Large Increase In Fluorescence Research Articles

Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Competitive fluorescence assay for Cd2+ based on aptamer structure-switching

Cadmium ion (Cd2+) is a heavy metal pollutant that causes serious effects on human health. Development of a simple and specific Cd2+ detection method is of great significance in food safety and environmental monitoring. In this work, we demonstrated a fluorescence assay for Cd2+ based on aptamer structure-switching. A short 17-mer aptamer against Cd2+ with fluorescein (FAM) labeled at the 5′ end was used as an affinity ligand, and a partially complementary DNA (cDNA) with black hole quencher 1 (BHQ1) labeled at the 3′ end was used as quencher. In the absence of Cd2+, the aptamer hybridized with the cDNA, forming a duplex helix structure that showed low fluorescence intensity as FAM was quenched by adjacent BHQ1. In the presence of Cd2+, the aptamer specially bound to Cd2+, and the FAM-labeled aptamer dissociated from the aptamer-cDNA duplex, showing high fluorescence intensity. We examined the effects of different lengths of cDNA, the composition of binding buffer, incubation time, and incubation temperature on the performance of the sensing system. Under the optimal experimental conditions, we achieved sensitive and selective detection of Cd2+ with a detection limit of 2.4 nM by using the 17-mer aptamer and 13-mer cDNA. The FAM-labeled aptamer exhibited a large fluorescence increase upon Cd2+, with a maximum change about 51.6-fold. The developed assay also showed good responses to Cd2+ in actual water samples, showing the potential for practical application.

Redox-Controlled Energy Transfer Quenching of Fluorophore-Labeled DNA SAMs Enables In Situ Study of These Complex Electrochemical Interfaces.

Interfaces modified by a molecular monolayer can be challenging to study, particularly in situ, requiring novel approaches. Coupling electrochemical and optical approaches can be useful when signals are correlated. Here we detail a methodology that uses redox electrochemistry to control surface-based fluorescence intensity for detecting DNA hybridization and studying the uniformity of the surface response. A mixed composition single-strand DNA SAM was prepared using potential-assisted thiol exchange with two alkylthiol-modified ssDNAs that were either labeled with a fluorophore (AlexaFluor488) or a methylene blue (MB) redox tag. A significant change in fluorescence was observed when reducing MB to colorless leuco-MB. In situ fluorescence microscopy on a single-crystal gold bead electrode showed that fluorescence intensity depended on (1) the potential controlling the oxidation state of MB, (2) the surface density of DNA, (3) the MB:AlexFluor488 ratio in the DNA SAM, and (4) the local environment around the DNA SAM. MB efficiently quenched AlexaFluor488 fluorescence. Reduction of MB showed a significant increase in fluorescence resulting from a decrease in quenching or energy transfer efficiency. Hybridization of DNA SAMs with its unlabeled complement showed a large increase in fluorescence due to MB reduction for surfaces with sufficient DNA coverage. Comparing electrochemical-fluorescence measurements to electrochemical (SWV) measurements showed an improvement in detection of a small fraction of hybridized DNA SAM for surfaces with optimal DNA SAM composition and coverage. Additionally, this coupled electrochemical redox-fluorescence microscopy method can measure the spatial heterogeneity of electron-transfer kinetics and the influence of the local interfacial environment.

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a Kd of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

