Stochastic sensing with nanopores is a versatile technology that can be used for the recognition and quantification of a wide range of substances (known as analytes) through the detection of individual molecules. Our partner company, Oxford Nanopore Technologies, has been incorporating stochastic sensing into next-generation hand-held devices. The most highly developed application at Oxford Nanopore is cheap, extremely rapid DNA sequencing, which promises to revolutionise numerous areas of biology including aspects of medicine, ancestry and forensics. Currently, a portable sequencer is being tested at hundreds of sites worldwide. In stochastic sensing, analytes are detected as they enter and leave a single narrow pore perturbing a current that flows through it. The diameters of the pores, known as nanopores, are similar to those of a small molecule, about one-fifty thousandth of the diameter of a human hair, providing the basis for detection by current perturbation. Typically current changes of the order of one trillionth of an ampere are measured. Analytes have included drug molecules and small molecules found in the body that act as markers for disease. In the case of DNA sequencing, individual bases are detected as an extended DNA strand is threaded through a nanopore. Protein pores are advantageous for stochastic sensing, because they can be modified for particular applications with atomic precision and prepared in near homogeneous form. Until now, very narrow protein pores have been used and therefore stochastic sensing has been limited to analytes of small size or, in the case of DNA, to extended polymer chains. In the proposed work, we will endeavour to make a new class of functional nanopores, DNA-Protein hybrid nanopores. These pores will be constructed from folded DNA, known as DNA origami, and protein components. The DNA will act as a scaffold for the protein, ensuring that the new pores are up to fifteen times larger in internal diameter than the pores used before. Further, each pore will be of identical size and no incompletes pores will be present, a goal that has not be achieved previously. Finally, it will be possible to modify the new pores at precisely determined sites, which cannot be done with competing technologies, such as solid-state pores. The DNA-protein hybrid nanopores will enable a critical step forward for stochastic sensing by allowing the detection of a wide range of large biological molecules that can enter the pores, including proteins, DNAs and polymeric sugars. Conversely, it will also be possible to lodge these large molecules within the hybrid pores, where they will act as binding sites for a variety of additional analytes. In a futuristic application, it may prove possible to sequence double-stranded DNAs with hybrid pores, which will provide a significant advantage over the manipulations currently required for nanopore sequencing. Our industrial partner, Oxford Nanopore, will evaluate and test our most promising DNA-protein hybrid nanopores in their hand-held sensing devices, which are capable of monitoring the outputs of hundreds of pores in parallel, offering the prospect of step changes in sensing technology in areas including biological warfare defense, food authentication, plant and animal breeding and medical diagnostics.

The overall aim of this project in to use a combination of in silico (biomolecular simulation) and experimental methods to explore bionanopore function via biomimetic design. This work, which will building on previous and existing collaborations between the Bayley and Sansom research groups, is of considerable importance to rational design of novel bionanopores for use in direct, electrical detection and analysis of single molecules, the latter being the primary aim of Oxford Nanopore Technologies. Previous studies of relevance have included: 1. Simulation studies of model nanopores in lipid bilayers, focussing on protein/lipid lipid interactions and on nanopore clustering (1). 2. Combined computational and experimental study of nanopore mutations and perturbation of transmembrane beta-barrel stability. 3. In silico electrophysiology via direct simulations of ion and water fluxes through alpha-hemolysin pores. 