Multiplexed Protein Mapping Using Nanopores and DNA Technology for Cancer Risk Stratification

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Cancer is still one of the most challenging diseases which can be inherited to its intra-tumour heterogeneity. Intra-tumour heterogeneity can be defined as the changes within cancer in individual patient. Understanding this heterogeneity can be tackled by detecting different proteins in individual tumour cells to provide a tumour protein profile, which is also known as protein mapping. A promising approach to provide a specific and sensitive mapping of multiple proteins is nanopore sensing. Nanopore sensing is based on resistive pulse sensing, where a nanopore is used to connect two chambers that have electrolyte solution. When a charge is applied, electrolytes move through the nanopore producing a base line current. This force can be used to drive molecules from one chamber to another, which would result in a partial blocking that induces a drop in current. The drop in current over time corresponds to the size and the charge of the molecule. This can be used to map and quantify structures on the DNA. DNA molecule with protein specific binding sites across its length can be used to identify the presence of certain proteins as each of the protein-protein-binding-site complex would result in a drop in current at a specific DNA location. These DNA constructs can be refereed as DNA carriers. Here we intend to design DNA carriers with at least 40 different protein binding sites on each carrier to improve upon current technologies that can identify up to 40 proteins; such as single-cell barcode chips and time-of-flight mass cytometry. To differentiate between each DNA carrier, different DNA structures embedded on one end of each DNA carrier can be used as barcoded to identify the different DNA carriers and their corresponding proteins.The aim of this is to enable mapping of the cancer-specific proteins. This comes with its own challenges, especially the stability of the binding in high salt concentration between the protein binding cites during the nanopore measurements. Stable protein binding will be ensured by the use of DNA-protein crosslinking, aptamer binding, DNA-antibody conjugates, or DNA sequence modification.To ensure the clinical reliability of the data, well-defined lung cancer cells and oesophagus cells from a potential collaborator will be obtained. Established single-cell isolation and recovery techniques in combination with magnetic separation are to be done to recover DNA carrier from individual cells. The analysis of the protein profiles via the DNA carrier will allow for the quantification of cell-specific proteins landscape.The outcome of this project will help in providing a clinically reliable risk stratification biosensing method that can aid in optimizing diagnostics and deciding the best course of treatment or intervention for different cancer types.