Visualization of Dynamic Mitochondrial Calcium Fluxes in Isolated Cardiomyocytes

BackgroundCardiomyocyte contraction requires a constant supply of ATP, which varies depending on work rate. Maintaining ATP supply is particularly important during excitation-contraction coupling, where cytosolic Ca2+ fluxes drive repeated cycles of contraction and relaxation. Ca2+ is one of the key regulators of ATP production, and its uptake into the mitochondrial matrix occurs via the mitochondrial calcium uniporter. Fluorescent indicators are commonly used for detecting cytosolic Ca2+ changes. However, visualizing mitochondrial Ca2+ fluxes using similar methods is more difficult, as the fluorophore must be permeable to both the sarcolemma and the inner mitochondrial membrane. Our aim was therefore to optimize a method using the fluorescent Ca2+ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes.MethodsHealthy, adult male Wistar rat hearts were isolated and enzymatically digested to yield rod-shaped, quiescent ventricular cardiomyocytes. The fluorescent Ca2+ indicator Rhod-2 was reduced to di-hydroRhod-2 and confocal microscopy was used to validate mitochondrial compartmentalization. Cardiomyocytes were subjected to various pharmacological interventions, including caffeine and β-adrenergic stimulation. Upon confirmation of mitochondrial Rhod-2 localization, loaded myocytes were then super-fused with 1.5 mM Ca2+ Tyrodes containing 1 μM isoproterenol and 150 μM spermine. Myocytes were externally stimulated at 0.1, 0.5 and 1 Hz and whole cell recordings of both cytosolic ([Ca2+]cyto) and mitochondrial calcium ([Ca2+]mito) transients were made.ResultsMyocytes loaded with di-hydroRhod-2 revealed a distinct mitochondrial pattern when visualized by confocal microscopy. Application of 20 mM caffeine revealed no change in fluorescence, confirming no sarcoplasmic reticulum compartmentalization. Myocytes loaded with di-hydroRhod-2 also showed a large increase in fluorescence within the mitochondria in response to β-adrenergic stimulation (P < 0.05). Beat-to-beat mitochondrial Ca2+ transients were smaller in amplitude and had a slower time to peak and maximum rate of rise relative to cytosolic calcium transients at all stimulation frequencies (P < 0.001).ConclusionMyocytes loaded with di-hydroRhod-2 revealed mitochondrial specific compartmentalization. Mitochondrial Ca2+ transients recorded from di-hydroRhod-2 loaded myocytes were distinct in comparison to the large and rapid Rhod-2 cytosolic transients, indicating different kinetics between [Ca2+]cyto and [Ca2+]mito transients. Overall, our results showed that di-hydroRhod-2 loading is a quick and suitable method for measuring beat-to-beat [Ca2+]mito transients in intact myocytes.

Enantioselective Synthesis and Application of Small and Environmentally Sensitive Fluorescent Amino Acids for Probing Biological Interactions.

Environmentally sensitive fluorescent amino acids (FlAAs) have been used extensively to probe biological interactions. However, most of these amino acids are large and do not resemble amino acid side chains. Here, we report the enantioselective synthesis of two small and environmentally sensitive fluorescent amino acids bearing 7-dialkylaminocoumarin side chains by alkylation of a Ni(II) glycine Schiff base complex. These amino acids exhibit a large increase in fluorescence as environment polarity decreases. One of these FLAAs was incorporated into a highly active analog of the cyclic lipopeptide antibiotic paenibacterin by Fmoc solid-phase peptide synthesis via a new and very efficient route. This peptide was used to probe the interaction of the antibiotic with model liposomes, lipopolysaccharides, and live bacteria.

Capped cadmium telluride quantum dots fluorescence enhancement by Se(IV) and its application to dietary supplements analysis

A thioglycolic capped CdTe probe for Se(IV) detection was optimised by nanocrystal size, pH, buffer composition and concentration, and the order of reagent addition. A large increase in fluorescence (>90%) was observed upon sequential addition of Se(IV), buffer, and CdTe probe, caused by surface passivation due to the presence of singly charged Se(IV) in ammoniacal buffer (0.01 mol L–1) at pH 8.4. This result indicated the possibility of inorganic Se speciation. The method presented a detection limit of 60.5 μg L–1 with RSD ≤ 3.5% (n = 8) and was successfully applied to dietary supplements analysis.

Michler’s hydrol blue elucidates structural differences in prion strains

Yeast prions provide self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that confer distinct phenotypes when introduced into cells that do not carry the prion. Classic dyes, such as thioflavin T and Congo red, exhibit large increases in fluorescence when bound to amyloids, but these dyes are not sensitive to local structural differences that distinguish amyloid strains. Here we describe the use of Michler's hydrol blue (MHB) to investigate fibrils formed by the weak and strong prion fibrils of Sup35NM and find that MHB differentiates between these two polymorphs. Quantum mechanical time-dependent density functional theory (TDDFT) calculations indicate that the fluorescence properties of amyloid-bound MHB can be correlated to the change of binding site polarity and that a tyrosine to phenylalanine substitution at a binding site could be detected. Through the use of site-specific mutants, we demonstrate that MHB is a site-specific environmentally sensitive probe that can provide structural details about amyloid fibrils and their polymorphs.

Next-generation GRAB sensors for monitoring dopaminergic activity in vivo.

Dopamine (DA) plays a critical role in the brain, and the ability to directly measure dopaminergic activity is essential for understanding its physiological functions. We therefore developed red fluorescent GPCR-activation–based DA (GRABDA) sensors and optimized versions of green fluorescent GRABDA sensors. In response to extracellular DA, both the red and green GRABDA sensors exhibit a large increase in fluorescence, with subcellular resolution, subsecond kinetics, and nanomolar to submicromolar affinity. Moreover, the GRABDA sensors resolve evoked DA release in mouse brain slices, detect evoked compartmental DA release from a single neuron in live flies, and report optogenetically elicited nigrostriatal DA release as well as mesoaccumbens dopaminergic activity during sexual behavior in freely behaving mice. Co-expressing red GRABDA with either green GRABDA or the calcium indicator GCaMP6s allows simultaneously tracking neuronal activity and dopaminergic signaling in distinct circuits in vivo.

