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PubMed2002

Applications of novel resonance energy transfer techniques to study dynamic hormone receptor interactions in living cells.

Many aspects of hormone receptor function that are crucial for controlling signal transduction of endocrine pathways can be monitored more accurately with the use of non-invasive, live cell resonance energy transfer (RET) techniques. Fluorescent RET (FRET), and its variation, bioluminescent RET (BRET), can be used to assess the real-time responses to specific hormonal stimuli, whilst preserving the cellular protein network, compartmentalization and spatial arrangement. Both FRET and BRET can be readily adapted to the study of membrane proteins. Here, we focus on their applications to the analysis of interactions involving the superfamily of hormone G-protein-coupled receptors. RET is also emerging as a significant tool for the determination of protein function in general. Such techniques will undoubtedly be of value in determining the functional identities of the vast array of proteins that are encoded by the human genome.

Energy Transfer↗
PubMed2003

Fraying and electron autodetachment dynamics of trapped gas phase oligonucleotides.

Sensitive methods recently developed to measure laser-induced fluorescence from trapped ions have been applied to study the dynamics of double- and single-stranded oligonucleotides. In this paper, the fraying of duplex terminal base pairs has been identified by measuring the donor fluorescence as a function of temperature from an oligonucleotide duplex labeled with a pair of FRET dyes. Comparison of the degree of dissociation of 14-mer duplexes observed in the mass spectra with the fluorescence intensity of the donor enables intermediate conformations of the unzipping duplex at the weaker binding end of the duplex to be identified. The autodetachment of electrons from double- and single-stranded oligonucleotide anions has been observed in a gas phase environment. To characterize this process, measurements were performed on 7-mers prepared without FRET fluorophores attached. The dependence of the decay rates of trapped anions have been measured as a function of charge state and temperature for various base compositions. An exceptionally strong dependence of the decay rate on base composition has been identified. The physical basis for this process will be discussed.

Boron Compounds↗
PubMed2002

Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast.

Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.

Bacterial Proteins↗
PubMed2001

High-throughput screening strategies for cardiac ion channels.

In this article we discuss new technologies for high-throughput screening (HTS) of cardiac ion channels. We review the current state of cell based HTS and discuss new technologies and approaches using voltage-sensing Fluorescence Resonance Energy Transfer (FRET) dyes and "native" cell lines. The advantages and disadvantages of a gene-centric approach in which a target protein is overexpressed in a non-native cell line are addressed and the role of such primary screens in the drug discovery process outlined. Primary and secondary screens using "native" type cell lines either endogenously expressing the ion channel of interest or overexpressing it are discussed with respect to HTS capacity and sensitivity for FRET voltage sensing dyes and other technologies. Finally the advantages and approaches of screening against multiple targets in an endogenous cardiac cell line are discussed.

Anti-Arrhythmia Agents↗
PubMed2001

Molecular distribution sensing in a fluorescence resonance energy transfer based affinity assay for glucose.

A newly developed method for determining molecular distribution functions is applied to a widely researched glucose affinity sensor. The reduction in fluorescence resonance energy transfer (FRET) to a malachite green (MG)-dextran complex from allophycocyanin (APC) bound to concanavalin A (ConA) due to displacement of the complex by glucose from ConA provides the basis of the assay. The higher sensitivity and specificity of a new approach to fluorescence decay analysis, over the methods based on conventional Forster-type models, is demonstrated and critical parameters in competitive binding FRET sensing derived.

Concanavalin A↗
PubMed2002

Fluorescence resonance energy transfer studies of U-shaped DNA molecules.

Fluorescence resonance energy transfer studies allow to determine global shape properties of nucleic acids and nucleoprotein complexes. In many DNA-protein complexes, the DNA is more or less bent and the degree of bending can be obtained by FRET. For example, the DNA in complex with the integration host factor (IHF) is kinked by approximately 160 degrees building a U-shaped structure. The two DNA helix ends come close to one another in space in a distance range easily measurable by FRET. The global DNA structure of this complex can be mimicked by introducing two regions with unpaired bases ('bulges') into the DNA each producing a sharp kink of approximately 80 degrees. These U-shaped DNA constructs were used to measure the electrostatic interaction of the two nearly parallel negatively charged DNA helix arms. The electrostatic repulsion between the helix arms, and as a consequence their distance, decreases with growing salt concentration of mono- or divalent cations. This experimental approach also allows the sensitive study of the local structure of DNA sequences positioned between the two bulges.

