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病毒通过ATP驱动电动机将其基因组包装到预制的procapsid中。 PHI29 DNA包装电动机的中心分量是由蛋白质GP10的十二份副本组成的门户连接器，该连接器形成了十二焦通道，该通道充当DSDNA易位的途径。它的直径在其最窄的末端为3.6 nm，它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，因此可以对PHI29连接器进行显式工程和孔修改，从而激发了将这种系统作为生物医学应用或纳米电视中的模块的使用。 该提案的短期目标是证明PHI29 DNA包装电机的脂质包裹通道的实用性，作为一种高度敏感的装置，用于使用电生理学分析以单分子分辨率捕获和指纹对化学物质和生物聚合物进行实时捕获和指纹。连接器将被重新设计以增加功能，并将采用各种方法来调节孔的尺寸。电荷分布的改变将以受控方式进行诱变，以确保最佳感应。该通道将被设计为充当随机生物传感器，并用识别组功能化，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，以促进单个孔的高吞吐量电生理测量，并提供快速有效的样品和缓冲液交换的规定。长期目的是将通道发展为可靠的感应装置，用于在许多污染物存在下以极低浓度的生物分子或化学物质在生物技术，早期疾病诊断，环境监测，定制隔离，药物测试和国家安全方面的广泛应用。 公共卫生相关性：噬菌体PHI29生物运动的膜嵌入纳米孔被重新设计，表征并发展为单个孔传感器，以检测生物分子和化学物质。实时检测单分子检测的捕获和指纹与多个鉴定参数的组合将使系统高度敏感地在许多污染物存在下以极低浓度检测分子。