Phi29 Motor Nanopore for Single Molecule Sensing

描述（由申请人提供）： 在生物系统中存在多种高度有序的大分子结构和有组织的纳米机器，用于运输多核酸和多肽。噬菌体phi29 DNA包装马达的巧妙设计是这一类中一个有趣的元素。所有的线性双链DNA病毒都通过一个ATP驱动的马达将它们的基因组包装到一个预先形成的原衣壳中，该马达涉及两种非结构酶或ATP酶。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，它形成了一个十二聚体通道，作为双链DNA易位的途径。其最西端的直径为3.6 nm，是已并入脂质膜的典型通道蛋白中最大的通道蛋白之一。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。 本申请的短期目标是证明phi29 DNA包装马达的脂质嵌入通道作为使用电生理学测定以单分子分辨率表征生物聚合物的高灵敏度装置的实用性。连接器将首先被重新设计以增加功能，并且将系统地研究调节连接器电导的各种方法。然后通过诱变以受控方式改变孔的尺寸和电荷分布，以进行特定的孔修饰，以确保最佳的传感。最后，该通道将被设计成作为一个随机生物传感器和功能化的修饰基团，以识别潜在的测序应用的双链DNA的核苷酸碱基。长期目标是将该通道开发成用于检测极低浓度的病原体和化学品的强大传感装置，用于疾病诊断和环境监测，并将其用作下一代双链DNA测序的高通量纳米孔装置。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装载、包装和基因递送至特定细胞以治疗癌症、病毒感染和遗传疾病的未来途径。 公共卫生相关性： 膜适应噬菌体phi29门户运动纳米孔蛋白将被修饰、表征和开发用于在极低浓度下对病原体和化学品的高灵敏度检测以及用于在单分子水平上表征双链DNA。该通道可以进一步开发为用于治疗性DNA包装和基因递送至特定细胞的工具，用于治疗癌症、病毒感染和遗传疾病。

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.