MFB: Dynamic Cellular RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Ralph Kleiner at Princeton University and Dr. Byron Purse at San Diego State University to develop a general and accessible method to track the synthesis, localization, and dynamics of RNA messages in living cells. In this project, synthetic ribonucleosides are introduced into cells, which can use its metabolic mechanisms to incorporate these nucleic acid building blocks into making fluorescent RNAs. Imaging these RNAs in living cells through fluorescence microscopy enables studies on the dynamics of RNA synthesis, along with how proteins bind to and disengage from RNAs in cellular structures. Such studies provide new insights into the organization of cellular structures that play important roles in biological and biotechnological processes and how they respond to environmental stimuli. This RNA imaging method will be made available to the broader scientific community to enable mechanistic investigation of cellular RNA biology across diverse systems. This project provides interdisciplinary training to graduate students and postdoctoral fellows in organic synthesis, chemical biology, RNA nucleotide metabolism, RNA mass spectrometry, and cellular microscopy. This proposal advances live-cell RNA imaging with fluorescent ribonucleosides from proof-of-concept to broad applicability by developing improved fluorescent ribonucleoside probes exhibiting increased brightness, photostability, and “turn-on” fluorescence, commensurate with established small molecule and protein-based fluorophores. Accordingly, new methods are developed to deliver enhanced fluorescent ribonucleosides using refined fluorophore designs and chemical prodrug strategies. Our methods are benchmarked in diverse mammalian cells by imaging RNAs in biomolecular condensates including the nucleolus and cytoplasmic RNA-protein granules. The three aims of the project are to 1) expand the toolkit of fluorescent ribonucleosides to maximize uptake and metabolic incorporation and optimize imaging sensitivity; 2) use masked, cell-permeable fluorescent ribonucleoside monophosphates to develop RNA labeling methods that are independent of the nucleoside kinase UCK2; and 3) establish the generality of fluorescent ribonucleosides for imaging biomolecular condensates in an expanded set of cell lines. 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.