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有机体的生存至关重要。蛋白激酶是在这些过程中发挥核心作用的酶，它将外部和内部信号传递到下游的细胞成分，从而调节植物的生长。因此，准确检测激酶活性对于了解植物对各种信号的反应机制至关重要，这可能会导致开发出具有更好的逆境适应性和生产力的新作物。这项拟议的研究的目标是开发一种新的分析方法，以便于检测植物中的激酶活性。这个项目不仅提供了一种新的方法来检测激酶活性，而且还使研究人员能够概述细胞在不同压力和生长条件下的动态变化。该项目还将为研究生和本科生提供研究培训，这些学生将获得结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，从而调节植物的生长、免疫和逆境反应。激酶活性的失调往往会导致植物整体适应性和逆境反应的严重缺陷。由于其在植物中的关键作用，它的活性可以用来推断信号转导状态和植物的逆境反应。在这方面，具有高灵敏度、高特异度、高性价比、易用性或多重复性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前提。然而，目前的大多数激酶分析仍然缺乏至少一些所需的功能。在药物发现研究中，新出现的基于DNA的酶活性测量在解决该领域对最佳激酶分析的未得到满足的需求方面表现出了希望，提供了具有所需方面的激酶工具箱。该方案的目的是利用基于DNA的酶活性测量的优势，建立一种激酶分析方法，并以植物激素乙烯和油菜素类固醇信号转导系统为模型系统，验证该方法用于活体植物动态组研究。该方法使用DNA编码的多肽底物作为活性探针，并基于定量聚合酶链式反应(QPCR)定量检测蛋白激酶活性。鉴于qPCR通过独特的DNA序列介导的底物多肽条形码来检测极低浓度的高特异性DNA的多功能性，研究人员推测，所提出的DNA编码的激酶分析应该能够克服现有激酶分析的局限性，成为植物信号转导研究中的一个有价值的工具。此外，通过使用分析和下一代DNA测序的多路复用能力，他们将开发DNA编码的多肽阵列，提供植物细胞磷酸化事件变化的同步定量动态概述。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.