Changes In Fluorescence Polarization Research Articles

Electrostatic-Mediated Binding of DNA to Lysozymes: Evaluation of Aptamer-Based Assays for Highly Positively Charged Targets.

Lysozymes are a highly popular protein target for the development of aptamer-based biosensors. Because a lysozyme is a polycation and DNA is a polyanion, it is essential to separate the contribution of nonspecific electrostatic interactions from specific aptamer binding. In this study, various factors affecting the binding of DNA and lysozymes, including the DNA sequence, DNA length, pH, and salt concentration, were explored using fluorescence polarization. We concluded that direct fluorescence polarization and fluorescence intensity changes are unlikely to be directly applicable for aptamer-based biosensors to detect lysozymes because all of the tested DNA sequences showed binding. These fundamental studies confirm the dominant role of electrostatic binding. We further evaluated three other methods, including label-free fluorescent detection using a DNA staining dye, label-free colorimetric detection using gold nanoparticles, and a fluorescent sensor based on the strand displacement reaction. In each case, we focused on a random DNA sequence that is not expected to bind to the lysozyme as an aptamer. Of all the methods, only the strand displacement strategy can be potentially used to evaluate aptamer binding, as the other methods all responded to nonaptamer sequences. This study provides valuable insights for assaying aptamer binding to cationic proteins that can exhibit a nonspecific attraction to DNA.

Probing Oxidant Effects on Superoxide Dismutase 1 Oligomeric States in Live Cells Using Single-Molecule Fluorescence Anisotropy.

The protein Cu/Zn superoxide dismutase (SOD1) is known to function as a dimer, but its concentration in cells (∼50 μM) and the dimerization constant (K d of 500 μM) results suggest that it exists in a monomer-dimer equilibrium. It is unclear how the oligomeric state of SOD1 changes when cells are initially exposed to high levels of extracellular oxidative stress. To address this problem, we introduced the single-molecule fluorescence anisotropy (smFA) assay to explore SOD1 oligomeric states in live COS7 cells. smFA specifically probes the fluorescence polarization changes caused by molecular rotations where the fast-rotating molecules (either due to smaller hydrodynamic volume or less viscous environments) deteriorate the emission polarization and thus lower the anisotropy. After validating that smFA is effective in distinguishing monomeric and dimeric fluorescence proteins, we overexpressed SOD1 in live COS7 cells and investigated how its oligomeric state changes under basal, 2 h, and 24 h 100 μM H2O2 treatments. We found that treating cells with H2O2 promotes SOD1 dimerization and decreases cellular viscosity in 2 h. Interestingly, prolonged H2O2 treatments show similar results as the basal conditions, indicating that cells return to a steady state similar to the basal state after 24 h, despite the presence of H2O2. Our results demonstrate that SOD1 changes its oligomeric state equilibrium in response to extracellular oxidative stresses. smFA will open new opportunities to explore the relationship between the SOD1 oligomer state and its H2O2-based signaling and transcription regulation roles.

In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy.

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.

Fluorometric determination of lipopolysaccharides via changes of the graphene oxide-enhanced fluorescence polarization caused by truncated aptamers

A broad-spectrum ssDNA aptamer containing 80 nucleotides (LA80) and capable of binding to four different sources of lipopolysaccharides (LPSs) was truncated. Two strategies are used to produce truncated aptamers of different length. The results show that LA27, a 27-nt aptamer, retained broad-spectrum capability and has a higher affinity (Kd = 46.2 ± 9.5nM). A graphene oxide based fluorescence polarization assay (excitation/emission wavelengths: 485/520nm) was worked out using FAM-labeled LA27. It can detect LPSs from Salmonella entericaserotype typhimurium, Pseudomonas aeruginosa 10 and Escherichia coli 055:B5 with enhanced performance (4.8 to 29-fold improvements) compared to LA80. The assay can be performed within 30min, and the detection limits are 38.7, 88.0 and 154ng·mL-1, respectively. Graphical abstract Schematic presentation of the assay: A shorter aptamer, with higher affinity than its original aptamer, was obtained by truncated strategies. This core aptamer lead to release easily and enhance the sensivity of the GO-based fluorescence polarization assay.

