only detect viral components, but also has the potential for the platform to be reusable and regeneratable. Central to these developments is the use of fluorous technology as a tool to immobilise elements which detect viral components. Much akin to Teflon, fluorous technology has the dual advantage as a method which can immobilise molecular components which have a complementary fluorous tag, and reduces non-specific binding to non-fluorous biomolecules, thus improving the sensitivity of the approach. Furthermore, the fluorous-directed immobilisation event is inherently reversible by a simple washing step with organic solvent. In this proposal, we will demonstrate the modularity of the strategy to detect viral RNA (by RT-PCR) or protein (by direct detection of intact viral particles). This will provide a powerful new tool for the biosciences which has the potential to be used for any application which requires rapid detection of pathogenic infections.

3D structure is fundamental to the biological function, level of activity and very nature of a protein. Key interactions between the protein and its ligand albeit a small molecule or another protein exploit specific structure. Variations in primary, secondary and tertiary structure can therefore result in significant changes in a protein's behaviour. These changes can range from a simple increase or decrease in enzymatic activity caused by alterations to its structure (caused by the presence of another molecular entity); through to the misfold pathogenesis observed in diseases such as Diabetes and Alzheimer's. Nature has developed control mechanisms to regulate structure/function in many biological systems. The big idea here is that nanoscale polymeric materials with exceptional selectivity, affinity and biocompatibility will act as biomimetics of these control mechanisms and influence protein behaviours. The vision is that these materials will act as role-specific artificial chaperones, opening a new field of bio-inspired materials with a single design process but multiple applications. The proposed programme of research is a unified design approach to the development of these artificial biomimetics using the principle of Molecular Imprinting. Molecular modelling techniques will identify target binding sites alongside compatible polymer components. These simple, elegant biomimetics incorporate binding sites bearing steric and chemical functionality complementary to a given target and as such represent a generic, versatile, scalable, cost-effective approach to the creation of synthetic molecular receptors. They currently are used in separation sciences, purification, sensors and catalysis; but this proposal will broaden their application, allowing the technology to reach its true potential. In activities 1 and 2, nanoscale MIPs including aptaMIPs (nucleic acid-hybrids in which the PI is a leading proponent) will be targeted towards specific binding sites (epitope or larger domain) with the aim to modulate the function of its target. The ability to enhance or inhibit enzymatic activity in relevant environments will be explored, all while building an understanding how these materials interact, and how the composition/target site generates the desired activity. In activities 3 and 4, the ability to guide the folding of protein into specific structures will be explored. By providing MIPs that favour binding a specific shape or conformation, we will look at the creation of misfolds to produce biomaterials for further use (tissue engineering). We will also explore the potential of these materials to reduce or reverse misfolding itself, providing proof-of-concept data for potential future therapeutics. Throughout commercial and clinically relevant targets are used to increase impact of the study, but also to show the power of the developed methodologies. The project will use facilities at DMU, and with an experienced project team, this interdisciplinary proposal which covers protein, polymer and analytical chemistry will take a deep-dive approach to MIP synthesis. It will build on existing proof-of-concept ideas, translating novel synthetic processes into viable options for artificial chaperones which can be exploited in multiple ways. The University of Auckland will host the PI on sabbatical who will study effects of MIPs on folding during this period. The host Dr Laura Domigan, as a visiting researcher, will visit the UK to learn MIP design prior to this, to best support the sabbatical goals. Project partners will support the program throughout, with experience in rational design, sensor application, circular dichroism expertise and folding experience. We will develop the synthetic methods to be scalable through clear step processes, with automation in mind. Potential commercialisation exists through UK based industrial project partners (MIP Diagnostics and Aptamer Group).

Regenerative medicine aims to develop biomaterial and cell-based therapies that restore function to damaged tissues and organs. It is a cornerstone of contemporary and future medicine that needs a multidisciplinary approach. There is a world-wide shortage in scientists with such skillsets, which was highlighted in 2012 by the Research Councils UK in their 'A Strategy for UK Regenerative Medicine" which promotes 'training programmes to build capacity and provide the skills-base needed for the field to flourish'. The major clinical need for regenerative medicine was highlighted by the Science and Technology Committee (House of Lords; July 2013), who identified that 'The UK has the chance to be a leader in [regenerative medicine] and this opportunity must not be missed', and that 'there is likely to be a £44-54bn NHS funding gap by 2022 and that management of chronic disease accounts for around 75% of all UK health costs'. Vascular diseases are the leading cause of death and disability worldwide, musculoskeletal diseases have a huge burden in pain and disability, diabetes may be the 7th leading cause of death by 2030, and peripheral nerve injuries impair mobility after traumatic injuries. There is a pressing need for commercial input into regenerative medicine. Whilst the next generation of therapies, such as stem cells and biomaterials, will be underpinned by cutting-edge biology and bioengineering, strong industrial-academic partnerships are essential for developing and commercialising these advances for clinical benefit. We have established strong industrial partnerships which will both enhance the CDT training experience and provide major added value to our industrial partners. Regenerative medicine is a top priority for the University of Manchester (UoM) which has excellence in interdisciplinary graduate training and a critical mass of internationally renowned researchers, including newly appointed world-leaders. Our regenerative medicine encompasses physical, chemical, biological and medical sciences; we focus on tissue regeneration and inflammation, engineering and fabrication of biomaterials, and in vivo imaging and clinical translation, all on our integrated biomedical campus. We propose a timely Centre for Doctoral Training in Regenerative Medicine in Manchester that draws on our exceptional multidisciplinary depth and breadth, and directly addresses the skills shortage in non-clinical and clinical RM scientists. Our expertise integrates tissue regeneration & repair, the design & engineering of biomaterials, and the clinical translation of both biological and synthetic constructs. Our centres of excellence and internationally-leading supervisors across this multidisciplinary spectrum (details in Case for Support and UoM Letter of Support) highlight the strength of our scientific training environment. Defining CDT features will be: integrated cohort-based multidisciplinary training; skills training in engineering, biomedical sciences and pre-clinical translation; imaging in national Large Facilities; medical problem-solving nature of clinically co-supervised PhD projects, including in vivo training; comprehensive instruction in transferable skills and commercialisation; outward-facing ethos with placements with UK Regenerative Medicine Platform hub partners (UoM is partner on all three funded hubs), industrial partners, and international exchanges with world-class similarly-orientated doctoral schools; presentations in seminars and conferences. In this way, we will deliver a cadre of multidisciplinary scientists to meet the needs of academia and industry, and ensure the UK's continuing international leadership in RM. Ultimately, through training this cadre of doctoral scientists in regenerative medicine, we will be able to improve wound healing, repair injured nerves, blood vessels, tendon and ligaments, treat joint disease and restore function to organs damaged by disease.