Phi29 Motor Nanopore for Single Molecule Sensing

描述（由申请人提供）：所有线性双链（ds） DNA病毒通过atp驱动马达将其基因组包装成预先形成的原衣壳。phi29 dna包装马达的核心组件是由12个gp10蛋白拷贝组成的入口连接器，它形成一个十二聚体通道，作为dsDNA易位的途径。它的最窄端直径为3.6纳米，是被纳入脂质膜的典型通道蛋白中最大的一个。由于phi29连接器的晶体结构，可以对其进行明确的工程和孔修改，从而激发了将该系统作为生物医学应用或纳米器件模块的使用。本提案的短期目标是证明phi29 DNA包装马达的脂质嵌入通道作为一种高灵敏度设备的实用性，用于使用电生理分析在单分子分辨率下实时捕获和识别化学物质和生物聚合物。连接器将重新设计以增加功能，并采用各种方法来调节孔的尺寸。电荷分布的改变将通过诱变以受控的方式进行，以确保最佳的传感。该通道将被设计成一个随机生物传感器，并通过识别基团进行功能化，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为一种强大的传感装置，用于在存在许多污染物的情况下检测极低浓度的生物分子或化学品，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等广泛应用。

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.