Generalizable Nanosensors for Probing Highly Specific Interactions of Protein Kinases

项目摘要 开发识别和量化瞬时蛋白质-蛋白质相互作用的新技术在 基础研究和医学生物技术。蛋白激酶是战略药物中的一个焦点 治疗多种血液系统恶性肿瘤和实体瘤的靶点。然而，创建高分辨率 传感器，用于检测、量化和分析广泛动态范围内的不同亲属组成员的可塑性 互动仍然很困难。这一挑战因激酶超家族成员的不同而加剧 它们的复杂性有很大提高。为了解决这一长期存在的技术缺陷，我们将制定， 开发和验证一种新的可推广和高度特异的纳米孔传感器(纳米传感器)，用于 激活剂分析。这一设计的关键创新方面是将通用蛋白质识别配体与 跨膜蛋白纳米孔。这种方法将使用由单一的 多肽实体，不需要额外的尾巴或其他外源标签。的绑定接口 蛋白质识别配体可互换以适应靶向激酶所需的特异性， 而纳米孔有助于产生报告电信号。一种蛋白激酶分析物 解决方案产生一个独特的电子签名，它随着其身份和数量的不同而变化。报告信号是 由纳米孔尖端的配体-激酶组装介导。在这些研究中，激酶识别事件将是 以单分子精度进行识别，无需使用复杂的数据分析算法。 这一工程策略大大拓宽了这些纳米传感器的应用范围，使其适用于各种 激酶及其相互作用。我们的初步研究证明了这种方法的力量，它创造了一个单一的- 分子纳米传感器平台，探测和量化结构和功能上不同的蛋白质 纳米孔内部传感的基本极限。此外，这种策略将使检测到 与同一识别配体竞争结合的激酶亚型的相互作用。这些是可概括的 纳米传感器允许集成到可扩展的设备中，代表了小分子的多种元素 抑制剂筛选和药物发现流水线。进一步的项目开发将旨在保持一个 这些纳米传感器在复杂的生物流体中的高性能。因此，可以利用它们来使用现实 样品，在分子诊断学中有应用前景。该项目的预期直接结果将是 用于受体酪氨酸超灵敏分析的高亲和力纳米传感器的发展 激酶(RTK)；(Ii)用于探测丝氨酸-苏氨酸激酶的基因编码纳米传感器的建立 (3)检测和分析多路传输环境和生物体液中的激活酶。这些研究将 通过提供生物传感器技术、合成生物学、 和单分子酶学。

Project Summary Developing novel technologies for identifying and quantifying transient protein-protein interactions is critical in basic research and medical biotechnology. Protein kinases represent a focal group among strategic drug targets for treating numerous hematological malignancies and solid tumors. Yet, creating high-resolution sensors to detect, quantify, and analyze the plasticity of diverse kinome members in a broad dynamic range of interactions remains difficult. This challenge is exacerbated because the kinase superfamily members vary drastically in their complexity. To address this long-standing technological shortcoming, we will formulate, develop, and validate a new class of generalizable and highly specific nanopore sensors (nanosensors) for kinase analytics. The key innovating aspect of this design is fusing a generic protein recognition ligand with a transmembrane protein nanopore. This approach will employ a robust nanostructure made of a single polypeptide entity with no requirement for an additional tail or other exogenous tags. The binding interface of the protein recognition ligand is interchangeable to accommodate the required specificity for a targeted kinase, whereas the nanopore facilitates the generation of a reporting electrical signal. A protein kinase analyte in solution produces a unique electrical signature that varies with its identity and quantity. The reporting signal is mediated by the ligand-kinase assembly at the nanopore tip. In these studies, kinase recognition events will be discriminated at single-molecule precision without the necessity of using complex data analysis algorithms. This engineering strategy substantially broadens the spectrum of applications of these nanosensors to various kinases and their interactions. Our preliminary studies prove the power of this approach by creating a single- molecule nanosensor platform that probes and quantifies structurally and functionally diverse proteins beyond the fundamental limit of sensing inside the nanopore. In addition, such a tactic will enable the detection of competing binding interactions of kinase isoforms against the same recognition ligand. These generalizable nanosensors permit integration into scalable devices, representing versatile elements for small-molecule inhibitor screening and drug discovery pipelines. Further project developments will be aimed at maintaining a high performance of these nanosensors in a complex biofluid. Therefore, they can be utilized using realistic samples, having prospects in molecular diagnostics. The expected immediate outcomes of this project will be the following: (i) the development of high-affinity nanosensors for ultrasensitive analysis of receptor tyrosine kinases (RTKs); (ii) the creation of genetically-encoded nanosensors for probing serine-threonine kinases (STKs); (iii) the detection and analysis of kinases in multiplexed settings and biofluids. These studies will impact healthcare by providing tools and a fundamental framework in biosensor technology, synthetic biology, and single-molecule enzymology.