Glyoxal-Based Caging for Temporal Control of Nucleic Acid Function

在化学系生命过程化学(CLP)项目的支持下，埃默里大学的詹妮弗·希姆斯特拉教授正在研究一种新的方法，可以可逆地将核酸序列关在笼子里，以控制它们的功能。核酸在细胞内的信息存储和催化中发挥着关键作用，控制这些功能的能力可以显著推动对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体的细胞室中。控制核酸功能的方法已经存在，但在什么类型的核酸可以被关在笼子里，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，乙二醛提供了一种多功能和成本效益高的方法来解决许多这些限制，拟议的研究将探索新的方法，以实现对笼子位置和重新激活速度的额外控制。这些方法随后将被应用于研究短RNA向生物分子凝聚体的运输。拟议的研究将通过解决与发展核酸传感器和新的潜在基因编辑方法有关的基本问题，推动生物技术的发展，从而对社会产生积极影响。这项研究有可能为研究生物分子凝聚体在强直性肌营养不良和肌萎缩侧索硬化症(ALS)等神经退行性疾病中的作用提供工具。该项目还将通过向STEM(科学、技术、工程和数学)学生提供专业发展和教育资源，促进科学队伍的发展。这项建议的总体目标是探索乙二醛和相关类似物，用于选择性笼化和对刺激反应的核酸降解。实现对核酸结构和功能的时间控制将为体外研究核酸的性质提供强有力的工具，目的是将这种方法应用于活细胞来研究生物功能。虽然已经报告了许多笼化方法，但在可被笼养的核酸的类型和可重新激活它们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，与其他方法不同，因为它通过与核苷酸碱基的沃森-克里克-富兰克林面反应，实现了核酸的后合成笼化。这会暂时扰乱碱基配对，导致结构变性和功能丧失。方便的是，笼中的寡核苷酸在室温下是稳定的，但在生理温度下，笼中的寡核苷酸在几个小时到几天的时间里是反向的。我们建议进一步发展这一方法，并应用我们的方法来理解RNA在液-液相分离(LLP)中形成生物分子凝聚体的作用。具体地说，我们将并行追求以下三个目标：(1)开发选择性地对长RNA的特定片段进行笼化的“化学光刻”；(2)探索用于掩盖DNA和XNAs碱基的改良双醛试剂；(3)利用乙二醛笼化来研究SnRNA结构和蛋白质结合在Cajal小体的形成和RNA运输中的作用。这项拟议的研究有望通过提供工具来推动生物技术的发展，并通过为学生创建和传播教育和职业发展资源来造福社会。该奖项反映了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。

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.