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Fluorescent Turn‐On Probes for the Development of Binding and Hydrolytic Activity Assays for mRNA Cap‐Recognizing Proteins
Author(s) -
Kasprzyk Renata,
Starek Beata J.,
Ciechanowicz Sylwia,
Kubacka Dorota,
Kowalska Joanna,
Jemielity Jacek
Publication year - 2019
Publication title -
chemistry – a european journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.687
H-Index - 242
eISSN - 1521-3765
pISSN - 0947-6539
DOI - 10.1002/chem.201900051
Subject(s) - fluorescence , chemistry , messenger rna , turn (biochemistry) , biochemistry , microbiology and biotechnology , biology , gene , quantum mechanics , physics
The m 7 G 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 m 7 G 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 m 7 GTP 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.