Acrylic Resins↗
PubMed2004

Detection of mitochondrial caspase activity in real time in situ in live cells.

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP-caspase 3 substrate-YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

Animals↗
PubMed2003

IL-2 or IL-4 mRNA as a potential flow cytometric marker molecule for selective collection of living T helper 1 or T helper 2 lymphocytes.

Flow cytometry has been widely used to analyze and sort out particular types of living cells that have specific marker molecules. In many cases, marker proteins are present on the cell surface and are detected by monoclonal antibodies against them. However, there are some cases in which cells do not have specific marker molecules on their surface. In this situation, it would be useful if mRNA that is expressed specifically in the particular cell could be used as a marker molecule. We previously reported that mRNA can be detected in living cells by hybridizing a pair of fluoreophore (donor or acceptor)-labeled oligonucleotides to adjacent locations on the target mRNA in the cytoplasm of cells (Tsuji, A.; Koshimoto, H.; Sato, Y.; Hirano, M.; Sei-Iida, Y.; Kondo, S.; Ishibashi, K. Biophys. J. 2000, 78, 3260-3274). On the formed hybrid of the two fluorescent oligonucleotides with the target mRNA, the distance between the two fluorophores becomes very close, which results in fluorescence resonance energy transfer (FRET). Combining this fluorescent labeling method for mRNA with flow cytometry, we have examined the isolation of living CD4+ T helper lymphocytes expressing IL-2 mRNA (Th1) or IL-4 mRNA (Th2). A pair of fluorescent oligonucleotides for hybridizing to IL-2 or IL-4 mRNA were introduced into activated CD4+ T lymphocytes by electroporation. The cells were applied to FACS and analyzed by FRET signals. Th1 or Th2 lymphocytes were exclusively sorted from their mixed populations in activated CD4+ T cells. Our results demonstrate that it is possible to use mRNA as marker molecules to analyze and isolate living cells in flow cytometry.

Biomarkers↗
PubMed2003

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons.

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.

Base Sequence↗
PubMed2003

Improving fluorescent DNAzyme biosensors by combining inter- and intramolecular quenchers.

A previously reported DNAzyme-based biosensor for Pb(2+) has shown high sensitivity and selectivity at 4 degrees C. In the system, the substrate and the enzyme strand of the DNAzyme are labeled with a fluorophore and a quencher, respectively. In the presence of Pb(2+), the substrate strand is cleaved by the enzyme strand, and the release of the cleaved fragment results in significant fluorescence increase. However, the performance of the sensor decreases considerably if the temperature is raised to room temperature because of high background fluorescence. A careful analysis of the sensor system, including measurement of the melting curve and fluorescence resonance energy-transfer (FRET) study of the free substrate, suggests that a fraction of the fluorophore-labeled substrate strand is dissociated from the enzyme strand, resulting in elevated background fluorescence signals at room temperature. To overcome this problem, we designed a new sensor system by introducing both inter- and intramolecular quenchers. The design was aided by the FRET study that showed the dissociated substrate maintained a random coil conformation with an end-to-end distance of approximately 39 A, which is much shorter than that of the fully extended DNA. With this new design, the background fluorescence was significantly suppressed, with 660% increase of fluorescence intensity as compared to 60% increase for the previous design. This suppression of background fluorescence signals was achieved without losing selectivity of the sensor. The new design makes it possible to use the sensor for practical applications in a wide temperature range. The design principle presented here should be applicable to other nucleic acid-based biosensors to decrease background fluorescence.

Biosensing Techniques↗
PubMed2005

One-step homogeneous immunoassay for small analytes.

We have developed a one-step, homogeneous noncompetitive immunoassay for small analytes using recombinant antibodies and morphine as the model analyte. A highly specific antibody against the immune complex (IC) formed between an anti-morphine antibody and morphine was selected from a naive scFv phage display library. The in vitro phage library selection procedure avoids the difficulties associated with the production of anti-IC antibodies by animal immunization. The anti-morphine and the anti-IC antibodies were labeled with a pair of fluorescence resonance energy transfer (FRET) fluorophores. In the FRET assay the labeled antibodies were incubated with saliva samples spiked with morphine, codeine, or heroin. Within 2 min, 5 ng/mL morphine, which is clearly under the recommended cutoff level, was detected without cross-reactivity to codeine or heroin. This assay principle is also widely applicable to other small analytes.

