Oligonucleotide (oligo) medicines work by modulating the expression of proteins and the functioning of genes. There are now 9 approved oligo drugs on the market and many more in development, and there is a growing need for an efficient manufacturing technology to make these high value molecules. This project will explore whether a new manufacturing concept for precise polymers, Nanostar Sieving, can be adapted to produce oligo molecules. Nature makes oligos by joining different monomers (nucelotides) in a prescribed sequence. The exact order of the nucleotides is absolutely crucial to the oligo function. Oligos are made industrially by sequential addition of monomers to growing oligos, taking care to remove residual, unreacted monomer before the next cycle, so that there are no errors in the sequence. This requires excellent separation at the end of each coupling cycle. A very effective way of doing this is to attach the growing oligo to a solid support, which is washed with clean solvents to remove residuals, before the next nucleotide is added. When oligo growth is complete, it is cleaved from the solid support. All other side chain protecting groups are then removed, and we proceed to test the purity of the final oligo - have all the required nucleotides been added? Often there are "missing" monomers because the reactions on the solid support did not go to completion, and it is typical to find 60-80% of the desired n-mer oligo, together with a "ladder" of n-1, n-2, n-3 mer shorter oligos which are missing 1, 2, 3 or more nucleotides. The ladder must be removed, and this requires extensive, and expensive, chromatography. Solid Phase Oligo Synthesis is a great tool for rapidly making lots of oligos in the lab, but has drawbacks for manufacturing hundreds of kg or even multi-ton quantities per year. The three major problems are: (i) one cannot know the extent of each reaction easily, because in-line analysis cannot be done on the solid phase; (ii) as the oligo grows, the space for the fresh nucleotides to diffuse in and react gets tight - leading to incomplete couplings and so n-1, n-2 errors; and, (iii) it is hard to scale up the solid beds. Research at Imperial College has pioneered Organic Solvent Nanofiltration (OSN), using membranes that are stable in organic solvents to separate small molecules from large molecules. These membranes have been commercialised, and are manufactured in the UK and employed globally in industries ranging from petrochemicals to pharmaceutical manufacture. Using OSN membranes, we have recently developed a new process, Nanostar Sieving. The key innovation is to use OSN membranes to separate a growing polymer from unreacted monomers. This is carried out in the liquid phase and analysis is relatively straightforward. By connecting three growing polymers to a central hub molecule, we create a large nanostar complex, enhancing membrane retention and promoting efficient separation. We have used Nanostar Sieving to produce PEG, a synthetic polymer used widely for medicines, with unprecedented control over purity. We have not yet been successful at making oligos using Nanostar Sieving, and to do so have to overcome a number of challenges. Here we seek to address these challenges - (i) to improve our membranes with surface modifying ligands; (ii) to use in-line analysis with UV-Vis and 31P NMR to optimise reactions end ensure they reach completion; and (iii) to maintain the solubility of the nanostar complex as the oligos grow in length, without the need for mixed solvents, by developing phosphoramidite monomers with new, solubility-enhancing side chain protecting groups. Our "stretch" goal will be to use the technology to attach targeting moieties to enhance drug delivery. If we are successful, the project will result in a new technology for oligo manufacture, and will lead to purer, and more cost-effective oligos becoming available at scale for applications in healthcare and beyond.

Approximately one in three proteins contains metal, probably the best known example being hemoglobin which binds the transition metal iron as a heme and is responsible for the red colour of blood. Generally a protein will contain one or more transition metals when its task is to pass electrons between other proteins or when its substrate is a small inorganic molecule. One example of a group of these small inorganic molecules is provided by the chemicals which are interconverted in the global nitrogen cycle. These includes the well known series of nitrate, nitrite, nitric oxide, nitrous oxide, nitrogen and ammonia. The dioxygen molecule which we breath in to live is another example. We may be interested in proteins extracted from soil bacteria which interconvert chemicals of the nitrogen cycle or we may study the cytochrome oxidase protein which binds the oxygen we breath and reduces it to water using electrons from our food. But in any case where a transition metal is involved we need specialised techniques to investigate how they work. One method to probe the nature of a metal site in a protein is to measure the absorption of light using electronic absorption spectroscopy. Obviously we can do this because the metals make the protein coloured. But the metals can also make the protein magnetic. If they are coloured AND magnetic then this allows us to use a technique called Magnetic Circular Dichroism spectroscopy, discovered by the British chemist Michael Faraday in 1845. This is similar to absorption spectroscopy but uses special polarised light and a strong magnetic field. In order to produce the strong magnetic field we use a superconducting magnet.

