A Modular Toolset for Imaging Cellular Metabolites Using Fluorogenic RNA Riboswitches

About This Grant

Metabolites such as sugar and fatty acids are essential for maintaining human health because they influence energy supply, growth, and cell signaling. Abnormal metabolic changes can lead to diseases such as diabetes, cancer, and heart conditions. Detecting metabolites in living systems in real-time is crucial, but conventional methods require obtaining and destroying samples, which means they can't be used to measure metabolites in individual cells. This project addresses the gap by developing fluorescent probes for highly sensitive and selective detection of key metabolites within living cells. These probes will enhance research in human health, nutrition, plant science, food safety, and environmental studies. Additionally, the project will support interdisciplinary scientist training and engage middle school students through workshops and lab tours. The specific goal of this project is to develop genetically encodable RNA-based fluorescent probes for metabolic imaging and profiling in living cells. These probes comprise three modular domains: a target-binding riboswitch, a fluorogenic RNA reporter, and a transducer. Fluorogenic RNA aptamers can selectively bind and activate the cellular fluorescence of chemical dyes. Riboswitches are natural metabolite-sensing RNA regulatory elements. About 60 distinct classes of riboswitches have been identified so far, covering a wide spectrum of important nucleotides, amino acids, ions, enzyme cofactors, signaling molecules, etc. Upon binding target metabolites, riboswitches can undergo rapid conformational change. The transducer here can be a simple RNA duplex or structural switching RNA sequence that will convert the conformational change of riboswitches into the activation of fluorogenic RNA signals. The major objectives are: (1) to engineer modular allosteric fluorogenic RNA “Broccoli”-based metabolite sensors with a duplex transducer and many-to-most existing class of riboswitches; (2) to develop a new in silico platform to further improve the throughput of probe design and their sensitivities; and (3) to obtain quantitative, multi-colored, and time-resolved imaging profiles of diverse metabolites in individual living bacterial and mammalian cells 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.