Amino Acid Sequence↗
PubMed2005

Self-assembled TNT biosensor based on modular multifunctional surface-tethered components.

We demonstrate a self-assembled reagentless biosensor based on a modular design strategy that functions in the detection of TNT and related explosive compounds. The sensor consists of a dye-labeled anti-TNT antibody fragment that interacts with a cofunctional surface-tethered DNA arm. The arm consists of a flexible biotinylated DNA oligonucleotide base specifically modified with a dye and terminating in a TNB recognition element, which is an analogue of TNT. Both of these elements are tethered to a Neutravidin surface with the TNB recognition element bound in the antibody fragment binding site, bringing the two dyes into proximity and establishing a baseline level of fluorescence resonance energy transfer (FRET). Addition of TNT, or related explosive compounds, to the sensor environment alters FRET in a concentration-dependent manner. The sensor can be regenerated repeatedly through washing away of analyte and specific reformation of the sensor assembly, allowing for subsequent detection events. Sensor dynamic range can be usefully altered through the addition of a DNA oligonucleotide that hybridizes to a portion of the cofunctional arm. The modular design of the sensor demonstrates that it can be easily adapted to detect a variety of different analytes.

Biosensing Techniques↗
PubMed2004

Femtomole mixer for microsecond kinetic studies of protein folding.

We have developed a microfluidic mixer for studying protein folding and other reactions with a mixing time of 8 mus and sample consumption of femtomoles. This device enables us to access conformational changes under conditions far from equilibrium and at previously inaccessible time scales. In this paper, we discuss the design and optimization of the mixer using modeling of convective diffusion phenomena and a characterization of the mixer performance using microparticle image velocimetry, dye quenching, and Forster resonance energy-transfer (FRET) measurements of single-stranded DNA. We also demonstrate the feasibility of measuring fast protein folding kinetics using FRET with acyl-CoA binding protein.

Acyl Coenzyme A↗
PubMed2004

A flow injection kinase assay system based on time-resolved fluorescence resonance energy-transfer detection in the millisecond range.

A flow injection analysis (FIA) system for biochemical assays using time-resolved fluorescence resonance energy transfer (TR-FRET) in the millisecond time scale was developed. As a model system, we studied a kinase assay, measuring the phosphorylation of poly(GT)-biotin (substrate) by a receptor tyrosine kinase (epidermal growth factor receptor). A streptavidin labeled with XL665 (SA-XL665)-the acceptor-was coupled to the biotin moiety, and an antiphosphotyrosine antibody labeled with europium cryptate (Ab-EuK)-the donor-was coupled to the phosphorylated tyrosine group(s). Long-lived FRET can only occur if the substrate is successfully phosphorylated. For the time-resolved detection of such long-lived luminescence phenomena in a flow system, the repetition rate of the excitation source plays a crucial role. Good results were obtained for a small-sized commercially available quadrupled Nd:YAG laser emitting at 266 nm with a repetition rate of 7.8 kHz and a pulse width of 0.3 ns. The long-lived emissions of the donor at 625 nm and that of the acceptor at 665 nm were monitored simultaneously with two photomultipliers, using a delay time of 50 micros and a gate time of 75 micros to exclude background fluorescence interferences. In the FIA experiments, the Ab-EuK concentration was 6 nM and the substrate concentration and SA-XL665 concentrations were 7 nM. By monitoring the intensity changes at 625 and 665 nm, the inhibition of tyrosine kinase by tyrphostin AG1478 was studied and an IC(50) value of 5.1 +/- 0.4 nM obtained.

Binding Sites↗
PubMed2005

Detection of single-base mutations by fluorogenic ribonuclease protection assay.

The ribonuclease protection assay is a generally applicable technique for the detection of known mutations. We have developed a simple and rapid method for mutation detection based on the ribonuclease protection assay using fluorescently labeled oligodeoxyribonucleotide probes. The fluorogenic ribonuclease protection (FRAP) assay uses two differently labeled oligodeoxyribonucleotides, a donor probe and an acceptor probe, to obtain a fluorescence resonance energy transfer (FRET) signal. We have utilized the FRAP assay for the detection of a single-base mutation in the YMDD motif of the hepatic B virus DNA polymerase gene. The occurrence of mismatch-selective RNA cleavage was successfully discriminated by measuring the FRET signal between the donor and acceptor probes. Moreover, mutation sensing was successfully visualized by a UV transillumination. This simple and rapid mutation sensing method should facilitate a high-throughput mutation analysis.