Resonant control of fluorescence from aluminium doped zinc oxide films

Moderate fluorescence from aluminium doped zinc oxide films is detected. It is shown that this defect-related fluorescence can be controlled for the films deposited on the gilded glass and attached to the semicylinder prism. Namely the changes in the spectral shape, intensity and polarization of fluorescence were detected for certain resonant angles in case of detection through the prism. Maxima in fluorescence intensity were obtained for detection angles corresponding to minima in light reflection. The strength of electric field inside the layered structure is calculated and analysed. On the basis of this analysis coupling of fluorescent light with hybrid waveguide-plasmonic and waveguide modes at resonant detection angles is considered as the cause for obtained directional fluorescence.

MnO2 nanosheet-assisted ligand-DNA interaction-based fluorescence polarization biosensor for the detection of Ag+ ions

Silver (Ag+) ions are highly toxic to aquatic organisms and accumulate in the human body via the food chain. Therefore, the development of sensitive and selective quantitative analytical methods for detecting trace amounts of these ions is necessary. In the present study, a MnO2 nanosheet-assisted, ligand-DNA interaction and fluorescence polarization-based method was developed, for the first time, for sensitive detection of Ag+. The addition of Ag+ to the preformed proflavine-DNA complex induced the release of proflavine, which elicited weak changes in fluorescence polarization. The subsequent addition of MnO2 nanosheets magnified the observed changes, making this a feasible method for the detection of Ag+. The calibration graphs indicated good linearity over the concentration ranges of 30–240nM for Ag+, with a detection limit (S/N=3) of 9.1nM. This method additionally exhibits high selectivity. The mechanism underlying the changes in fluorescence polarization caused by the addition of Ag+ in the presence of MnO2 nanosheets was further explored in this study. These findings demonstrate that the present MnO2 nanosheet-assisted fluorescence polarization biosensor may represent a promising tool for the detection of Ag+ ions. The results for practical detecting Ag+ proved that this biosensor can be applied to environmental water sample analysis.

Probes for biomolecules detection based on RET-enhanced fluorescence polarization

Fluorescent probes based on the principle of resonance energy transfer (RET) or the principle of fluorescence polarization (FP) are already used to detect biomolecules independently. However, there were no in-depth studies about the impact of RET on FP. Also, very few studies gave a comprehensive analysis on how to effectively design such a fluorescent probe. Based on the principle of resonance energy transfer (RET), we constructed fluorescent probes (SA-488-sub-nanogold) using streptavidin labeled Alexa488 (SA-488), nanogold and biotinylated substrate peptide (biotin-subpeptide). The influence of the structure and the ingredients of the substrate peptide were discussed. After SA-488 was combined with the biotin-subpeptide and the nanogold, its fluorescence intensity (FI) would be suppressed due to the energy transfer, leading to an increase in its volume and mass. The suppression of the FI led to a decrease in SA-488's effective concentration, and the increase in the volume or mass prolonged the SA-488's rotational relaxation time. Both changes increased SA-488's polarization in the solution. Therefore, the FP performance of the probe is enhanced by the RET. Using the probe, trypsin and biotin were detected by the change in both fluorescence intensity and fluorescence polarization, showing higher reliability, higher sensitivity, and a lower detection limit.

Preliminary investigations into surface molecularly imprinted nanoparticles for Helicobacter pylori eradication

This paper reports investigations into the preparation and characterization of surface molecularly imprinted nanoparticles (SMINs) designed to adhere to Helicobacter pylori (H. pylori). Imprinted nanoparticles were prepared by the inverse microemulsion polymerization method. A fraction of Lpp20, an outer membrane protein of H. pylori known as NQA, was chosen as template and modified with myristic acid to facilitate its localization on the surface of the nanoparticles. The interaction between these SMINs with the template NQA were evaluated using surface plasmon resonance (SPR), change in zeta potential and fluorescence polarization (FP). The results were highly consistent in demonstrating a preferential recognition of the template NQA for SMINs compared with the control nanoparticles. In vitro experiments also indicate that such SMINs are able to adhere to H. pylori and may be useful for H. pylori eradication.

Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5′ overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z′ factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

High-throughput 1,536-well fluorescence polarization assays for α(1)-acid glycoprotein and human serum albumin binding.

Two major plasma proteins in humans are primarily responsible for drug binding, the α1-acid-glycoprotein (AGP) and human serum albumin (HSA). The availability of at least a semiquantitative high-throughput assay for assessment of protein binding is expected to aid in bridging the current gap between high-throughput screening and early lead discovery, where cell-based and biochemical assays are deployed routinely to test up to several million compounds rapidly, as opposed to the late-stage candidate drug profiling methods which test at most dozens of compounds at a time. Here, we describe the miniaturization of a pair of assays based on the binding- and displacement-induced changes in fluorescence polarization (FP) of fluorescent small molecule probes known to specifically target the drug-binding sites of these two proteins. A robust and reproducible assay performance was achieved in ≤4 µL assay volume in 1,536-well format. The assays were tested against a validation set of 10 known protein binders, and the results compared favorably with data obtained using protein-coated beads with high-performance liquid chromatography analysis. The miniaturized assays were taken to a high-throughput level in a screen of the LOPAC1280 collection of 1,280 pharmacologically active compounds. The adaptation of the AGP and HSA FP assays to a 1,536-well format should allow their use in early-stage profiling of large-size compound sets.

A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1

The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X-linked disease caused by mutations of the inositol-5-phosphatase OCRL1. OCRL1 is a membrane-bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab-binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α-helical structure elements, the Rab-binding interface of OCRL1 consists mainly of the IgG-like β-strand structure of the ASPM-SPD-2-Hydin domain as well as one α-helix. Our results give a deeper structural understanding of disease-causing mutations of OCRL1 affecting Rab binding.

Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E (BoNT/A and BoNT/E). As detailed in that article, the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein (BFP) and green fluorescent protein (GFP) moieties linked via residues 134–206 of SNAP-25 (synaptosome-associated protein of 25 kDa), the protein substrate for BoNT/A and BoNT/E. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is very low due to depolarization following energy transfer from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance, the polarization is high due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows an assay, termed DARET (depolarization after resonance energy transfer), that is robust and sensitive. In this article, we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarizations, and lifetimes.

A High-Throughput, Homogeneous, Fluorescence Polarization Assay for Inhibitors of Hedgehog Protein Autoprocessing

Hedgehog (Hh) signaling plays an important role in embryonic patterning and adult stem cell renewal but has recently been found also to be involved in certain stem cell cancers. One of the first steps in Hh signaling is the autoprocessing of Hh protein, in which the C-terminal domain (Hh-C) catalyzes a cholesterol-dependent autocleavage reaction that leads to the production of the cholesterol ester of the N-terminal Hh domain (Hh-N), thereby yielding a signaling molecule that activates the Hh pathway by binding to the Patched receptor. This article describes an in vitro, homogeneous assay system that measures changes in fluorescence polarization that accompany the cholesterol-dependent autocleavage of Hh protein. The assay system makes use of a modified Hh protein in which Hh-N, which is not essential for autocleavage, is replaced by a 25-residue peptide containing a tetracysteine motif, complexed with a bisarsenical fluorophore. The assay is quite robust and easily adapted to high-throughput screening in 384-well plates with Z' factors above 0.8. It has been used to screen the National Institutes of Health Clinical Collection, which has led to the identification of 2 compounds that inhibit the cholesterol-dependent autocleavage of Hh protein at micromolar concentrations.

High‐affinity binding of phosphatidylinositol 4‐phosphate by Legionella pneumophila DrrA

The DrrA protein of Legionella pneumophila is involved in mistargeting of endoplasmic reticulum-derived vesicles to Legionella-containing vacuoles through recruitment of the small GTPase Rab1. To this effect, DrrA binds specifically to phosphatidylinositol 4-phosphate (PtdIns(4)P) lipids on the cytosolic surface of the phagosomal membrane shortly after infection. In this study, we present the atomic structure of the PtdIns(4)P-binding domain of a protein (DrrA) from a human pathogen. A detailed kinetic investigation of its interaction with PtdIns(4)P reveals that DrrA binds to this phospholipid with, as yet unprecedented, high affinity, suggesting that DrrA can sense a very low abundance of the lipid.