Molecular Mechanism and Functional Role of Hypoxia‐induced Reactive Oxygen Species in Pulmonary Arterial Endothelial Cells

Hypoxia‐induced (Group 3) pulmonary hypertension (PH) is a common devastating lung disease. Pulmonary artery endothelial cells (PAECs) are a major contributor in hypoxic PH due to possible roles of reactive oxygen species (ROS). However, the production up‐and‐down, molecular mechanisms and functional roles of hypoxia‐induced ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed‐derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2−) production. Our assays showed that hypoxia largely increased O2− production. Hypoxia also enhanced H2O2 production in isolated mitochondria from PAECs. In support, using the genetically encoded H2O2 sensor HyPer, we revealed that hypoxia caused a large increase in HyPer‐derived fluorescence (ROS production) in PAECs, which was blocked by the mitochondrial inhibitor rotenone or myxothiazol. Hypoxia largely increased proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. The hypoxic cell proliferation was blocked by rotenone or myxothiazol. Like rotenone and myxothiazol, lentiviral shRNA‐mediated knockdown of Rieske iron‐sulfur protein nicotine (RISP), known to be a primary molecule in mitochondrial ROS generation, inhibited the hypoxic proliferation as well. Hypoxic exposure evoked a significant increase in migration of PAECs, evaluated by wound healing assay. Pharmacological and genetic inhibition of mitochondrial ROS generation with myxothiazol and RISP knockdown both diminished the hypoxic cell migration. Taken together, we conclude that hypoxia significantly increases intracellular ROS generation in human PAECs, the hypoxia‐induced intracellular ROS generation primarily occur in mitochondria, and RISP serves as an essential molecule in controlling the hypoxic mitochondrial ROS generation. Equally Importantly, and RISP‐dependent mitochondrial proliferation that is likely to be ROS‐dependent. Conceivably, hypoxia remarkably causes proliferation and migration of human PAECs, which is predominantly mediated by RISP‐dependent mitochondrial ROS, thereby making an essential contribution to hypoxic pulmonary hypertension.

Efficient Detection of Early Events of α-Synuclein Aggregation Using a Cysteine Specific Hybrid Scaffold.

In this study, we have designed and synthesized a new hybrid ligand (SCG) that can selectively detect cysteine in the free and protein-bound states within minutes at the subnanomolar level. Photoinduced electron transfer was responsible for the visible color change as well as a large increase in steady state fluorescence. This detection was validated by using multiple model protein systems with differing cysteine environments and spatial arrangements. SCG was able to monitor the early events of the folding/aggregation kinetics of α-synuclein, a protein involved in the pathology of Parkinson's disease. The early events consisted of conformational fluctuations between different forms of the protein and oligomer formation. SCG was found to be effective in detecting early isomers of α-syn in vitro and in live cell environments.

A Genetically Encoded Fluorescent Sensor Enables Rapid and Specific Detection of Dopamine in Flies, Fish, and Mice

Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here, we report the development of genetically encoded GPCR-activation-based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA, GRABDA sensors exhibit large fluorescence increases (ΔF/F0 ∼90%) with subcellular resolution, subsecond kinetics, nanomolar to submicromolar affinities, and excellent molecular specificity. GRABDA sensors can resolve a single-electrical-stimulus-evoked DA release in mouse brain slices and detect endogenous DA release in living flies, fish, and mice. In freely behaving mice, GRABDA sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.

Optical determination of the electronic coupling and intercalation geometry of thiazole orange homodimer in DNA.

Sequence-selective bis-intercalating dyes exhibit large increases in fluorescence in the presence of specific DNA sequences. This property makes this class of fluorophore of particular importance to biosensing and super-resolution imaging. Here we report ultrafast transient anisotropy measurements of resonance energy transfer (RET) between thiazole orange (TO) molecules in a complex formed between the homodimer TOTO and double-stranded (ds) DNA. Biexponential homo-RET dynamics suggest two subpopulations within the ensemble: 80% intercalated and 20% non-intercalated. Based on the application of the transition density cube method to describe the electronic coupling and Monte Carlo simulations of the TOTO/dsDNA geometry, the dihedral angle between intercalated TO molecules is estimated to be 81° ± 5°, corresponding to a coupling strength of 45 ± 22 cm-1. Dye intercalation with this geometry is found to occur independently of the underlying DNA sequence, despite the known preference of TOTO for the nucleobase sequence CTAG. The non-intercalated subpopulation is inferred to have a mean inter-dye separation distance of 19 Å, corresponding to coupling strengths between 0 and 25 cm-1. This information is important to enable the rational design of energy transfer systems that utilize TOTO as a relay dye. The approach used here is generally applicable to determining the electronic coupling strength and intercalation configuration of other dimeric bis-intercalators.

