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

使用纳米孔和 DNA 技术进行多重蛋白质图谱分析以进行癌症风险分层

• 批准号: 2258738
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2258738
• 关键词: Multiplexed; Protein; Mapping; Using; Nanopores

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.

癌症仍然是最具挑战性的疾病之一，它可以遗传到其肿瘤内的异质性。肿瘤内异质性可以定义为个体患者癌症内的变化。了解这种异质性可以通过检测单个肿瘤细胞中的不同蛋白质来解决，以提供肿瘤蛋白质谱，这也被称为蛋白质图谱。提供多种蛋白质的特异性和灵敏性映射的有前途的方法是纳米孔感测。纳米孔感测基于电阻脉冲感测，其中纳米孔用于连接具有电解质溶液的两个腔室。当施加电荷时，电解质移动通过纳米孔，产生基线电流。这种力可以用来将分子从一个腔室驱动到另一个腔室，这将导致部分阻断，从而引起电流下降。电流随时间的下降对应于分子的大小和电荷。这可以用于绘制和量化DNA上的结构。在其整个长度上具有蛋白质特异性结合位点的DNA分子可用于鉴定某些蛋白质的存在，因为每个蛋白质-蛋白质-结合位点复合物将导致特定DNA位置处的电流下降。这些DNA构建体可以被称为DNA载体。在这里，我们打算设计DNA载体，每个载体上至少有40个不同的蛋白质结合位点，以改进目前可以识别多达40种蛋白质的技术;例如单细胞条形码芯片和飞行时间质谱仪。为了区分每种DNA载体，嵌入在每种DNA载体一端的不同DNA结构可以用作条形码，以识别不同的DNA载体及其相应的蛋白质。其目的是能够绘制癌症特异性蛋白质。这伴随着其自身的挑战，特别是在纳米孔测量期间蛋白质结合位点之间在高盐浓度下的结合的稳定性。通过使用DNA-蛋白质交联、适体结合、DNA-抗体缀合物或DNA序列修饰来确保稳定的蛋白质结合。为了确保数据的临床可靠性，将从潜在合作者获得明确的肺癌细胞和食管细胞。建立单细胞分离和回收技术与磁分离相结合，以从单个细胞中回收DNA载体。通过DNA载体对蛋白质图谱的分析将允许对细胞特异性蛋白质景观进行定量。该项目的结果将有助于提供临床可靠的风险分层生物传感方法，可以帮助优化诊断并决定不同癌症类型的最佳治疗或干预方案。