Spectroscopic Characterization of Depolarization Via FRET in a BFP-GFP BoNT/A Assay

The assay is based on depolarization due to Forster Resonance Energy Transfer (FRET) between Blue Fluorescent Protein (BFP) and Green Fluorescent Protein (GFP) moieties linked by a peptide containing residues 134-206 of SNAP-25, the protein substrate for BoNT/A's proteolytic activity. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is −0.04 due to depolarization following FRET from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the FRET distance, the polarization increases significantly to ∼0.4, due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows for an assay (termed DARET for Depolarization After Resonance Energy Transfer) that is robust and sensitive. In this report we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarization and time-resolved methods. Our results suggest that the donor and acceptor dipoles are at a large angle (72°) with respect to one another but that the BFP and GFP are in direct contact in the intact substrate. Evidence is also provided to demonstrate the direct interaction of BFP and GFP after cleavage. We have used this assay to determine the enzyme kinetic parameters (Km, Kcat and Vmax) for BoNT/A proteolysis of the assay substrate. Our conclusions bear on the issue of the common choice of 2/3 for κ2 for FRET studies in general and on fluorescent proteins in particular. This work was supported by Allergan Inc.

Fluorescence Emission and Polarization for Analyzing Binding of Ruthenium Metalloglycocluster to Lectin and Tetanus Toxin C-Fragment

We have designed and synthesized ruthenium complexes bearing clustered galactose Ru(bpy-2Gal)(3) and glucose Ru(bpy-2Glc)(3). Changes in fluorescence emission (FE) and fluorescence polarization (FP) of the metalloglycoclusters were measured by adding each lectin (peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), concanavalin A (ConA), or wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA and ConA, the FE spectra of Ru(bpy-2Gal)(3) and Ru(bpy-2Glc)(3) showed new emission peaks, respectively. In addition, Ru(bpy-2Gal)(3) and Ru(bpy-2Glc)(3) exclusively increased the FP values by addition of PNA and ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were confirmed by the FE and FP measurement. From the FP analyses, the dissociation constants (K(d)) of Ru(bpy-2Gal)(3) to PNA and Ru(bpy-2Glc)(3) to ConA were calculated to be ca. 6.1 x 10(-6) M and 1.8 x 10(-5) M. Furthermore, the FP analyses proved specific binding of Ru(bpy-2Gal)(3) to TCF.

A fluorescence polarization immunoassay for the detection of zearalenone in corn

The aim of this study was to develop a fluorescence polarization immunoassay (FPIA) that is based on the change in fluorescence polarization of fluorescently labeled small antigen when bound by a specific antibody, for use as a screening test for zearalenone (ZEN) in cereals and their products. Syntheses of fluorescein-labeled ZEN tracers containing three linkers of different lengths (2, 3 and 6-carbon bridge), ethylenediamine, 1,2-diaminopropane and hexamethylenediamine, were explored and their binding response with ZEN-specific antibody was evaluated. A fluoresceinthiocarbamyl hexamethylenediamine-labeled ZEN conjugate (ZEN-HMDF), which contain a 6-carbon bridge, was found to be the most sensitive FPIA for detection of ZEN. When tested on corn matrix the FPIA using the ZEN-HMDF tracer showed a detection range for ZEN of 150–1000 μg kg −1 with a detection limit of 137 μg kg −1, and required less than 2 min per sample to carry out, excluding extraction time. The average recovery from spiked corn samples was 106.4 ± 12.5%. Comparative analyses of 70 naturally occurring cereals and their products using FPIA, enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC) showed that the coefficients of determination ( r 2) between FPIA and ELISA, and between FPIA and HPLC were 0.76 and 0.72, respectively. These results suggest that the ZEN-HMDF tracer is suitable for FPIA, which has potential as a screening tool for ZEN in grains without the need for a complicated clean-up procedure.

