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（申请人提供）：通过对细胞的复杂调节过程提供机械理解，确定其与疾病的关系，加速蛋白质生物标志物的发现以及协助药物设计，从而在基本和临床生物医学诊断中阐明相互作用网络至关重要。理性膜蛋白设计，化学修饰，生物分子识别和单分子科学的进步将用于创建一种新方法，以在高时间和空间分辨率以及单个蛋白质的检测，探索和表征上样本蛋白质 - 蛋白质相互作用。这些提出的研究旨在工程蛋白纳米孔基于含配体的柔性系数。羟氨酸铁甲酸铁的摄取成分A（FHUA）的充分重新设计，一种具有出色的有利性状的单体B桶蛋白，例如鲁棒性，多功能性和易处理性，将导致独特的纳米结构具有独特的纳米结构，并具有单个有的蛋白质蛋白质DNA dna aptamer ligage tagration aptamer ligander ninan straltain anan ninan ninan nan nnan ninan sate nnan nnan ninan ninan ninan ninan ninan sate。基于FHUA的脚手架是这项任务的一个吸引人的选择，因为它是开放状态，安静的电流在异常广泛的检测环境中长期保持稳定。这些益处将用于各种生物传感方案，其中单个蛋白质 - 蛋白质蛋白和蛋白-DNA识别事件将在可移动束缚的构象动力学中产生可检测，离散和可逆的变化，从而诱导单个通道电签名的改变。预期的即时结果将是以下内容：（i）在平衡和非平衡条件下检查蛋白质 - 蛋白质相互作用的传感元素； （ii）为蛋白质生物标志物的高度特异性基于纳米孔的传感元件的发展； （iii）更好地理解可调和约束系数对蛋白质伴侣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）蛋白质蛋白界面的单分子检测的灵敏度的提高，推动了纳米孔技术以脱离弱蛋白质蛋白质相互作用的范围； （v）工程蛋白纳米孔的模块化和可扩展性的扩展以及它们与合成膜的整合，改善了它们的机械，热，电和化学稳定性。这些不寻常的纳米结构具有可移动臂对综合微型芯片平台的适应，将提供新一代的研究工具，以敏感，特定和定量的方式探索蛋白质蛋白质识别事件的分子基础。