Engineered Nanopores for Single-Molecule Stochastic Sensing

描述(由申请人提供)：将采用合理的膜蛋白设计、分子识别和单分子技术的进步，以实现高时间和空间分辨率的生化采样，以及对单个生物分子的检测、探索和表征。我们将使用异羟甲酸铁摄取成分A(FhuA)，它是细菌外膜蛋白超家族的成员之一。FhuA蛋白的分子工程将被用于单分子随机传感，因为该系统显示出一系列显著的优势特征，包括其单体结构、稳健性、多功能性、易操纵性以及其高分辨率晶体结构的可用性。我们的研究将致力于开发基于纳米孔的生物传感器，这种传感器具有更宽的孔径，以适应巨大的生物聚合物，包括蛋白质、双链DNA及其与相互作用配体的络合物。将单个分析物分割成基于FhuA的工程纳米孔将通过瞬时单通道电流阻断进行检测，其性质取决于几个因素，这些因素将由蛋白质工程和单分子设计很好地控制。获得的数据将通过建立的单分子电检测、宏观电流和分析分析物产生的电流噪声波动的既定方案进行进一步处理。预期的近期结果如下：(1)通过将关键的共价和非共价分子内接触放置在孔腔内的关键位置，使基于FhuA的工程纳米孔异常稳定；(2)高度特异的HIV-1适配子的单分子随机传感；(3)通过获得对动力学和热力学常数的熵和热能贡献，确定DNA适配子-HIV-1核衣壳蛋白相互作用的精确性质，提供关于DNA-蛋白质相互作用中哪个过程占主导地位的关键信息；(4)折叠蛋白质及其与相互作用配体的复合体的单分子随机传感；(5)通过设计内部静电陷阱来提高基于纳米孔的蛋白质设备的检测能力；(6)开发药物-DNA复合体的无标记诊断方法。将这些方法应用于微制造芯片平台，不仅将为定量研究复杂识别事件的细节提供新一代纳米医学研究工具，而且将代表着为生物医学分子诊断、环境监测和国土安全设计基于纳米孔的生物传感器和高通量设备的关键一步。 公共卫生相关性：工程纳米孔将代表着设计用于生物医学分子诊断、生物治疗和生物传感技术的高通量设备的关键一步。它们还将提供新一代纳米医学研究工具，用于定量检查复杂识别事件的细节。

DESCRIPTION (provided by applicant): Advances in rational membrane protein design, molecular recognition, and single-molecule technology will be employed to enable biochemical sampling at high temporal and spatial resolution, as well as the detection, exploration, and characterization of individual biomolecules. We will use Ferric hydroxamate uptake component A (FhuA), one of the members of the superfamily of bacterial outer membrane proteins. Molecular engineering of the FhuA protein will be used in single-molecule stochastic sensing, because this system exhibits a remarkable array of advantageous characteristics, including its monomeric structure, robustness, versatility, tractability, and the availability of its high-resolution crystal structure. Our studies will be aimed at developing engineered nanopore-based biosensors that feature a wider pore diameter to accommodate bulky biopolymers, including proteins, double-stranded DNA, and their complexes with the interacting ligands. The partitioning of a single analyte into an engineered FhuA-based nanopore will be detected by a transient single- channel current blockade, the nature of which dependents on several factors that will be well-controlled by protein engineering and single-molecule design. The obtained data will be further processed through established protocols of single-molecule electric detection, macroscopic currents, and the analysis of current noise fluctuations produced by the analyte. The expected immediate outcomes will be the following: (1) the unusual stabilization of engineered FhuA-based nanopores by placing critical covalent and noncovalent intra- molecular contacts at strategic positions within the pore lumen; (2) the single-molecule stochastic sensing of highly specific HIV-1 aptamers; (3) the determination of the precise nature of the DNA aptamer-HIV-1 nucleocapsid protein interactions by obtaining the entropic and enthalpic contributions to the kinetic and thermodynamic constants, providing key information about which process in the DNA-protein interaction is dominant; (4) the single-molecule stochastic sensing of folded proteins and their complexes with the interacting ligands; (5) the improvement of the detection capabilities of the nanopore-based devices for proteins by engineering internal electrostatic traps; (6) the development of label-free diagnostic assays for drug-DNA complexes. The adaptation of these approaches to a microfabricated chip platform not only will provide a new generation of research tools in nanomedicine for examining the details of complex recognition events in a quantitative manner, but also will represent a crucial step in designing nanopore-based biosensors and high- throughput devices for biomedical molecular diagnosis, environmental monitoring, and homeland security. PUBLIC HEALTH RELEVANCE: Engineered nanopores will represent a crucial step in the design of high-throughput devices for biomedical molecular diagnosis, biotherapeutics, and biosensing technology. They will also provide a new generation of research tools in nanomedicine for examining the details of complex recognition events in a quantitative manner.