Fluorescent Branched RNAs for High-Throughput Analysis of Dbr1 Enzyme Kinetics and Inhibition.

We have developed fluorescent 2',5' branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5' arm of the bRNA that is quenched by a dabcyl moiety on the 2' arm. Dbr1-mediated hydrolysis of the 2',5' linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2',5'-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC50 values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.

Pyrrolidinyl peptide nucleic acid terminally labeled with fluorophore and end-stacking quencher as a probe for highly specific DNA sequence discrimination

Peptide nucleic acid (PNA) labeled with a dye/quencher pair is useful as a high-performance fluorescence probe for DNA sequence determination. Previous work has mainly focused on placing the dye/quencher at different ends of the PNA strand. In this work, we propose a new pyrrolidinyl PNA probe bearing a fluorescent dye and an anthraquinone quencher at the same end of the PNA molecule. The single-stranded PNA probe exhibits a weak fluorescence as a result of close contact between the dye and the quencher. End-stacking of the anthraquinone to the terminal base pair of the PNA·DNA duplex disrupts the fluorophore-quencher interaction, resulting in a large fluorescence increase in the presence of complementary DNA. The high specificity of the present PNA probe allows multicolor, multiplex detection of single mismatched DNA targets. In addition, direct detection of double stranded DNA target by double duplex invasion was demonstrated.

Nucleotide-free kinesin motor domains reversibly convert to an inactive conformation with characteristics of a molten globule

Nucleotide-free kinesin motor domains from several kinesin families convert reversibly to a refractory conformation that cannot rapidly rebind ADP. In the absence of glycerol, the refractory conformation of Drosophila kinesin motor domains is favored by 50-fold with conversion of the active to the refractory species at ∼0.052 s−1 and reactivating in the presence of ADP at ∼0.001 s−1. This reactivation by ADP is due to conformational selection rather than induced fit because ADP is not bound to the refractory species at concentrations of ADP that are sufficient to saturate the rate of reactivation. Glycerol stabilizes the active conformation by reducing the rate of inactivation, while having little effect on the reactivation rate. Circular dichroism indicates a large conformational change occurs on formation of the refractory species. The refractory conformation binds ANS (8-anilino-1-napthalenesulfonic acid) with a large increase in fluorescence, indicating that it has molten globule character. High ANS binding is also observed with the refractory forms of Eg5 (a kinesin-5) and Ncd (a kinesin-14), indicating that a refractory conformation with molten globule characteristics may be a common feature of nucleotide-free kinesin motor domains.

The aptamer DNA-templated fluorescence silver nanoclusters: ATP detection and preliminary mechanism investigation

Two general and reliable fluorescence sensors were proposed in this work utilizing aptamer DNA-templated silver nanoclusters (Ag NCs). Both DNA-AgNCs could be used for label-free detecting of ATP with the limits of detection of 0.44 and 0.65mM. One of them was further applied to monitor the activity of adenosine deaminase (ADA). In our effort to elucidate the light-up mechanism, we studied a total of six Ag NCs prepared by different DNA sequences, and found that they showed different sensitivity to ATP. Both BT3T3- and BT3T3(R)-templated Ag NCs were chose to make particular studies by UV–vis, TEM, fluorescence, and TCSPC methods. The results showed that when DNA-Ag NCs was kept for 1.5h and presented a strong fluorescence, the addition of ATP failed to cause a large change of fluorescence intensity; on the contrary, after Ag NCs was kept for 24h and emitted a weak fluorescence, adding ATP was able to result in the large fluorescence enhanced of 43 and 33 times for BT3T3- and BT3T3(R)-templated Ag NCs, respectively. The possible mechanism was also suggested that ATP binding to aptamer segment of template induced the change of the DNA secondary structure, which made the aggregated Ag nanoparticles disperse into Ag NCs with an average diameter of about 2nm that were responsible for the large fluorescence increase. Moreover, ATP could protect the fluorescence intensity of BT3T3(R)-templated Ag NCs from quenching for at least 9h.

