Fluorescent Turn-On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap-Recognizing Proteins |
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Authors: | Renata Kasprzyk Beata J Starek Sylwia Ciechanowicz Dorota Kubacka Joanna Kowalska Jacek Jemielity |
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Institution: | 1. Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097 Warsaw, Poland;2. Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Pasteura 5, 02-093 Warsaw, Poland |
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Abstract: | The m7G cap is a unique nucleotide structure at the 5′-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m7G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m7GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells. |
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Keywords: | dyes/pigments fluorescent probes protein expression pyrene RNA structures |
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