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

EAGER：开发 DNA 编码的植物激酶检测系统

• 批准号: 2006017
• 负责人: Gyeong Mee Yoon
• 金额: $ 29.99万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2006017&HistoricalAwards=false
• 关键词: EAGER; Development; DNA; encoded; Kinase

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.

对环境胁迫或发育线索的适当反应对于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组分，从而相应地调节植物生长。因此，激酶活性的准确检测对于理解植物对各种信号的反应机制至关重要，这可能导致开发具有更好的抗逆性和生产力的新作物。这项研究的目的是开发一种新的检测方法，有助于检测植物中的激酶活性。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，但也使研究人员能够概述动态细胞变化响应于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，他们将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点的磷酸化状态，从而调节植物生长、免疫和应激反应。激酶活性的失调常常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测定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。在药物发现研究中，新兴的基于DNA的酶活性测量在解决该领域对最佳激酶测定的未满足需求方面显示出希望，提供了具有所需方面的激酶工具箱。本提案的目标是开发一种激酶测定法，利用基于DNA的酶活性测量的优势，并通过使用植物激素乙烯和油菜素类固醇信号传导作为模型系统来验证用于体内植物激酶组研究的测定法。拟定的激酶试验使用DNA编码的肽底物作为活性探针，并基于qPCR定量激酶活性。鉴于qPCR通过独特的DNA序列介导的底物肽条形码在检测极低浓度的DNA方面具有高特异性的多功能性，研究人员假设所提出的DNA编码激酶测定应该能够克服当前激酶测定的局限性，并成为植物信号转导研究中有价值的工具。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编码的肽阵列，提供植物细胞磷酸化事件变化的同步定量动态概览。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。