Phi29 Motor Nanopore for Single Molecule Sensing

用于单分子传感的 Phi29 马达纳米孔

• 批准号: 8107966
• 负责人: PEIXUAN GUO
• 金额: $ 22.82万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8107966
• 关键词: Affinity; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Antibodies; Antigens; Area; Automobile Driving; Bacteriophages; Binding; Biological; Biological Assay; Biological Models; Biopolymers; Biosensor; Biotechnology; Biotin; Buffers; Caliber; Cells; Characteristics; Charge; Chemicals; Complex; Custom; Cysteine; DNA; DNA Packaging; Data; Detection; Devices; Dimensions; Disulfide Linkage; Double Stranded DNA Virus; Engineering; Ensure; Event; Exhibits; Fingerprint; Frequencies; Genome; Goals; Housing; Infection; Ions; Kinetics; Length; Life; Ligands; Lipid Bilayers; Lipids; Maleimides; Measurement; Membrane; Membrane Lipids; Methods; Modification; Molecular Conformation; Monitor; Motor; Mutagenesis; Nanotechnology; National Security; Nature; Nickel; Pathway interactions; Pattern; Peptides; Phage Display; Property; Proteins; Quarantine; RNA; RNA-Protein Interaction; Reaction; Regulation; Resolution; Sampling; Shapes; Signal Transduction; Streptavidin; Structure; System; Time; Transportation; Virion; base; chemical fingerprinting; design; disease diagnosis; drug testing; ds-DNA; instrument; interest; macromolecule; nanodevice; nanopore; novel; patch clamp; protein protein interaction; proteoliposomes; research study; sensor; single molecule; thioester; time use

DESCRIPTION (provided by applicant): All linear double-stranded (ds) DNA viruses package their genome into a preformed procapsid via an ATP-driving motor. The central component of the phi29 DNA-packaging motor is the portal connector composed of twelve copies of the protein gp10, which form a dodecamer channel that acts as a pathway for the translocation of dsDNA. With a diameter of 3.6 nm at its narrowest end, it is one of the largest among typical channel proteins that have been incorporated into lipid membranes. Explicit engineering and pore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiring the use of such a system as a module in biomedical applications or nanodevices. The short-term objective of this proposal is to demonstrate the utility of the lipid-embedded channel of the phi29 DNA packaging motor as a highly sensitive device for capture and fingerprinting of chemicals and biopolymers in real time at single molecule resolution using electrophysiological assays. The connector will be reengineered for added functionality and various approaches will be undertaken to modulate the dimensions of the pore. Alteration of the charge distributions will be made by mutagenesis in a controlled fashion to ensure optimal sensing. The channel will be designed to act as a stochastic biosensor and functionalized with recognition groups to bind various analytes of interest. Methods to increase the stability and lifetime of the lipid/connector complex will be developed to facilitate high throughput electrophysiological measurement of a single pore with provisions for rapid and efficient sample and buffer exchange. The long-term objective is to develop the channel into a robust sensing device for detecting biomolecules or chemicals at extremely low concentrations in the presence of many contaminants for a wide range of applications in biotechnology, earlier disease diagnosis, environmental surveillance, custom quarantine, drug testing and national security. PUBLIC HEALTH RELEVANCE: The membrane embedded nanopore of bacteriophage phi29 biomotor will be reengineered, characterized and developed into a single pore sensor for detection of biomolecules and chemicals. The combination of capture and fingerprinting with multiple identification parameters for single molecule detection in real time will make the system highly sensitive to detect molecules at extremely low concentrations in the presence of many contaminants.

描述（由申请人提供）：所有线性双链 (ds) DNA 病毒都通过 ATP 驱动电机将其基因组包装到预先形成的衣壳中。 phi29 DNA 包装马达的核心组件是由 12 个蛋白质 gp10 拷贝组成的门户连接器，形成十二聚体通道，充当 dsDNA 易位的途径。它的最窄端直径为 3.6 nm，是已掺入脂质膜的典型通道蛋白中最大的之一。由于其可用的晶体结构，phi29 连接器的显式工程和孔隙修改是可能的，从而激发了将此类系统用作生物医学应用或纳米设备中的模块。 该提案的短期目标是证明 phi29 DNA 包装电机的脂质嵌入通道作为高灵敏度设备的实用性，用于使用电生理测定以单分子分辨率实时捕获化学物质和生物聚合物并进行指纹识别。连接器将被重新设计以增加功能，并且将采取各种方法来调节孔隙的尺寸。电荷分布的改变将以受控方式通过诱变进行，以确保最佳传感。该通道将被设计用作随机生物传感器，并通过识别基团进行功能​​化以结合各种感兴趣的分析物。将开发提高脂质/连接器复合物的稳定性和寿命的方法，以促进单个孔的高通量电生理测量，并提供快速有效的样品和缓冲液交换。长期目标是将通道开发成强大的传感装置，用于在存在多种污染物的情况下检测极低浓度的生物分子或化学物质，广泛应用于生物技术、早期疾病诊断、环境监测、定制检疫、药物测试和国家安全。 公共健康相关性：噬菌体phi29生物马达的膜嵌入纳米孔将被重新设计、表征并开发成用于检测生物分子和化学物质的单孔传感器。将捕获和指纹识别与实时单分子检测的多个识别参数相结合，将使系统高度灵敏，能够在存在许多污染物的情况下检测极低浓度的分子。