EAGER: Development of DNA-encoded Kinase Assay System for Plants

对环境压力或发育线索的适当反应对包括植物在内的所有生物的生存至关重要。蛋白激酶是在这些过程中发挥核心作用的酶，通过将外部和内部信号转导到下游细胞成分，从而相应地调节植物的生长。因此，准确检测激酶活性对于了解植物对各种信号的响应机制至关重要，这可能导致开发具有更好的逆境抗性和生产力的新作物。本研究的目的是开发一种新的检测方法，以促进植物中激酶活性的检测。该项目不仅提供了一种新的方法来检测激酶活性在一个简单的方式，而且使研究人员能够概述动态的细胞变化，以响应各种压力和生长条件。该项目还将为研究生和本科生提供研究培训，使他们获得整合分子生物学和化学分析的跨学科技术。蛋白激酶调节细胞靶标的磷酸化状态，从而调节植物生长、免疫和应激反应。激酶活性的失调通常会导致植物整体适应性和胁迫反应的严重缺陷。由于其在植物中的重要作用，激酶活性可以用来推断信号转导的状态和植物的胁迫反应。在这方面，具有高灵敏度、特异性、成本效益、易用性或多路复用能力的激酶活性分析是研究人员准确测量体内和体外激酶活性的关键先决条件。然而，大多数目前的激酶测定仍然缺乏至少一些期望的能力。新兴的基于dna的酶活性测量在药物发现研究中显示出了解决该领域未满足的最佳激酶测定需求的希望，提供了一个具有所需方面的激酶工具箱。本提案的目标是利用基于dna的酶活性测量的优势，开发一种激酶测定方法，并通过使用植物激素乙烯和油菜素内酯信号作为模型系统，验证该方法在体内植物激酶研究中的有效性。提出的激酶测定使用dna编码的肽底物作为活性探针和基于qpcr的激酶活性定量。考虑到qPCR通过独特的DNA序列介导的底物肽条形码检测极低浓度DNA的多功能性和高特异性，研究人员假设提出的DNA编码激酶检测应该能够克服当前激酶检测的局限性，成为植物信号转导研究的有价值的工具。此外，通过利用该检测的多路复用能力和下一代DNA测序，他们将开发DNA编码肽阵列，提供植物细胞磷酸化事件变化的同时定量动态概述。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Appropriate responses to environmental stresses or developmental cues are critical for the survival of all organisms including plants. Protein kinases are enzymes that play a central role during these processes by transducing external and internal signals to downstream cellular components resulting in the modulation of plant growth accordingly. Thus, the accurate detection of kinase activity is critical for understanding the mechanism governing the plant’s response to various signals, which may lead to the development of new crops with better stress resilience and productivity. The goal of this proposed study is to develop a new assay that facilitates the detection of kinase activity in plants. This project not only provides a novel approach to detect kinase activity in a facile manner but also enables the researcher to overview dynamic cellular changes in response to various stresses and growth conditions. The project will also provide research training to graduate and undergraduate students who will acquire interdisciplinary techniques integrating molecular biology and chemical analysis. Protein kinases modulate the phosphorylation status of cellular targets, thereby regulating plant growth, immunity, and stress responses. Dysregulation of kinase activity often leads to severe defects in the overall fitness of plants and stress responses. Because of its critical roles in plants, kinase activity can be used to deduce the status of signal transduction and the plant’s stress response. In this respect, kinase activity assays with high sensitivity, specificity, cost-efficiency, ease of use, or multiplexing capability are critical prerequisites for researchers to accurately measure in vivo and in vitro kinase activity. However, most current kinase assays still lack at least some desired capabilities. Emerging DNA-based activity measurements of enzymes in drug discovery research have shown promise in addressing the unmet need for optimal kinase assays in the field, providing a kinase toolbox with the desired aspects. The goal of this proposal is to develop a kinase assay, taking advantage of the strength of DNA-based enzyme activity measurement, and to validate the assay for in vivo plant kinome studies by using the plant hormone ethylene and brassinosteroid signaling as a model system. The proposed kinase assay uses DNA-encoded peptide substrates as activity probes and qPCR-based quantification of kinase activity. Given the versatility of qPCR in detecting extremely low concentrations of DNA with high specificity through unique DNA sequence-mediated barcoding for substrate peptides, the investigators hypothesize that the proposed DNA-encoded kinase assay should be able to overcome the limitations of current kinase assays and become a valuable tool in plant signal transduction research. Furthermore, by using the multiplexing capability of the assay and next-generation DNA sequencing, they will develop DNA-encoded peptide arrays that provide a simultaneous quantitative dynamic overview of the changes in cellular phosphorylation events in plants.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.