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)： 在用于运输多核酸和多肽的生物系统中，存在着各种各样的高度有序的大分子结构和有组织的纳米机器。噬菌体phi29 DNA包装马达的巧妙设计是这一类别中一个耐人寻味的元素。所有的线状dsDNA病毒都通过一个ATP驱动的马达将它们的基因组包装成一个预先形成的前壳，其中包括两个非结构酶或ATPase。Phi29马达的中心组件是由12个拷贝的GP10蛋白组成的门户连接器，它形成一个十二聚体通道，作为双链DNA转位的途径。它最窄的一端直径为3.6 nm，是已被整合到脂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，phi29连接器的显式工程和孔修饰是可能的，从而激发了其生物医学应用。这项应用的短期目标是证明phi29 DNA包装马达的脂质嵌入通道作为一种高灵敏度的设备，用于使用电生理分析在单分子分辨率下表征生物聚合物。首先将重新设计连接器以增加功能性，并将系统地研究调节连接器电导的各种方法。然后，孔的尺寸和电荷分布将通过可控的特定孔修改的诱变来改变，以确保最佳的感测。最后，该通道将被设计为一个随机生物传感器，并通过修饰基团来识别双链DNA的核苷酸碱基，以用于潜在的测序应用。长期目标是将该通道开发成一个强大的传感设备，用于在极低浓度下检测病原体和化学物质，用于疾病诊断和环境监测，并将其用作高通量纳米孔设备，用于下一代双链DNA测序。该通道还可进一步用作分析生物分子和复合体的结构和功能的工具，以及未来治疗癌症、病毒感染和遗传疾病的治疗性DNA负载、包装和基因输送到特定细胞的途径。 公共卫生相关性： 膜适应噬菌体phi29门脉电机纳米孔蛋白将被修饰、表征和开发，用于在极低浓度下高灵敏地检测病原体和化学物质，以及在单分子水平上表征双链DNA。该通道还可以进一步开发为治疗性DNA包装和将基因输送到特定细胞的工具，用于治疗癌症、病毒感染和遗传性疾病。