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Genetically Encoded FRET-Based Probes for Live Cell Imaging


Biosensors based on the principle of Förster resonance energy transfer (FRET) have proven to be powerful tools for biological research. Aided by the plethora of color variants of green fluorescent proteins, numerous FRET-based probes have been developed for study of molecular interactions, enzyme activities, and small molecules in live mammalian cells with high spatial and temporal resolution. In this dissertation, we focus on the development of genetically encoded FRET-based probes for cellular processes related to hydrogen sulfide and hypoxic signaling.

Hydrogen sulfide (H2S) has been recently identified as an important gasotransmitter playing crucial roles in cell signaling. We developed the first, genetically encoded, FRET-based biosensor, hsCY, for imaging live-cell H2S. We utilized a blue- (EBFP) and a green- (cpGFP) fluorescent proteins, and genetically modified the probe with p-azidophenylalanine as a H2S-sensory element. We validated hsCY in vitro and mammalian cells and demonstrated the use of hsCY for selective, ratiometric imaging of H2S. hsCY thus represents a valuable addition to the toolbox for H2S detection and imaging.

Oxygen is vital for all aerobic life forms. Oxygen-dependent hydroxylation of hypoxia-inducible factor (HIF)-1α by prolyl hydroxylase domain enzymes (PHDs) is an important step for controlling the expression of oxygen-regulated genes in metazoan species, thereby constituting a molecular mechanism for oxygen sensing and response. Herein, we report a genetically encoded dual-emission ratiometric fluorescent sensor, ProCY, which responds to PHD activities in vitro and in live cells. We demonstrated that ProCY could monitor hypoxia in mammalian cells. By targeting this novel genetically encoded biosensor to the cell nucleus and cytosol, we determined that, under normoxic conditions, the HIF-prolyl hydroxylase activity was mainly confined to the cytosol of HEK 293T cells. The results collectively suggest broad applications of ProCY on evaluation of hypoxia and PHD activities and understanding of pathways for the control of hypoxic responses.

As HIF-prolyl hydroxylases have emerged as promising drug targets for a variety of diseases, such as myocardial infarction, stroke, cancer, diabetes, and severe anemia, we also explored the use of ProCY in high-throughput assays to identify inhibitors or activators of PHDs. Our preliminary study supports the feasibility of this approach. A lentiviral system has been developed for transduction of HEK 293T and establishment of ProCY-expressing stable cells.

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