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Fluorescent turn-on probes for the development of binding and hydrolytic activity assays for mRNA cap-recognising proteins
摘要： 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 essential for cell growth and function. To provide small molecular probes to study important cap-recognising proteins, we synthesized m7G nucleotides labelled 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-labelled compounds behaved as sensitive turn-on probes. A pyrene-labelled m7GTP analogue showed up to 8-fold 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.
关键词： eIF4E,fluorescence assay,cap,pyrene,DcpS
更新于2025-11-14 15:32:45
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Green fluorescent carbon quantum dots functionalized with polyethyleneimine, and their application to aptamer-based determination of thrombin and ATP
摘要： Brightly fluorescent carbon quantum dots coated with polyethylenimine (PEI-CDs) were prepared using malic acid and PEI as the precursors. The PEI-CDs have a high quantum yield (41%) and green emission (peaking at 502 nm under 430 nm excitation), both of which are not affected by high ionic strength. The PEI-CDs have a positive charge at physiological pH values and can electrostatically bind aptamers with their negative charge. This is shown for aptamers binding thrombin or ATP. Binding of aptamers results in quenching of fluorescence. If thrombin or ATP are introduced, the respective aptamer will bind them, and the complex is then released from the PEI-CDs. Fluorescence increases in proportion to the analyte concentration. Under optimized conditions, thrombin and ATP can be sensitively and selectively detected by fluorometry with lower detection limits of 1.2 and 13 nM, respectively. The assay was successfully applied to the determination of thrombin and of ATP in spiked serum samples.
关键词： Aptamer sensor,Thrombin detection,Serum analysis,Green luminescent,PEI-CDs,Malic acid,Polyethylenimine,Fluorescence assay,ATP detection,Hydrothermal reaction
更新于2025-09-19 17:13:59