4. Free energy profiles for porins, enabling a quantitative comparison of phosphate and chloride interactions with the OprP pore (2). 5. Design of biomimetic nanopores based on OprP, exploring the importance of sidechain flexibility in smoothing free energy landscapes for permeant ions (Pongprayoon et al., ms. in preparation). Ongoing studies include: 1. Exploration of the mechanism and energetics of porins for hydrophobic/aromatic solutes. Systems being studied include OpdK (selective for vanillate; Pongprayoon et al., ms. in preparation); and TodX (selective for toluene and its derivatives) The current project will exploit and extend these studies in a more synthetic fashion. Specifically, the project will involve: 1. Analysis of existing bionanopores: a semi-quantitative survey of the relationship between structure and function (solute specificity) in bacterial outer membrane proteins (OMPs) using electrostatics and related analytical methods based on lessons learned from the above studies. 2. De novo design of nanopore functionality: using the free energy profile and related methods developed in the studies of biomimetic nanopores based on OprP. 3. Design of existing and novel selectivity functionality into existing templates (e.g. OmpG (3), alpha-hemolysin). 4. Combine our understanding of selectivity for phosphate (OprP) and for aromatics (OpdK; TodX) to design a pore selective for DNA and related polymers. 5. Focus on minimum requirements for single stranded DNA recognition which are consistent with optimal DNA transport rates. 6. Use our understanding of OmpG and of stability of 'featureless' beta-barrels to combine these requirements with a stable nanopore template. 7. Experimental evaluation of design and refinement by combined simulation and experimental studies. References (1) Klingelhoefer, J., Carpenter, T., and Sansom, M. S. P. (2009) Peptide nanopores and lipid bilayers: Interactions by coarse-grained molecular dynamics simulations. Biophys. J. 96, 3519-3528. (2) Pongprayoon, P., Beckstein, O., Wee, C. L., and Sansom, M. S. P. (2009) Simulations of anion transport through OprP reveal the molecular basis for high affinity and selectivity for phosphate. Proc. Natl. Acad. Sci. USA 106, 21614-21618. (3) Chen, M., Khalid, S., Sansom, M. S. P., and Bayley, H. (2008) Outer membrane protein G: engineering a quiet pore for biosensing. Proc. Natl. Acad. Sci. USA 105, 6272-6277.

Determining antibody levels in humans is crucial for monitoring immunity against Covid-19 and tackling the national crisis. Antibody levels report about a previous infection and help decide whether people can return to work or live with others without spreading the disease. To be effective for national screening, antibody testing should deliver accurate results to individuals ideally within minutes, and be portable as well as high-throughput. Existing techniques based on immunosorbent assays do not deliver these benefits due to the need for multiple liquid handling steps, signal amplification, insufficient accuracy, or read-out with bulky optical equipment. This project will deliver fast, portable, high-throughput and accurate antibody sensing by pioneering step-changing sensor nanopores from the lead PI at University College London Chemistry (UCLC), and by integrating them into memory-stick-sized on-the-market kits from industrial partner and biotech unicorn Oxford Nanopore Technologies (ONT). These MinION analysis kits have ushered in a revolution in portable DNA sequencing and are currently used for unravelling the Covid-19 sequence. The PI has a strong working relationship with the company and has licensed sequencing pore technology which has been one key component to make the MinION a success. In this project, the technology will be adapted with wider nanopores tailored for Covid- 19 antibodies. The new sensor pores can be plugged into the existing MinION kits without the need for redesigning the device, thereby ensuring production to scale. The devices will be clinically tested and benchmarked by Co-PI and intensive care and monitoring specialist Prof. Mervyn Singer at UCL Medicine (UCLM). All project partners have previously successfully worked together in joint grants, for publications, or via technology licensing contracts.

