Rational Design of FRET-Based Ratiometric Chemosensors for in Vitro and in Cell Fluorescence Analyses of Nucleoside Polyphosphates

Abstract

Ratiometric fluorescence sensing is a useful technique for the precise and quantitative analysis of biological events occurring under complex conditions, such as those inside cells. We report herein the design of new ratiometric chemosensors for nucleoside polyphosphates such as ATP that are based on binding-induced modulation of fluorescence resonance energy transfer (FRET) coupled with a turn-on fluorescence-sensing mechanism. We designed these new FRET-based ratiometric chemosensors by utilizing spectral overlap changes to modulate the FRET efficiency. Introduction of coumarin fluorophores as the FRET donors into a binuclear zinc complex as the FRET acceptor provided the ratiometric chemosensors. These chemosensors exhibited a clear dual-mission signal change upon binding with strong affinity (K(app) ≈ 10(6)-10(7) M(-1)) to nucleoside polyphosphates in aqueous solution, whereas no detectable emission change was observed with monophosphates and phosphodiester species or various other anions. These chemosensors were used for real-time fluorescence monitoring of enzyme reactions such as saccharide synthesis by glycosyltransferase and phosphorylation by protein kinase, both of which involve nucleoside polyphosphates as substrates. The utility of ratiometric sensing by chemosensors was further demonstrated in a fluorescence-imaging study of the nucleoside polyphosphates inside living cells, wherein we ratiometrically visualized the stimulus-responsive concentration change of ATP, an indicator of the cellular energy level.

Ternary complex formation and competition quench fluorescence of ZnAF family zinc sensors

“…The remaining fluorescence of zinc-free sensors, corrected for dilution, was expressed as fraction of the Zn(sensor) complex. Additionally, the Zn 2+ content of the 0.1 M Hepes buffer was controlled by ICP-MS, with a satisfactory agreement 10 with the results obtained using sensors. The published conditional Zn(II) binding constants of ZnAF-1 and ZnAF-2F were determined by competitive fluorescence titrations with NTA.…”

“…22 5 ATP is known to require Mg 2+ ions for its biological activity, and the association constant of the Mg(ATP) complex is sufficiently high to assure that the majority of ATP molecules will be complexed in vivo if adequate amounts of Mg 2+ ions are available. 21 On the other hand, the levels of ATP may be much 10 higher from those of Mg 2+ ions locally. 24 Therefore, experiments with ATP were performed in the absence and presence of equimolar amounts of MgCl 2 .…”

“…Titrations of ZnAF-1 with His and ATP were also followed by 10 UV-vis spectroscopy. A higher, 10 µM sensor concentration was chosen for better detection of the spectrum.…”

CR1-mediated ATP Release by Human Red Blood Cells Promotes CR1 Clustering and Modulates the Immune Transfer Process

“…Visualization of CR1-mediated ATP Release Using a Fluorescent ATP-sensing Probe-To directly image ATP release promoted by RBC CR1 ligation, we employed a novel fluorescent nucleotide-sensing probe, 2-2Zn(II), which binds the extracellular side of the plasma membrane, and upon interaction with ATP, fluoresces in an ATP concentration-dependent manner (13,19,20). First, to test the specificity of the probe for ATP, 2-2Zn(II)-treated RBC were incubated at room temperature (5 min) in the presence or absence of ATP diphosphohydrolase (apyrase) and subsequently analyzed by flow cytometry.…”

Pillararene-based fluorescent indicator displacement assay for the selective recognition of ATP

“…Since the nucleotides, especially ATP is pivotal in living systems [38],numerous fluorescent chemosensors have been developed for their detection [39][40][41][42][43], involving direct sensing [44,45], cation-based recognition units [46][47][48][49][50][51][52] and also some indicator displacement assays [53][54][55][56][57].…”