Phi29 Motor Nanopore for Single Molecule Sensing

用于单分子传感的 Phi29 马达纳米孔

• 批准号: 8435052
• 负责人: PEIXUAN GUO
• 金额: $ 8.2万
• 依托单位: UNIVERSITY OF KENTUCKY
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435052
• 关键词: Phi29; Motor; Nanopore; Single; Molecule

DESCRIPTION (provided by applicant): A wide variety of highly-ordered structures of macromolecules and organized nanomachines exist in biological systems for transporting polynucleic acids and polypeptides. The ingenious design of the bacteriophage phi29 DNA packaging motor is an intriguing element in this category. All linear dsDNA viruses package their genome into a preformed procapsid via an ATP-driving motor involving two nonstructural enzymes or ATPases. The central component of the phi29 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 double-stranded DNA. 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 in lipid membranes. Explicit engineering and pore modifications of the phi29 connector are possible due to its available crystal structure, thus inspiring its biomedical applications. The short-term objective of this application is to demonstrate the utility of the lipid-embedded channel of the phi29 DNA packaging motor as a highly sensitive device for characterizing biopolymers at single molecule resolution using electrophysiological assays. The connector will first be reengineered for added functionality and various approaches to modulate the conductance of the connector will be systematically investigated. The dimensions of the pore and charge distributions will then be altered by mutagenesis for specific pore modifications in a controlled fashion to ensure optimal sensing. Finally, the channel will be designed to act as a stochastic biosensor and functionalized with modifying groups to recognize nucleotide bases of double stranded DNA for potential sequencing applications. The long-term objective is to develop the channel into a robust sensing device for detecting pathogens and chemicals at extremely low concentrations for disease diagnosis and environmental monitoring, and to utilize it as a high throughput nanopore-based apparatus for the next generation sequencing of double stranded DNA. The channel can further be used as a tool to assay the structure and function of biological molecules and complexes as well as future avenues for therapeutic DNA loading, packaging, and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases. PUBLIC HEALTH RELEVANCE: The membrane adapted bacteriophage phi29 portal motor nanopore protein will be modified, characterized and developed for highly sensitive detection of pathogens and chemicals at extremely low concentrations as well as for characterizing double-stranded DNA at the single molecule level. The channel can further be developed as a tool for therapeutic DNA packaging and gene delivery to specific cells for the treatment of cancers, viral infections, and genetics diseases.

描述（由申请人提供）： 生物系统中存在多种大分子和有组织的纳米机器的多种高序结构，用于运输多核酸和多肽。噬菌体PHI29 DNA包装电机的巧妙设计是该类别中有趣的元素。所有线性DSDNA病毒通过涉及两个非结构酶或ATPases的ATP驱动电动机将其基因组包装到预制的procapsid中。 PHI29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，该通道充当双链DNA易位的途径。它的直径在其最窄的末端为3.6 nm，它是已掺入脂质膜中的典型通道蛋白中最大的之一。由于其可用的晶体结构，可以激发其生物医学应用，因此可以对PHI29连接器进行显式工程和孔修改。 该应用的短期目的是证明PHI29 DNA包装电机的脂质包装通道的实用性，作为一种高度敏感的装置，用于使用电生理测定法以单分子分辨率表征生物聚合物。连接器将首先重新设计以增加功能，并系统地研究连接器电导的各种方法。然后，孔隙和电荷分布的尺寸将通过诱变以受控的方式改变特定的孔隙修饰，以确保最佳感应。最后，该通道将被设计为充当随机生物传感器，并通过修饰组功能化，以识别双链DNA的核苷酸碱基，以实现潜在的测序应用。长期目标是将通道开发为可靠的感应装置，用于以极低的浓度检测病原体和化学物质，以进行疾病诊断和环境监测，并将其用作基于高吞吐量的纳米孔的较高的基于双吞吐量的设备滞留的DNA。该通道可以进一步用作分析生物分子和复合物的结构和功能的工具，以及用于治疗性DNA负荷，包装和基因递送到特定细胞的未来途径，以治疗癌症，病毒感染和遗传学疾病。 公共卫生相关性： 膜适应的噬菌体PHI29门户运动纳米孔蛋白将经过修改，表征和开发，以高度敏感的病原体和化学物质在极低的浓度下以及在单分子水平上表征双链DNA。该通道可以作为治疗性DNA包装和转移至特定细胞的基因递送的工具，用于治疗癌症，病毒感染和遗传学疾病。