Generalizable Nanosensors for Probing Highly Specific Interactions of Protein Kinases

项目概要 开发识别和量化瞬时蛋白质-蛋白质相互作用的新技术对于 基础研究和医学生物技术。蛋白激酶代表战略药物中的焦点群体 targets for treating numerous hematological malignancies and solid tumors.然而，创建高分辨率 传感器可在广泛的动态范围内检测、量化和分析不同激酶组成员的可塑性 互动仍然困难。由于激酶超家族成员各不相同，这一挑战变得更加严峻 其复杂性极大。为了解决这个长期存在的技术缺陷，我们将制定， 开发并验证一类新型通用且高度特异性的纳米孔传感器（纳米传感器） 激酶分析。 The key innovating aspect of this design is fusing a generic protein recognition ligand with a 跨膜蛋白纳米孔。这种方法将采用由单一材料制成的坚固的纳米结构 不需要额外尾部或其他外源标签的多肽实体。的绑定接口 the protein recognition ligand is interchangeable to accommodate the required specificity for a targeted kinase, 而纳米孔有利于报告电信号的生成。蛋白激酶分析物 解决方案产生独特的电子签名，该签名随其身份和数量而变化。报告信号是 由纳米孔尖端的配体-激酶组装介导。 In these studies, kinase recognition events will be discriminated at single-molecule precision without the necessity of using complex data analysis algorithms. 这种工程策略大大拓宽了这些纳米传感器的应用范围到各种 激酶及其相互作用。我们的初步研究通过创建一个单一的方法证明了这种方法的力量 分子纳米传感器平台，可探测和定量结构和功能多样的蛋白质 the fundamental limit of sensing inside the nanopore. In addition, such a tactic will enable the detection of 激酶亚型针对相同识别配体的竞争性结合相互作用。这些可以概括的 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 这些纳米传感器在复杂的生物流体中具有高性能。因此，可以使用它们来使用现实的 samples, having prospects in molecular diagnostics. The expected immediate outcomes of this project will be 以下内容：（i）开发用于受体酪氨酸超灵敏分析的高亲和力纳米传感器 激酶（RTK）； (ii) 创建用于探测丝氨酸-苏氨酸激酶的基因编码纳米传感器 （STK）； (iii) the detection and analysis of kinases in multiplexed settings and biofluids.这些研究将 impact healthcare by providing tools and a fundamental framework in biosensor technology, synthetic biology, 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.