The Challenge: Schizophrenia (SZ), bipolar disorder (BD), and schizoaffective disorder (SAD) are core severe mental illnesses (SMIs) and place an enormous burden on individuals, their carers and wider society. They account for more than a quarter of the ~£118Bn annual UK costs of mental illness and are associated with a reduction in life expectancy similar to some of the most serious chronic illnesses in medicine. Yet, unlike other areas of medicine, no truly novel treatments have been developed for SMIs in decades and consequently real-world outcomes and recovery rates are little changed since the mid-twentieth century. The fundamental cause for this stasis is a lack of causal understanding. Consequently, we are unable to classify patients according to the biological, psychological or social causes of their symptoms, or in any way that is informed by why they have developed their condition which hampers the discovery of new mechanistically informed treatments. Instead, we rely on symptom-based diagnoses that show poor correspondence to underlying physiology. This approach has been unfavourably compared to cancer treatment prior to precision treatments where non-specific, modestly effective treatments, with severe side effects, were selected based largely on the tissue origin rather than patient-specific cancer profiling. There must me another way. Our vision is that by combining rich phenotypic data acquired at scale across multiple domains (e.g. clinical, cognitive, developmental, immune and metabolic, genomic and brain imaging) with sophisticated analysis, we will be able to move towards a new causally and mechanistically informed diagnostic approach for SMIs that will advance psychiatry and improve the lives of people with these disorders. To achieve this, we have created an outstanding interdisciplinary network of early-, mid- and senior-career researchers and people with lived experience from South Wales (Cardiff Swansea) and South-West England (Bath, Bristol, Exeter). Drawing on our world-leading cohorts of more than 12,000 patients with BD-SAD-SZ our SW2 hub will build an unprecedentedly detailed cohort of 600 representative participants. We will also access the Welsh SAIL Databank to incorporate rich environmental and developmental data, interrogate the biological correlates of these factors with our world-leading expertise in advanced epigenetic. Drawing on our expertise in machine-learning and big data analysis, we will then apply advanced analyses improve how we group patients across the SZ-BD spectrum. Underpinned by the application of cutting-edge genomics we will access the dark genome for the first time at scale in severe mental illness. Sharing of data and availability of replication cohorts has been critical to the success of psychiatric genetics and will be essential to the wider application of machine-learning models. The diverse range of phenotypic data generated and curated by the SW2 Hub will be made openly available to the academic community providing a unique data resource of exceptional value to international researchers to develop new methods, for replication of results and combining of datasets for more powerful outputs that will inform better ways of diagnosing people. These programs of work will create a rich resource for the field that will support training and the delivery of excellent research as a key facet of the UKRI Mental Health Research Platform but more importantly will offer a route to advancing psychiatry and improving the lives of people with some of our most serious mental illnesses.

Proteins are of paramount importance in our lives. They carry out the main functions in our bodies and control our movement, energy conversion, immune defence, and thinking. In medicine, functionally aberrant proteins cause disease. The proteins can, however, be targeted by drugs to cure cells. Enzymes are also of biotechnological importance in the cost-efficient synthesis of drug molecules or for the energy-saving cleaning of fabrics. Detecting and analysing proteins is the first step towards predicting diseases, developing cures, and engineering proteins for industry. The analysis of proteins improves our understanding their structure, dynamics, and function. Ideally, sensing of proteins should be simple and fast and be conducted using inexpensive and portable equipment. This increase research efficiency and opens up point-of-care sensing in diagnosis and homeland security. This project will provide a new way to sense proteins in a portable yet scientifically accurate fashion thereby overcoming problems of existing approaches. Classical approaches have issues such as the requirement to label the proteins with a fluorescent tag which can interfere with the structure and function of the proteins. Optical detection can also increase the weight and cost of the analytical device. Another limitation of conventional sensing approaches is that they average over millions of molecules and have difficulties to detect biologically important sub-groups. We will develop a new approach to sense proteins in a label and optics-free electrical fashion using portable equipment capable of uncovering proteins down to the level of individual molecules. The proposed strategy is currently being used for DNA strand sequencing. Our industry partner Oxford Nanopore Technologies has developed a hand-held device for genome sequencing. The analytes are detected when individual strands pass through nanoscale pores in a thin membrane. The temporary blockade of the pores alters the electronic read-out signal similar to the reduction of water flow when a stone is inside a tube. We will be able to sense proteins which are wide enough to accommodate proteins. The new pores will be composed of DNA stands, thereby exploiting the exquisite ability of DNA to function as a nanoscale construction material. Chemical modification will be key to achieve the functional performance of the pores. To maximise the benefit for academia, industry and society, we will strongly collaborate with our commercial partner to test the new pores in the portable electrical sensing devices.