Nucleic acid specific, photoswitchable fluorophores for monitoring RNAs in live cells
Final Report Abstract
Nucleic acid-templated chemical reactions can occur at low concentrations of reagents (<10 -9 moles/L). Therefore, they can be applied for highly sensitive detection of nucleic acids in cell free settings and in cells. Photochemical reactions of this type offer additional advantages of accurate temporal and spatial control. However, majority of them are triggered by toxic to cells light at wavelengths < 550 nm that limits their broad applications. The goal of this project was to develop template reactions triggered by nontoxic red light, allow detecting < 10 -10 moles/L nucleic acids and compatible with live cells. We tested a variety of substrates for such reactions based on conjugates of oligonucleotides and their chemically modified analogues with derivatives of anthracene, azobenzene as well as arylalkylselenides. Moreover, we optimized a nucleic acid-triggered fluorogenic response by a variation of substrate structure and by using quenchers for both substrates and catalysts. Finally, we investigated the applicability of a series of commercially available as well as experimental transfection agents for enabling cell membrane permeability of substrates and catalysts of templated reactions. The best combination of substrates, catalysts and quenchers led to the templated reaction that allows detecting down to 10 -11 moles/L nucleic acids that is a 200-fold improvement over previously known red light triggered photochemical templated reactions. We confirmed that the optimized reaction is compatible with live cells and can be used to detect abundant mRNAs like ß-actin-mRNA. However, less abundant RNAs could not be detected due to insufficient transfection efficiency of substrates and catalysts.
Publications
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Cu(II)-complexes as quenchers of photocatalytic activity of visible light-absorbing photosensitizers: an application in detection of nucleic acids. Inorg. Chim. Acta, 2016, 452, 118-124
Schikora, M.; Mokhir, A.
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DNA-dye-conjugates: conformation and spectra of fluorescence probes. PLOS ONE, 2016, 11(7), e0160229
Beierlein, F. R.; Palomo, M. P.; Sharapa, D.I.; Zozulia, O.; Mokhir, A.; Clark, T.
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Endoperoxides revealed as origin of toxicity of graphene oxide. Angew. Chem. Int. Ed. 2016, 55(1), 405-407
Pieper, H.; Chercheja, S.; Eigler, S.; Halbig, C.; Filipovic, M. R.; Mokhir, A.
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Oxo-functionalized graphene as cell membrane carrier of nucleic acids probes controlled by aging. Chem. Eur. J., 2016, 22(43), 15389-15395
Pieper, H.; Halbig, C. E.; Kovbasyuk, L.; M. R. Filipovic, Eigler, S.; Mokhir, A.
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[1,10]Phenanthroline based cyanine dyes as fluorescent probes for ribonucleic acids in live cells. Methods and Applications in Fluorescence, 2017, 5(4), 045002
Kovalska, V.; Kuperman, M.; Kryvorotenko, D.; Kinski, E.; Schikora, M.; Varzatskii, O.; Janko, C.; Alexiou, C.; Yarmoluk, S.; Mokhir, A.
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Hybrids of a 9- Anthracenyl Moiety and Fluorescein as Chemodosimeters for Detection of Singlet Oxygen in Live Cells. Org. Biomol. Chem., 2019, 17, 9883-9891
Chercheja, S.; Daum, S.; Xu, H.; Beierlein, F.; Mokhir, A.
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Red Light Triggered Fluorogenic Reaction with Picomolar Sensitivity Towards Nucleic Acids. Bioconjugate Chem., 2019, 30(7), 2023-2031
Zozulia, O.; Bachmann, T.; Mokhir, A.
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Red Light-Triggered Nucleic Acid-Templated Reaction Based on Cyclic Oligonucleotide Substrates. Chem. Comm., 2019, 55(72), 10713-10716
Zozulia, O.; Bachmann, T.; Deussner-Helfmann, N.; Beierlein, F.; Heilemann, M.; Mokhir, A.
