Engineered Nanopores for Single-Molecule Stochastic Sensing

描述（由申请人提供）：通过提供对细胞复杂调控过程的机制理解，确定它们与疾病的关系，加速蛋白质生物标志物的发现，并协助药物设计，揭示功能蛋白之间的相互作用网络在基础和临床生物医学诊断中是必不可少的。在合理膜蛋白设计、化学修饰、生物分子识别和单分子科学方面的进展将被用于创建一种新的方法，以高时间和空间分辨率采样蛋白质-蛋白质相互作用，以及单个蛋白质的检测、探索和表征。这些建议的研究旨在工程蛋白纳米孔传感装置的特点是配体含有柔性系绳。铁羟酸盐摄取成分A （FhuA）是一种单体b桶蛋白，具有一系列显著的优势特性，如稳健性、多功能性和可追踪性，对其进行充分的重新设计，将产生一种独特的纳米结构，在纳米孔的战略位置上具有单个栓系蛋白DNA适体配体。基于fhua的支架是这项任务的一个有吸引力的选择，因为它的开放状态，安静的电流在异常广泛的检测环境中保持长时间稳定。这些优点将用于各种生物传感方案，其中单个蛋白质-蛋白质和蛋白质- dna识别事件将在可移动系绳的构象动力学中产生可检测的，离散的和可逆的变化，诱导单通道电签名的改变。预期的直接结果将如下：(i)创建用于检查平衡和非平衡条件下蛋白质-蛋白质相互作用的传感元件；（ii）开发高度特异性的基于纳米孔的蛋白质生物标志物传感元件；（iii）更好地理解可调系链和限制性系链对蛋白质伴侣之间分子间作用力的影响，这对锚定蛋白质结构域产生的复杂识别事件的体内背景具有重要意义；(4)提高了单分子检测蛋白-蛋白界面的灵敏度，推动了纳米孔技术用于解缠弱蛋白-蛋白相互作用；(v)扩展工程蛋白纳米孔的模块化和可扩展性，以及它们与合成膜的集成，提高它们的机械、热、电和化学稳定性。这些不寻常的纳米结构与可移动臂的适应集成微加工芯片平台将提供新一代的研究工具，以敏感，特异性和定量的方式探索蛋白质-蛋白质识别事件的分子基础。

DESCRIPTION (provided by applicant): Unraveling the interaction networks among functional proteins is essential in fundamental and clinical biomedical diagnostics by providing a mechanistic understanding of the complex regulatory processes of the cell, identifying their relationships to diseases, accelerating protein biomarker discovery, and assisting drug design. Advances in rational membrane protein design, chemical modification, biomolecular recognition, and single-molecule science will be used in concert for the creation of a new methodology to sample protein-protein interactions at high temporal and spatial resolution, as well as for the detection, exploration, and characterization of individual proteins. These proposed studies are aimed at engineering protein nanopore- based sensing devices featured by ligand-containing flexible tethers. Ample redesign of ferric hydroxamate uptake component A (FhuA), a monomeric b-barrel protein with a remarkable array of advantageous traits, such as robustness, versatility, and tractability, will result in a unique nanostructure with a single tethered proteinor DNA aptamer ligand at a strategic positioning of the nanopore. The FhuA-based scaffold is an attractive choice for this task, because it's open-state, quiet current remains stable for long periods within an unusually broad range of detection circumstances. These benefits will be used in various biosensing schemes, in which individual protein-protein and protein-DNA recognition events will produce detectable, discrete and reversible changes in the conformational dynamics of the movable tether, inducing alterations in the single- channel electrical signature. The expected immediate outcomes will be the following: (i) the creation of sensing elements for examining protein-protein interactions under equilibrium and non-equilibrium conditions; (ii) the development of highly specific nanopore-based sensing elements for a protein biomarker; (iii) a better understanding of the impact of tunable and constraining tethers on the intermolecular forces among protein partners, which has implications for the in vivo contexts of complex recognition events produced by anchored protein domains; (iv) the improvement in the sensitivity of the single-molecule detection of protein-protein interfaces, pushing forward the nanopore technology for the disentanglement of weak protein-protein interactions; (v) the expansion of the modularity and scalability of engineered protein nanopores as well as their integration with a synthetic membrane, improving their mechanical, thermal, electrical, and chemical stability. The adaptation of these unusual nanostructures with movable arms to an integrated microfabricated chip platform will provide a new generation of research tools for exploring the molecular basis of protein-protein recognition events in a sensitive, specific and quantitative fashion.