Base Pair Mismatch↗
PubMedDate not supplied

Fluorescent nanometer microspheres as a reporter for sensitive detection of simulants of biological threats using multiplexed suspension arrays.

We succeeded in using 40 nm FRET (fluorescence resonance energy transfer) microspheres conjugated to antibodies as the fluorescent reporters to perform the multiplexing suspension array measurements on two simulants of biological threats, ricin (A chain) and a crude spore preparation of Bacillus globigii (Bg). The microspheres were impregnated with two types of fluorophores in equal number (approximately 140 fluorophores in total per microsphere) and displayed bright PE-like fluorescence via a fluorescence resonance energy transfer mechanism. Activated microspheres (aldehyde groups) were directly coupled to antibodies and used to form sandwich-type immunoassays in a suspension array. For the crude preparations of Bg, the assay sensitivity using antibody-conjugated microspheres is an order of magnitude higher than that using the conventional fluorescent reporter, R-phycoerythrin (PE). Using the microspheres, Bg at the concentration of 5 ng/mL can be easily detected. For ricin, the assay sensitivity was similar to that obtained using PE as the reporter, but washing the reaction mixtures resulted in the fluorescence signals that were 2-3 times higher compared to those using PE. Ricin at a concentration of 1 ng/mL can be readily identified. Importantly, the two simulants do not interfere with each other in the multiplexing experiments. The 40 nm FRET microspheres are a new sensitive alternative as fluorescent reporters for detection in suspension arrays.

Bacillus↗
PubMedDate not supplied

Orientation and affinity of HIV-1 Tat fragments in Tat-TAR complex determined by fluorescence resonance energy transfer.

The human immunodeficiency virus (HIV-1) encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis. Tat interacts with the transactivation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all HIV mRNAs. Tat-derived peptides that contain the basic arginine-rich region of Tat are able to form in vitro complexes with TAR RNA, and these peptides provide a well-characterized system for understanding the mechanism of RNA-protein recognition. It is not known how RNA-binding Tat peptides are folded or docked in the Tat-TAR complex, and to what extent structural reorganization occurs upon TAR binding. To address these questions, we developed a fluorescence resonance energy transfer (FRET) system to analyze the interactions between TAR RNA and a Tat protein fragment (aa 38-72) uniquely labeled with donor and acceptor dye molecules, respectively. Using this FRET assay, we determined the binding affinity of Tat (47-58) and Tat (38-72) for TAR RNA under physiological conditions. We also delineated the distance between the N- and C-terminus of Tat (38-72) and the distance between the two termini and the 5' end of TAR when Tat (38-72) is bound to TAR. Our results suggest that the N- and C-termini of Tat (38-72) are close to each other when the peptide is folded and that the peptide does not go through a large structural change upon TAR binding.

Amino Acid Sequence↗
PubMedDate not supplied

Design consideration and probes for fluorescence resonance energy transfer studies.

Spectroscopic properties of two newly synthesized water-soluble thiol-reactive fluorescent probes, 7-(iodoacetamido)-coumarin-4-carboxylic acid (I-Cca) and N-iodoacetyl-beta-(2-naphthyl)alanine (I-Nal), were characterized using single cysteine mutants of Escherichia coli adenylate kinase. Together with two known water-soluble thiol-reactive dyes (Lucifer yellow iodoacetamide and 5-iodoacetamidosalicylic acid) and as well, tryptophan residues (either native or inserted into a protein by site directed mutagenesis), these probes can be arranged pairwise in a molecular tool set for studies of structural transitions in proteins by means of fluorescence resonance energy-transfer (FRET) experiments. A set of seven donor/acceptor pairs which allow determination of intramolecular distances and their distributions over the range 10-40 A in labeled protein derivatives is described. The charged groups present in the probes facilitate the conjugation reaction and improve postlabeling purification. General considerations for design of charged probes and site-directed labeling for applications of FRET methods in studies of protein structure and dynamics are presented.

Acetamides↗