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, photostabi