Glyoxal-Based Caging for Temporal Control of Nucleic Acid Function

用于核酸功能时间控制的乙二醛封闭

• 批准号: 2306047
• 负责人: Jennifer Heemstra
• 金额: $ 44.48万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2025-09-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2306047&HistoricalAwards=false
• 关键词: Glyoxal; Based; Caging; Temporal; Control

With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Professor Jennifer Heemstra of Emory University is studying a new approach to reversibly “cage” nucleic acid sequences in order to control their function. Nucleic acids play a critical role in information storage and catalysis within cells and the ability to control these functions can significantly advance the study of important biological questions, such as when and how nucleic acids are sequestered in cellular compartments known as biomolecular condensates. Methods exist for controlling nucleic acid function, but still have limitations with regard to what types of nucleic acids can be caged and the time scales for decaging and restoration of activity. The Heemstra lab has found that glyoxal provides a versatile and cost-effective approach to addressing many of these limitations, and the proposed research will explore new methods for achieving additional control over the location of caging and speed of reactivation. These methods will subsequently be applied to study the trafficking of short RNAs to biomolecular condensates. The proposed research will positively impact society by advancing biotechnologies by addressing fundamental questions related to the development of nucleic acid sensors and new potential approaches to gene editing. The research has the potential to provide tools to study the role of biomolecular condensates in neurodegenerative diseases such as myotonic dystrophy and amyotrophic lateral sclerosis (ALS). This project will also contribute to the development of the scientific workforce by providing professional development and educational resources to STEM (science, technology, engineering and mathematics) students.The overarching objective of this proposal is to explore glyoxal and related analogues for selective caging and stimuli-responsive decaging of nucleic acids. Achieving temporal control over the structure and function of nucleic acids would provide a powerful tool to study their properties in vitro with the goal of deploying such methods in living cells to study biological function. While numerous caging methods have been reported, significant limitations remain with regard to the types of nucleic acids that can be caged and the time scales upon which they can be reactivated. The approach taken here, using glyoxal, is distinct from other approaches in that it enables post-synthetic caging of nucleic acids through reaction with the Watson-Crick-Franklin face of nucleobases. This temporarily disrupts base-pairing, leading to denaturation of structure and loss of function. Conveniently, caged oligonucleotides are stable at room temperature, but caging is reversed over hours to days at physiological temperature. We propose to further develop this approach and apply our method to understand the role of RNA in liquid-liquid phase separation (LLPS) to form biomolecular condensates. Specifically, we will pursue the following three objectives in parallel: (1) Develop “chemical lithography” for selective caging of specific segments of long RNAs; (2) Explore modified bis-aldehyde reagents for nucleobase cloaking of DNA and XNAs; (3) Utilize glyoxal-caging to study the role of snRNA structure and protein binding in the formation and RNA trafficking of Cajal bodies. The proposed research is expected to benefit society by providing tools to advance biotechnology and through the creation and dissemination of educational and professional development resources for students.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.

在化学系生命过程化学（CLP）项目的支持下，埃默里大学的Jennifer Heemstra教授正在研究一种可逆“笼”核酸序列的新方法，以控制其功能。核酸在细胞内的信息存储和催化中起着至关重要的作用，控制这些功能的能力可以显著推进重要生物学问题的研究，例如核酸何时以及如何被隔离在称为生物分子凝聚物的细胞隔间中。控制核酸功能的方法已经存在，但在哪些类型的核酸可以被笼化以及降解和恢复活性的时间尺度方面仍然存在局限性。Heemstra实验室发现乙二醛提供了一种通用且经济的方法来解决许多这些限制，并且提出的研究将探索新的方法来实现对笼化位置和再激活速度的额外控制。这些方法将随后应用于研究短rna到生物分子凝聚体的运输。该研究将解决与核酸传感器开发和基因编辑新方法相关的基本问题，从而推进生物技术，对社会产生积极影响。该研究有可能为研究生物分子凝聚物在神经退行性疾病如肌强直性营养不良和肌萎缩侧索硬化症（ALS）中的作用提供工具。该项目还将通过为STEM（科学、技术、工程和数学）学生提供专业发展和教育资源，促进科学劳动力的发展。本提案的总体目标是探索乙二醛及其相关类似物对核酸的选择性笼化和刺激反应性老化的作用。实现对核酸结构和功能的时间控制将为在体外研究其特性提供一个强大的工具，目标是在活细胞中部署这种方法来研究生物功能。虽然已经报道了许多笼化方法，但在可以笼化的核酸类型和它们可以重新激活的时间尺度方面仍然存在重大限制。这里采用的方法，使用乙二醛，不同于其他方法，因为它可以通过与核碱基的沃森-克里克-富兰克林面反应来实现核酸的合成后笼化。这会暂时破坏碱基配对，导致结构变性和功能丧失。方便的是，笼化寡核苷酸在室温下是稳定的，但在生理温度下，笼化在数小时到数天内会发生逆转。我们建议进一步发展这种方法，并应用我们的方法来理解RNA在液-液相分离（LLPS）中形成生物分子凝聚体的作用。具体而言，我们将并行追求以下三个目标：(1)开发“化学光刻”技术，用于选择性地保存长rna的特定片段；(2)探索修饰双醛试剂用于DNA和XNAs的核碱基掩盖；(3)利用乙二醛笼化技术研究snRNA结构和蛋白质结合在Cajal小体形成和RNA转运中的作用。这项拟议的研究可望提供工具，以促进生物科技的发展，并为学生创造和传播教育和专业发展资源，从而造福社会。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。