Development of a FRET-based imaging method for the study of endogenous and viral RNA in Single Live Cells Phil Santangelo Georgia Tech/Emory Department of Biomedical Engineering   Cellular biophysics is the study of macromolecular events within cells, emphasizing interactions and organization that illuminate function. One such macromolecule that is gaining in interest and importance is ribonucleic acid (RNA). Even with renewed interest in this class of molecules, attempts at detecting, measuring and tracking endogenous RNA in live cells have been relatively unsuccessful until recently. In this presentation I will discuss a general approach based on fluorescence imaging, for the detection and study of endogenous, intracellular RNA in live cells. First, I will introduce a class of nanoscale-photonic switches called molecular beacons. Molecular beacons are dual-labeled oligonucleotide probes designed to form a stem-loop structure in the absence of a complementary target so that fluorescence of the fluorophore is quenched. Hybridization with a target nucleic acid changes the conformation of the probe allowing a fluorescence signal to be emitted upon excitation. The structure-function properties of beacons will be discussed, as will relevant details regarding their use in live cells, such as cellular delivery. Second, I will present imaging data with single and multiple beacons, utilizing fluorescence resonance energy transfer (FRET), in order to measure changes in the quantity and localization of two cancer-related, endogenous messenger RNAs in live cells. Using this method, it was observed directly, and for the first time, that these messenger RNAs colocalize with the mitochondria. In addition, beacons were used to image the genomic RNA of an RNA virus in live cells with signal-to-noise ratios over 50. The replication of the virus in live cells was imaged with the beacon via fluorescence microscopy over 7 days, and the measurement sensitivity was shown by imaging the beacon signal in live cells in response to serial dilutions of virus stock. Using this approach a new view of the organization of viral factories has been observed. Basic biophysical studies, such as the study of viral, life-cycle dynamics, viral RNA interactions with the cytoskeleton, and viral RNA space-function correlations, will also be introduced as will the application of sub-diffraction limited microscopy technology to the study of RNA.