Engineered Nanopores for Single-Molecule Stochastic Sensing

用于单分子随机传感的工程纳米孔

• 批准号: 8760824
• 负责人: LIVIU MOVILEANU
• 金额: $ 28.64万
• 依托单位: SYRACUSE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-28 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760824
• 关键词: Affinity; Applications Grants; Area; Attention; Bacillus amyloliquefaciens barstar protein; Bacillus amyloliquefaciens ribonuclease; Binding; Binding Proteins; Biological Markers; Biosensing Techniques; Biosensor; Cancer Detection; Cell physiology; Characteristics; Chemicals; Chemistry; Clinical; Complex; Coupled; DNA; Detection; Development; Devices; Diagnostic; Dimensions; Disease; Drug Design; Elements; Engineering; Environmental Monitoring; Equilibrium; Event; Exhibits; Generations; Generic Drugs; Genetic Engineering; Genomics; Goals; Hybrids; Individual; Kinetics; Knowledge; Ligands; Mechanics; Membrane; Membrane Proteins; Methodology; Modification; Molecular; Nanostructures; Nature; Outcome; Pharmaceutical Preparations; Phase; Positioning Attribute; Process; Property; Prostate-Specific Antigen; Protein Analysis; Protein Engineering; Proteins; Proteomics; Research; Resolution; Ribonucleases; Sampling; Scaffolding Protein; Scheme; Science; Sensitivity and Specificity; Specificity; Staging; Structure; Technology; Tertiary Protein Structure; Thermodynamics; Time; Work; aptamer; aqueous; arm; base; chemical stability; design; detector; flexibility; hydroxamate; improved; in vivo; inhibitor/antagonist; nanodevice; nanopore; nanoscale; outcome forecast; protein complex; protein protein interaction; public health relevance; response; scaffold; sensor; single molecule; tool; trait; uptake

DESCRIPTION (provided by applicant): Unraveling the interaction networks among functional proteins is essential in fundamental and clinical biomedical diagnostics by providing a mechanistic understanding of the complex regulatory processes of the cell, identifying their relationships to diseases, accelerating protein biomarker discovery, and assisting drug design. Advances in rational membrane protein design, chemical modification, biomolecular recognition, and single-molecule science will be used in concert for the creation of a new methodology to sample protein-protein interactions at high temporal and spatial resolution, as well as for the detection, exploration, and characterization of individual proteins. These proposed studies are aimed at engineering protein nanopore- based sensing devices featured by ligand-containing flexible tethers. Ample redesign of ferric hydroxamate uptake component A (FhuA), a monomeric b-barrel protein with a remarkable array of advantageous traits, such as robustness, versatility, and tractability, will result in a unique nanostructure with a single tethered proteinor DNA aptamer ligand at a strategic positioning of the nanopore. The FhuA-based scaffold is an attractive choice for this task, because it's open-state, quiet current remains stable for long periods within an unusually broad range of detection circumstances. These benefits will be used in various biosensing schemes, in which individual protein-protein and protein-DNA recognition events will produce detectable, discrete and reversible changes in the conformational dynamics of the movable tether, inducing alterations in the single- channel electrical signature. The expected immediate outcomes will be the following: (i) the creation of sensing elements for examining protein-protein interactions under equilibrium and non-equilibrium conditions; (ii) the development of highly specific nanopore-based sensing elements for a protein biomarker; (iii) a better understanding of the impact of tunable and constraining tethers on the intermolecular forces among protein partners, which has implications for the in vivo contexts of complex recognition events produced by anchored protein domains; (iv) the improvement in the sensitivity of the single-molecule detection of protein-protein interfaces, pushing forward the nanopore technology for the disentanglement of weak protein-protein interactions; (v) the expansion of the modularity and scalability of engineered protein nanopores as well as their integration with a synthetic membrane, improving their mechanical, thermal, electrical, and chemical stability. The adaptation of these unusual nanostructures with movable arms to an integrated microfabricated chip platform will provide a new generation of research tools for exploring the molecular basis of protein-protein recognition events in a sensitive, specific and quantitative fashion.

描述（由申请人提供）：通过提供对细胞复杂调节过程的机械理解、确定其与疾病的关系、加速蛋白质生物标志物发现并协助药物，阐明功能蛋白之间的相互作用网络对于基础和临床生物医学诊断至关重要设计。合理膜蛋白设计、化学修饰、生物分子识别和单分子科学方面的进步将共同用于创建一种新的方法，以高时间和空间分辨率对蛋白质-蛋白质相互作用进行采样，并用于检测、单个蛋白质的探索和表征。这些拟议的研究旨在工程化基于蛋白质纳米孔的传感装置，其特征在于含有配体的柔性系链。异羟肟酸铁吸收成分 A (FhuA) 是一种单体 b 桶蛋白，具有一系列显着的优势特性，例如稳健性、多功能性和易处理性，对异羟肟酸铁吸收成分 A (FhuA) 进行充分重新设计，将产生具有单一束缚蛋白或 DNA 适体配体的独特纳米结构纳米孔的战略定位。基于 FhuA 的支架是这项任务的一个有吸引力的选择，因为它处于开放状态，安静电流在异常广泛的检测环境下长时间保持稳定。这些优点将用于各种生物传感方案，其中单个蛋白质-蛋白质和蛋白质-DNA 识别事件将在可移动系绳的构象动力学中产生可检测的、离散的和可逆的变化，从而引起单通道电特征的改变。预期的直接成果如下：（i）创建用于检查平衡和非平衡条件下蛋白质-蛋白质相互作用的传感元件； (ii) 开发用于蛋白质生物标志物的高度特异性的基于纳米孔的传感元件； (iii) 更好地了解可调和约束系链对蛋白质伙伴之间分子间力的影响，这对锚定蛋白质结构域产生的复杂识别事件的体内环境具有影响； (iv) 提高蛋白质-蛋白质界面单分子检测的灵敏度，推动纳米孔技术解开弱蛋白质-蛋白质相互作用； (v) 工程蛋白质纳米孔的模块化和可扩展性的扩展以及它们与合成膜的集成，提高它们的机械、热、电和化学稳定性。将这些具有可移动臂的不寻常纳米结构适应集成微加工芯片平台将为以灵敏、特异和定量的方式探索蛋白质-蛋白质识别事件的分子基础提供新一代研究工具。