This strategic equipment proposal is for a new class of instrument, a Bionanofabrication suite, to link together the worlds of precision devices with that of biomolecules and drugs to make new classes of biomedical devices. The world of electronic equipment has been revolutionised by the precise fabrication techniques of the semiconductor industry. In the past electrical circuits were hand assembled from discrete highly variable electrical components. The advent of microfabrication techniques first enabled the robust combinations of components to make integrated devices such as transistors and amplifiers. Continued development of processes has led to the current advanced state where we each carry a super-computer in our pockets - we just call it a mobile phone. This proposal seeks to enable the same transformation for biomedical measurement and therapy delivery devices. From the patient perspective, the devices used to measure molecular biomarkers of disease or injury are largely unchanged over the last 20 years. Blood or other body fluid samples are taken, processed in a central laboratory or maybe in the ward and the results logged in a chart. Similarly, drugs are delivered by mouth or by venous injection. Ultimately, even in intensive care measurements are made on an hourly basis. We will develop technologies to build new biomedical / bioelectronics devices that measure from cells and tissue continuously, or target therapy in a controlled way at the site of action. Potentially, we can envisage implantable devices that deliver therapy in response to the tissue signals measured by the device. This would allow truly individualised therapy. The atom-based building and etching instruments that have been continuously refined by the modern semiconductor industry can also be used to make the sensing surfaces, channels and detectors required for a measurement device. However, current manufacturing processes run at high temperatures with a very limited range of silicon-based materials. The Bionanofabrication suite will bring together for the first time, in the same instrument, atom-based building and etching processes that are capable of running at room temperature with a wide range of final surface chemistries. The Bionanofabrication suite will operate within a quality management system, addressing an important hurdle for the early clinical testing of new medical devices. Devices that are subsequently shown to be successful clinically, could be put into production using the fabrication techniques developed within this grant without the need for changing production methods.

The FIB/SEM instrument proposed combines various components to provide a powerful tool for a range of advanced nanoscale science. An accelerated ion beam focused to a spot size as small as 5 nanometres can be used to mill and slice materials with extreme precision, while an electron beam and various detectors provide means for nanoscale imaging and characterisation of the surfaces produced. A nanomanipulator probe allows samples to be rotated in-situ and for nanoscale slices of material to be lifted out for further study or use in devices. We will use this instrument in two main ways: 1) Fabrication of micro-optical components In Oxford we have in the past six years pioneered the use of focused ion beams to fabricate surfaces on materials such as fused silica or silicon with nanometre precision and sub-nanometre roughness. This allows us to create devices in which light is stored and manipulated with ultra-low scattering losses, and in which the interaction between light and matter is controlled with exquisite accuracy. We have already had considerable success with this technique on a small scale but are limited in the size of features we can produce. In this new instrument the sample can be moved with extremely high accuracy allowing larger surfaces to be patterned and enabling more complex and extended optical devices that reveal new physics and can be used as key components in a range of technologies. Photonics underpins a diverse range of industry in the UK and we anticipate that our work will lead to innovations in advanced information technologies and sensor systems for defence, healthcare and environmental monitoring, as well as the new field of Quantum Technologies in which the government is currently investing significant resources. 2) Characterisation of Materials Oxford Materials department has long been a world-leading centre for materials characterisation, with particular contributions in electron microscopy and the microstructure of metals. It maintains a wide range of state-of-the-art instruments that are used both as high end scientific tools and as platforms for developing new techniques in microanalysis. This instrument will be used in both ways. It offers leading edge capabilities is 3D characterisation of material defects and impurities at the nanoscale that will enable new techniques aimed at understanding materials with unprecedented detail, and will be applied to solving key problems in the fields of nuclear materials, aerospace alloys, catalysis, and high temperature superconductors. Many of these projects are carried out in collaboration with industry, providing excellent routes towards commercial and societal impact as well as development of new knowledge. In collaboration with a local company (Oxford Instruments) we will try out prototype detector systems to accelerate instrument development and maintain our position at the forefront of this important field. As well as the projects described above, a percentage of time on the instrument will be made available to outside users who will be able to find out about the instrument via our website and annual open days, and apply for instrument time to carry out their own research. The Oxford Materials department has extensive instrument support and user training programmes to ensure that all users can obtain the best from their instrument time. To ensure that the scientific projects pursued are of the highest quality, the use of the instrument and time allocation will be carried out by a steering board of experts who will meet at quarterly intervals.