Calcium-Regulated Conformational Changes in the COOH-terminus of Troponin I

The troponin complex plays an essential role in the calcium regulation of skeletal and cardiac muscle contractions. Of the three subunits of troponin (TnC, TnI and TnT), TnI is the inhibitory subunit that responds to the binding of Ca2+ to TnC during the activation of contraction. The COOH-terminal region of TnI is a highly conserved structure implying a fundamental function. Previous studies using reconstituted troponin or myofilaments suggested that the COOH-terminal domain of TnI undergoes epitopic and positional changes in the presence or absence of calcium. Here we tested the calcium-induced conformational changes in the COOH-terminal region of TnI by engineering a unique Cys at the COOH terminus of TnI for the addition of a reporting label. Monoclonal antibody epitope analysis and protein binding assays indicated that this modification and the replacement of two internal Cys residues (C81I and C98S) in a cardiac TnI core structure (McTnI-ND29-Cys) did not affect the COOH-terminal conformation of TnI and preserved binding to TnT and TnC. McTnI-ND29-Cys purified from bacterial culture was fluorescently labeled with the Alexa Fluor 532 dye and used to reconstitute troponin complex. After verifying the ratio of fluorophore to protein conjugation by spectrophotometer and SDS-PAGE, Ca2+-titrations were performed for fluorescence intensity and polarization changes. The results demonstrated Ca2+ regulated conformational/environmental changes as well as flexibility change in the COOH terminus of TnI. Further experiments are performed to measure the Ca2+-induced structural changes in reconstituted myofilaments to understand the function of TnI COOH terminal domain in calcium-regulation of muscle contraction.

The Template-Directed Dye-Incorporation Assay with Fluorescence Polarization Detection (FP-TDI)

INTRODUCTIONThe template-directed dye-incorporation assay with fluorescence polarization detection (FP-TDI) is an economical and robust SNP genotyping method that is easy to optimize and implement in both single-marker and moderate-throughput studies. It utilizes unlabeled, unpurified PCR-grade primers in a dideoxy chain-terminating DNA-sequencing reaction to ascertain the identity of one base immediately 3' to an unlabeled primer, which is annealed immediately upstream of the polymorphic site on the target DNA. The FP-TDI protocol consists of four key steps, all of which can be carried out in the same microtiter plate without further separation or purification. First, PCR amplification of genomic DNA produces the template for the primer extension reaction. Second, a PCR clean-up enzyme cocktail with pyrophosphatase is added directly into the PCR products to remove excess PCR primers, deoxynucleoside triphosphates, and inorganic pyrophosphates. Third, single-base primer extension is carried out using a SNP primer annealed to the target DNA one base upstream from the polymorphic site. Finally, the end product is scanned with an FP plate reader to determine the changes in fluorescence polarization. With the incorporation of pyrophosphatase in the protocol and the use of quenching to analyze the fluorescence data, up to 95% of the assays work well even without optimization.

Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes

Aptamers are single-stranded DNA or RNA molecules that can bind target molecules with high affinity and specificity. The conformation of an aptamer usually changes upon binding to its target analyte, and this property has been used in a wide variety of sensing applications, including detection based on fluorescence intensity, polarization, energy transfer, electrochemistry or color change. Colorimetric sensors are particularly important because they minimize or eliminate the necessity of using expensive and complicated instruments. Among the many colorimetric sensing strategies, metallic nanoparticle-based detection is desirable because of the high extinction coefficients and strong distance-dependent optical properties of the nanoparticles. Here, we describe a protocol for the preparation of aptamer-linked gold nanoparticle purple aggregates that undergo fast disassembly into red dispersed nanoparticles upon binding of target analytes. This method has proved to be generally applicable for colorimetric sensing of a broad range of analytes. The time range for the entire protocol is approximately 5 d, including synthesis and functionalization of nanoparticles, preparation of nanoparticle aggregates and sensing.