Phi29 Motor Nanopore for Single Molecule Sensing

描述(申请人提供)：所有线性双链(DS)DNA病毒通过三磷酸腺苷驱动的马达将它们的基因组包装成预制的前壳蛋白。Phi29 DNA包装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dsDNA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phi29连接器具有可用的晶体结构，因此可以对其进行显式工程和孔修饰，从而鼓励将这种系统用作生物医学应用或纳米设备中的模块。这项提议的短期目标是展示phi29 DNA包装马达的脂质嵌入通道作为一种高灵敏设备的用途，利用电生理分析在单分子分辨率下实时捕获化学品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，并将采取各种方法来调节孔的尺寸。电荷分布的改变将通过可控的诱变方式进行，以确保最佳的感测。该通道将被设计成一个随机生物传感器，并通过识别基团来结合各种感兴趣的分析物。将开发提高脂质/连接器复合体的稳定性和寿命的方法，以促进单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。长期目标是将该通道发展成一种坚固的传感设备，用于在存在许多污染物的情况下以极低的浓度检测生物分子或化学品，用于生物技术、早期疾病诊断、环境监测、海关检疫、药物测试和国家安全的广泛应用。 公共卫生意义：噬菌体phi29生物摩托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多个识别参数相结合，用于实时检测单分子，将使该系统在存在许多污染物的情况下，对极低浓度的分子检测具有高度的灵敏度。

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.