JS1-3 - Rapid in vivo Cancer Imaging by Topically Spraying a Newly Designed Activatable Fluorescence Probe

ABSTRACT Fluorescence imaging is one of the most powerful techniques currently available for continuous observation of dynamic responses in living cells and animals. For highly efficient development of novel fluorescence probes, we have established several rational strategies for controlling the properties of visible light to NIR-excitable fluorophores based on the intramolecular photoinduced electron transfer. These strategies are quite powerful and versatile and, indeed, based on these strategies, we have succeeded to develop a series of fluorescence probes for reactive oxygen species, ions, and various enzymatic activities. Recently, we have established another strategy to control the fluorescent properties of various fluorophores based on intramolecular spirocyclization. For example, if one of the amino groups of hydroxymethyl rhodaminegreen (HMRG), a new rhodaminegreen derivative bearing a hydroxymethyl group instead of the original carboxy group, is amidated with an amino acid, the resultant product is colorless in neutral pH buffer due to the preferred spirocyclization. This compound can be a highly sensitive fluorescence probe for the target aminopeptidase, because it is selectively reactive towards the target enzyme to yield highly fluorescent HMRG. Based on these HMRG-based fluorescence probes, we have succeeded to establish a novel strategy for in vivo tumor imaging by utilizing upregulated enzymatic activities in cancer cells. Gamma-glutamyltranspeptidase (GGT) is well known to be significantly overexpressed in several human tumors, and we have developed a novel fluorescence probe (gGlu-HMRG) for GGT based on HMRG scaffold, which is quickly activated by GGT and yields an over 350-fold increase in fluorescence signal compared with the quenched state. gGlu-HMRG showed a large fluorescence increase in several cancer cell lines, but not in a normal cell line. Indeed, tumor cells in a mouse model of peritoneal metastases were successfully visualized with high signal contrast between the tumor and background. The activation occurred within 1 min of spraying the probe, and the signal was strong enough to be detected even with our naked eyes. This probe is believed to be practical for clinical application during surgical or endoscopic procedures.

Visualization of Subunit-Specific Delivery of Glutamate Receptors to Postsynaptic Membrane during Hippocampal Long-Term Potentiation

An increase in the number of AMPA-type glutamate receptors (AMPARs) is critical for long-term potentiation (LTP), synaptic plasticity regarded as a basal mechanism of learning and memory. However, when and how each type of AMPAR reaches the postsynaptic membrane remain unclear. We have developed experimental methods to form postsynaptic-like membrane (PSLM) on a glass surface to precisely visualize the location and movement of receptors. We observed fluorescence-labeled AMPAR subunits (GluA1-3) around PSLM with total internal reflection fluorescence microscopy. The increases of GluA1, 2, and 3 in PSLM showed different time courses after LTP induction. GluA1 increased first, and was exocytosed to the periphery of PSLM soon after LTP induction. GluA2 and GluA3 initially decreased, and then increased. Exocytosis of GluA2 and GluA3 occurred primarily in non-PSLM, and later than exocytosis of GluA1. Thus, GluA1-3 appear to increase in the postsynaptic membrane through distinct pathways during LTP.

Fluorescent, Reagentless Biosensors for Phosphate: Altering Properties, Expanding Usefulness

Two forms of biosensor for inorganic phosphate (Pi) have been developed, based on the phosphate binding protein of Escherichia coli, and are now widely used for time-resolved measurements of enzymes that produce Pi. One form is an adduct with a single coumarin fluorophore. For the second version, two cysteine mutations were introduced and labeled with a rhodamine. When physically close to each other and appropriately orientated, two rhodamine dyes can interact to form a non-covalent dimer. In this state they have little or no fluorescence, unlike the high fluorescence intensity of monomeric rhodamine. The labeling sites were so placed that the distance and orientation between the rhodamines changes with the conformation change associated with Pi binding. This movement alters the extent of interaction between rhodamines and gives a large fluorescence increase as Pi binds. In both these forms, Pi binds rapidly and tightly, making them good probes for rapid reaction assays, where all Pi can be bound to the biosensor: that is, the probe must be at least stoichiometric with the Pi. The rhodamine version has a large advantage over coumarin in terms of sensitivity and photobleaching. However, to make the probe more useful for different assay formats, such as high-throughput, the biosensor would best be used sub-stoichiometrically. This is more efficient in terms of usage amounts and has the potential of measuring over a wider range of Pi concentrations. To achieve this, several strategies were examined to locate mutations on this two domain protein to weaken the Pi binding while maintaining the fluorescence response.