Carbonic anhydrase (CA) is universally responsible for catalyzing the rapid interconversion between carbon dioxide to bicarbonate. It plays a key role in respiration, ion transport, and pH balance. The type of CA isoform and its specific degree of expression both contribute to diseases such as glaucoma and cancer. Our goal is to target and map out the zinc-bound CA present in cells with a customizable fluorescent molecule design. Three components comprise our fluorescent probe: (a) a commonly available fluorophore like fluorescein that governs organelle localization, (b) a fluorescein quencher that also carries the sulfonamide metal binding group (MBG) for CA, and (c) a linker that connects the fluorophore to the quencher/MBG. The linker length is key in bringing the quencher close to the fluorophore and thus keeping the fluorescence quenched or in its ‘off’ state. Consequently, the probe is turned back “on” once the MBG binds to CA and the ends come apart. This turn ‘off-on’ mechanism works mainly via static quenching and FRET spectral overlap. This design is governed by suitable fluorophore-quencher system which is specific for CA interaction. Our target fluorophore-quencher combinations allow efficient quenching (up to 80%), high fluorescence quantum yields, good solubility and biological sensing of CA. While quenched, molecules have previously been employed for nucleic acid sensing, their application in protein sensing have been very limited- mainly due to the need for tuning for every metalloprotein. The interaction of our molecules with CA is characterized spectroscopically using UV-Vis spectroscopy and fluorimetry. Several biochemical assays for fluorescence, quantum yield, and NPA are being conducted to investigate the interactions between the probe and CA. Ongoing work is also focused on conducting confocal microscopy to visualize and track carbonic anhydrase in mammalian cells using the synthesized fluorophores.

Quenched-fluorophore design for carbonic anhydrase sensing

2019 Fall National Meeting

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