Nucleic acid specific, photoswitchable fluorophores for monitoring RNAs in live cells

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Photochemical, nucleic acid-dependent (templated) reactions can potentially be applied in biological research to study endogenous ribonucleic acids in live cells with spatial and temporal resolution. For example, photoreduction of arylazide-based immolative linkers in the presence of Ru(II)-bipyridine complexes upon irradiation with 455 nm was applied to detect miR-21 in BT474 cells. Few other templated reactions have been reported, which are controlled by UV-light and short-wavelength visible light not exceeding 532 nm. However, such triggers are not ideally suitable for cellular applications. In particular, UV-light causes DNA mutations and is cytotoxic, whereas < 550 nm - visible light is efficiently absorbed by endogeneous riboflavins, thereby generating their excited triplet state. The latter state is quenched in cells with formation of singlet oxygen, superoxide anion radical and other reactive species, which induce DNA mutations, lead to depletion of riboflavins and damage of other biomolecules. These side effects limit cellular applications of photochemical templated reactions. In contrast, since cells are practically insensitive to light at wavelengths exceeding 650 nm, reagents responsive to this trigger are expected to be well compatible with live cells. The objective of this project is to develop caged fluorophores, which are activated by non-toxic red light in the presence of specific ribonucleic acids in live cells. All required components of the system including a caged fluorophore (substrate) and a photocatalyst, will be assembled on the RNA target due to highly specific hybridization reaction. Exposure of this associate to red light will lead to generation of singlet oxygen on the catalyst in close proximity to the substrate. The latter moiety will react with singlet oxygen leading to generation of a bright and photostable fluorophore, which can generate over 1000 photons upon its excitation. This great amplification effect will allow detecting a single dye by using fluorescence microscopy or single molecule fluorescence imaging. Based on this reaction a method for detection, localization and monitoring of rare endogenous RNAs (down to one sequence per cell) in live cells will be developed. The photocatalyst moiety will be optimized in such a way that it generates sufficient amount of the mediator to fully activate the caged fluorophore. However, after this desired reaction is complete, the photocatalyst will be quickly bleached thereby limiting the possible toxic effect of singlet oxygen.