Oligonucleotide (oligo) medicines work by modulating the expression of proteins and the functioning of genes. There are now 9 approved oligo drugs on the market and many more in development, and there is a growing need for an efficient manufacturing technology to make these high value molecules. This project will explore whether a new manufacturing concept for precise polymers, Nanostar Sieving, can be adapted to produce oligo molecules. Nature makes oligos by joining different monomers (nucelotides) in a prescribed sequence. The exact order of the nucleotides is absolutely crucial to the oligo function. Oligos are made industrially by sequential addition of monomers to growing oligos, taking care to remove residual, unreacted monomer before the next cycle, so that there are no errors in the sequence. This requires excellent separation at the end of each coupling cycle. A very effective way of doing this is to attach the growing oligo to a solid support, which is washed with clean solvents to remove residuals, before the next nucleotide is added. When oligo growth is complete, it is cleaved from the solid support. All other side chain protecting groups are then removed, and we proceed to test the purity of the final oligo - have all the required nucleotides been added? Often there are "missing" monomers because the reactions on the solid support did not go to completion, and it is typical to find 60-80% of the desired n-mer oligo, together with a "ladder" of n-1, n-2, n-3 mer shorter oligos which are missing 1, 2, 3 or more nucleotides. The ladder must be removed, and this requires extensive, and expensive, chromatography. Solid Phase Oligo Synthesis is a great tool for rapidly making lots of oligos in the lab, but has drawbacks for manufacturing hundreds of kg or even multi-ton quantities per year. The three major problems are: (i) one cannot know the extent of each reaction easily, because in-line analysis cannot be done on the solid phase; (ii) as the oligo grows, the space for the fresh nucleotides to diffuse in and react gets tight - leading to incomplete couplings and so n-1, n-2 errors; and, (iii) it is hard to scale up the solid beds. Research at Imperial College has pioneered Organic Solvent Nanofiltration (OSN), using membranes that are stable in organic solvents to separate small molecules from large molecules. These membranes have been commercialised, and are manufactured in the UK and employed globally in industries ranging from petrochemicals to pharmaceutical manufacture. Using OSN membranes, we have recently developed a new process, Nanostar Sieving. The key innovation is to use OSN membranes to separate a growing polymer from unreacted monomers. This is carried out in the liquid phase and analysis is relatively straightforward. By connecting three growing polymers to a central hub molecule, we create a large nanostar complex, enhancing membrane retention and promoting efficient separation. We have used Nanostar Sieving to produce PEG, a synthetic polymer used widely for medicines, with unprecedented control over purity. We have not yet been successful at making oligos using Nanostar Sieving, and to do so have to overcome a number of challenges. Here we seek to address these challenges - (i) to improve our membranes with surface modifying ligands; (ii) to use in-line analysis with UV-Vis and 31P NMR to optimise reactions end ensure they reach completion; and (iii) to maintain the solubility of the nanostar complex as the oligos grow in length, without the need for mixed solvents, by developing phosphoramidite monomers with new, solubility-enhancing side chain protecting groups. Our "stretch" goal will be to use the technology to attach targeting moieties to enhance drug delivery. If we are successful, the project will result in a new technology for oligo manufacture, and will lead to purer, and more cost-effective oligos becoming available at scale for applications in healthcare and beyond.