Generalizable Nanosensors for Probing Highly Specific Interactions of Protein Kinases

项目摘要 开发新的技术来识别和定量瞬时蛋白质-蛋白质相互作用是至关重要的， 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组 用于治疗多种血液恶性肿瘤和实体瘤的靶点。然而，创建高分辨率 传感器，以检测，量化和分析不同的激酶组成员在广泛的动态范围内的可塑性， 互动仍然很困难。这种挑战加剧，因为激酶超家族成员的变化 在它们的复杂性上有很大的不同。为了解决这个长期存在的技术缺陷，我们将制定， 开发并验证一类新的可通用且高度特异性的纳米孔传感器（纳米传感器）， 激酶分析。该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。这种方法将采用由单一纳米结构制成的坚固的纳米结构。 在一些实施方案中，所述多肽是多肽实体，不需要额外的尾或其它外源标签。的绑定接口 蛋白质识别配体是可互换的以适应靶向激酶所需的特异性， 而纳米孔促进报告电信号的产生。蛋白激酶分析物， 溶液产生随其身份和数量而变化的唯一电子签名。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，激酶识别事件将被 以单分子精度进行区分，而不需要使用复杂的数据分析算法。 这种工程策略大大拓宽了这些纳米传感器的应用范围， 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，通过创建一个单一的- 分子纳米传感器平台，探测和量化结构和功能多样的蛋白质， 纳米孔内传感的基本极限。此外，这种战术将使检测 激酶同种型对相同识别配体的竞争结合相互作用。这些可概括的 纳米传感器允许集成到可扩展的设备中，代表小分子的多功能元件 抑制剂筛选和药物发现管道。进一步的项目发展将旨在保持 这些纳米传感器在复杂的生物流体中的高性能。因此，可以使用现实的 样品，在分子诊断中具有应用前景。该项目的预期直接成果将是 以下内容：（i）用于受体酪氨酸超灵敏分析的高亲和力纳米传感器的开发 激酶（RTK）;（ii）创建用于探测丝氨酸-苏氨酸激酶的遗传编码纳米传感器 （iii）在多重设置和生物流体中检测和分析激酶。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.