Recent studies from many labs have uncovered great complexity in cellular RNA biology, and critically important connections of RNA to human health. It is becoming increasingly apparent that RNA biology, like protein biology, is not merely peripheral, but rather central to cellular phenotypes and pathologies. Unfortunately, methods for study of RNA, such as tools for functionalization, labeling and control, lag well behind those used widely for proteins. Preliminary experiments have established the promise of a suite of novel molecular strategies for study of RNAs, based on multifunctional acylating agents that react at the 2'-OH group. This started with the development of the first cell-permeable acylating agents, based on a nicotinyl scaffold, that react with accessible 2'-OH groups in RNAs. These reagents allow unprecedented measurement of RNA structure and protein-RNA interactions in vivo at nucleotide resolution. In unpublished work, studies have shown that an azide functional handle can be employed on these acylating scaffolds to enable mild, bioorthogonal reversal of the acylation by Staudinger reduction. Excitingly, experiments show that this acylation/deacylation strategy can be used to block and initiate hybridization of RNA. Moreover, the data establishes that a label can be incorporated into such an acylating agent, enabling one-step, reversible fluorescent labeling of native RNA. These preliminary experiments suggest a suite of new acylating reagents as tools to isolate, immobilize, label, and analyze RNAs, and a range of molecular strategies to control their biological activities with chemical or optical signals. During the term of this project, the development of reversible protecting reagents for stabilizing and capturing RNAs from biological samples are proposed. Reagents for covalent delivery and release of RNAs into cells are also described. Further, new fluorescent acylating agents and methods will be developed and employed to measure protein-RNA interactions. Finally, a novel range of unprecedented chemical caging and release strategies will be developed for controlling biological function of RNAs in living systems, enabling initiation of mRNA expression, RNA folding, and gene editing in time and space. This work is significant because it will develop enabling molecular technologies that will greatly enhance the study of RNA biology and biomedicine. This new premise of multifunctional acylation will lead to universal and easy-to-use reagents that will markedly improve the isolation, analysis, delivery, and control of RNAs for researchers worldwide. Unlike previous methods, these reagents will function with large and native RNAs, and are simple enough that non-chemists can apply them. The research program is innovative because it develops a suite of new molecular probes and novel molecular strategies, making use of the concept of reversible labeling and functionalization of RNA via new selective bond-forming and ?breaking strategies.
/ Public Health Relevance Statement Our proposed research is aimed at developing new chemical tools and methods for studying RNA molecules, many of which are directly linked to disease. We have identified a new and general molecular strategy for linking molecules to RNAs in a reversible way. This work will develop these molecular designs into a suite of chemical tools for isolating, detecting, analyzing, and controlling RNAs, thus enabling new science on their roles in human health.
