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: 8136461
• 负责人: LIVIU MOVILEANU
• 金额: $ 28.04万
• 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 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8136461
• 关键词: Anions; Antibodies; Bacillus amyloliquefaciens ribonuclease; Bacterial Outer Membrane Proteins; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biological Response Modifier Therapy; Biopolymers; Biosensing Techniques; Biosensor; Biotechnology; Caliber; Characteristics; Charge; Complex; Data; Detection; Development; Devices; Diagnostic; Discrimination; Disulfides; Electrostatics; Engineering; Environmental Monitoring; Event; Exhibits; Experimental Designs; Foundations; Generations; Goals; HIV-1; Individual; Kinetics; Label; Laboratories; Ligands; Measurement; Medical; Membrane Proteins; Methodology; Methods; Molecular; Molecular Diagnosis; Molecular Probes; N-terminal; Nature; Noise; Nucleic Acids; Nucleocapsid Proteins; Outcome; Pharmaceutical Preparations; Positioning Attribute; Process; Property; Protein Engineering; Proteins; Protocols documentation; RNA; Research; Resolution; Ribonucleases; Sampling; Scaffolding Protein; Scheme; Security; Signal Transduction; Sodium Chloride; Spectrum Analysis; Structure; System; Techniques; Technology; Tertiary Protein Structure; Therapeutic; Thermodynamics; Time; Variant; Work; aptamer; base; conformer; design; drug testing; ds-DNA; extracellular; functional group; hydroxamate; improved; interest; member; molecular recognition; nanomedicine; nanopore; novel; nucleic acid binding protein; polypeptide; protein folding; protein structure function; public health relevance; sensor; single molecule; tool; uptake

DESCRIPTION (provided by applicant): Advances in rational membrane protein design, molecular recognition, and single-molecule technology will be employed to enable biochemical sampling at high temporal and spatial resolution, as well as the detection, exploration, and characterization of individual biomolecules. We will use Ferric hydroxamate uptake component A (FhuA), one of the members of the superfamily of bacterial outer membrane proteins. Molecular engineering of the FhuA protein will be used in single-molecule stochastic sensing, because this system exhibits a remarkable array of advantageous characteristics, including its monomeric structure, robustness, versatility, tractability, and the availability of its high-resolution crystal structure. Our studies will be aimed at developing engineered nanopore-based biosensors that feature a wider pore diameter to accommodate bulky biopolymers, including proteins, double-stranded DNA, and their complexes with the interacting ligands. The partitioning of a single analyte into an engineered FhuA-based nanopore will be detected by a transient single- channel current blockade, the nature of which dependents on several factors that will be well-controlled by protein engineering and single-molecule design. The obtained data will be further processed through established protocols of single-molecule electric detection, macroscopic currents, and the analysis of current noise fluctuations produced by the analyte. The expected immediate outcomes will be the following: (1) the unusual stabilization of engineered FhuA-based nanopores by placing critical covalent and noncovalent intra- molecular contacts at strategic positions within the pore lumen; (2) the single-molecule stochastic sensing of highly specific HIV-1 aptamers; (3) the determination of the precise nature of the DNA aptamer-HIV-1 nucleocapsid protein interactions by obtaining the entropic and enthalpic contributions to the kinetic and thermodynamic constants, providing key information about which process in the DNA-protein interaction is dominant; (4) the single-molecule stochastic sensing of folded proteins and their complexes with the interacting ligands; (5) the improvement of the detection capabilities of the nanopore-based devices for proteins by engineering internal electrostatic traps; (6) the development of label-free diagnostic assays for drug-DNA complexes. The adaptation of these approaches to a microfabricated chip platform not only will provide a new generation of research tools in nanomedicine for examining the details of complex recognition events in a quantitative manner, but also will represent a crucial step in designing nanopore-based biosensors and high- throughput devices for biomedical molecular diagnosis, environmental monitoring, and homeland security. PUBLIC HEALTH RELEVANCE: Engineered nanopores will represent a crucial step in the design of high-throughput devices for biomedical molecular diagnosis, biotherapeutics, and biosensing technology. They will also provide a new generation of research tools in nanomedicine for examining the details of complex recognition events in a quantitative manner.

描述（由申请人提供）：将利用合理膜蛋白设计、分子识别和单分子技术的进步来实现高时间和空间分辨率的生化采样，以及单个生物分子的检测、探索和表征。我们将使用异羟肟酸铁吸收成分 A (FhuA)，它是细菌外膜蛋白超家族的成员之一。 FhuA蛋白的分子工程将用于单分子随机传感，因为该系统表现出一系列显着的有利特性，包括其单体结构、稳健性、多功能性、易处理性以及高分辨率晶体结构的可用性。我们的研究旨在开发基于工程纳米孔的生物传感器，其具有更宽的孔径，以容纳大体积的生物聚合物，包括蛋白质、双链 DNA 及其与相互作用配体的复合物。单个分析物分配到基于 FhuA 的工程化纳米孔中将通过瞬时单通道电流阻断来检测，其性质取决于几个因素，这些因素将通过蛋白质工程和单分子设计得到很好的控制。获得的数据将通过已建立的单分子电检测、宏观电流以及分析物产生的电流噪声波动的分析方案进行进一步处理。预期的直接结果如下：（1）通过在孔腔内的战略位置放置关键的共价和非共价分子内接触，使工程化的基于 FhuA 的纳米孔异常稳定； (2) 高特异性HIV-1适体的单分子随机传感； (3) 通过获得熵和焓对动力学和热力学常数的贡献，确定DNA适体-HIV-1核衣壳蛋白相互作用的精确性质，提供关于DNA-蛋白质相互作用中哪个过程占主导地位的关键信息； (4) 折叠蛋白及其与相互作用配体的复合物的单分子随机传感； （5）通过设计内部静电陷阱来提高基于纳米孔的蛋白质检测装置的能力； (6)药物-DNA复合物无标记诊断分析的开发。这些方法对微加工芯片平台的适应不仅将为纳米医学领域提供新一代研究工具，用于以定量方式检查复杂识别事件的细节，而且也代表着设计基于纳米孔的生物传感器和高通量纳米技术的关键一步。 - 用于生物医学分子诊断、环境监测和国土安全的吞吐量设备。 公共健康相关性：工程纳米孔将代表生物医学分子诊断、生物治疗和生物传感技术的高通量设备设计的关键一步。他们还将提供新一代纳米医学研究工具，用于以定量方式检查复杂识别事件的细节。