Glyoxal-Based Caging for Temporal Control of Nucleic Acid Function

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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.