Comparative analysis of fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA)

Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.     

A high-throughput fluorescence resonance energy transfer-based assay for DNA ligase

DNA ligase is the enzyme that catalyzes the formation of the backbone phosphodiester bond between the 5'-PO(4) and 3'-OH of adjacent DNA nucleotides at single-stranded nicks. These nicks occur between Okazaki fragments during replication of the lagging strand of the DNA as well as during DNA repair and recombination. As essential enzymes for DNA replication, the NAD(+)-dependent DNA ligases of pathogenic bacteria are potential targets for the development of antibacterial drugs. For the purposes of drug discovery, a high-throughput assay for DNA ligase activity is invaluable. This article describes a straightforward, fluorescence resonance energy transfer-based DNA ligase assay that is well suited for high-throughput screening for DNA ligase inhibitors as well as for use in enzyme kinetics studies. Its use is demonstrated for measurement of the steady-state kinetic constants of Haemophilus influenzae NAD(+)-dependent DNA ligase and for measurement of the potency of an inhibitor of this enzyme.     

A high-throughput, homogeneous, fluorescence resonance energy transfer-based assay for phospho-N-acetylmuramoyl-pentapeptide translocase (MraY)

Peptidoglycan biosynthesis is an essential process in bacteria and is therefore a suitable target for the discovery of new antibacterial drugs. One of the last cytoplasmic steps of peptidoglycan biosynthesis is catalyzed by the integral membrane protein MraY, which attaches soluble UDP-N-acetylmuramoyl-pentapeptide to the membrane-bound acceptor undecaprenyl phosphate. Although several natural product-derived inhibitors of MraY are known, none have the properties necessary to be of clinical use as antibacterial drugs. Here we describe a novel, homogeneous, fluorescence resonance energy transfer-based MraY assay that is suitable for high-throughput screening for novel MraY inhibitors. The assay allows for continuous measurement, or it can be quenched prior to measurement.     

Fluorescence resonance energy transfer (FRET)-based subcellular visualization of pathogen-induced host receptor signaling

Background  

Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET) to visualize pathogen-initiated signaling events in human cells.     

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.     

A homogeneous, high-throughput fluorescence resonance energy transfer-based DNA polymerase assay

A homogeneous, fluorescence resonance energy transfer (FRET)-based DNA polymerase assay that is suitable for high-throughput screening for inhibitors, and can also be used for steady-state kinetic investigations, is described. The activity, kinetic mechanism, and processivity of the isolated alpha subunit of DNA polymerase III, the product of the dnaE gene, from the gram-negative pathogen Haemophilus influenzae were investigated using the FRET assay.     

DNA probes using fluorescence resonance energy transfer (FRET): designs and applications.

Fluorescence resonance energy transfer (FRET) is widely used in biomedical research as a reporter method. Oligonucleotides with a DNA backbone and one or several chromophore tags have found multiple applications as FRET probes. They are especially advantageous for the real-time monitoring of biochemical reactions and in vivo studies. This paper reviews the design and applications of various DNA-based probes that use FRET The approaches used in the design of new DNA FRET probes are discussed.     

Intracellular oligonucleotide hybridization detected by fluorescence resonance energy transfer (FRET).

Fluorescence resonance energy transfer (FRET) was used to study hybrid formation and dissociation after microinjection of oligonucleotides (ODNs) into living cells. A 28-mer phosphodiester ODN (+PD) was synthesized and labeled with a 3' rhodamine (+PD-R). The complementary, antisense 5'-fluorescein labeled phosphorothioate ODN (-PT-F) was specifically quenched by addition of the +PD-R. In solution, the -PT-F/+PD-R hybrid had a denaturation temperature of 65 +/- 3 degrees C detected by both absorbance and FRET. Hybridization between the ODNs occurred within 1 minute at 17 microM and was not appreciably affected by the presence of non-specific DNA. The pre-formed hybrid slowly dissociated (T1/2 approximately 3 h) in the presence of a 300-fold excess of the unlabeled complementary ODN and could be degraded by DNAse I. Upon microinjection into the cytoplasm of cells, pre-formed fluorescent hybrids dissociated with a half-time of 15 minutes, which is attributed to the degradation of the phosphodiester. Formation of the hybrid from sequentially injected ODNs was detected by FRET transiently in the cytoplasm and later in the cell nucleus, where nearly all injected ODNs accumulate. This suggests that antisense ODNs can hybridize to an intracellular target, of exogenous origin in these studies, in both the cytoplasm and the nucleus.     

A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activation

This report describes the development, optimization, and implementation of a miniaturized cell-based assay for the identification of small-molecule insulin mimetics and potentiators. Cell-based assays are attractive formats for compound screening because they present the molecular targets in their cellular environment. A fluorescence resonance energy transfer (FRET) cell-based assay that measures the insulin-dependent colocalization of Akt2 fused with either cyan fluorescent protein or yellow fluorescent protein to the cellular membrane was developed. This ratiometric FRET assay was miniaturized into a robust, yet sensitive 3456-well nanoplate assay with Z' factors of approximately 0.6 despite a very small assay window (less than twofold full activation with insulin). The FRET assay was used for primary screening of a large compound collection for insulin-receptor agonists and potentiators. To prioritize compounds for further development, primary hits were tested in two additional assays, a biochemical time-resolved fluorescence resonance energy transfer assay to measure insulin-receptor phosphorylation and a translocation-based imaging assay. Results from the three assays were combined to yield 11 compounds as potential leads for the development of insulin mimetics or potentiators.     

Live cell imaging of protein interactions in poliovirus RNA replication complex using fluorescence resonance energy transfer (FRET)

Poliovirus RNA replication is directed by a replication complex on the rosette-like arrangement of membranous vesicles. Proteins derived from the p3 region of the polioviral genome, such as 3D, 3AB, and 3B (VPg), play key roles in the formation and function of the replication complex. In the present study, by using an acceptor photobleaching protocol for fluorescence resonance energy transfer (FRET) imaging, we visualized the interactions of 3D, 3AB, and VPg in living cells. The interaction of 3AB-VPg was determined by live cell FRET analysis. Quantitative analyses showed that the FRET efficiencies of 3AB-3D, VPg-3D, and 3AB-VPg were 3.9 ± 0.4% (n = 36), 4.5 ± 0.4% (n = 39), and 8.3 ± 0.6% (n = 44), respectively, in the cell cytoplasm where viral replication complexes are formed and function. Poliovirus infection enhanced the protein interactions of VPg-3D and 3AB-3D, with FRET efficiencies in the virus-infected cells of 10.7 ± 1.1% (n = 39) and 9.0 ± 0.9% (n = 37), respectively. This method of live cell analysis of protein interactions in the poliovirus RNA replication complex lays the foundation for further understanding of the real-time process of poliovirus RNA replication.     

Fluorescence resonance energy transfer (FRET): theory and experiments

Fluorescence resonance energy transfer (FRET) is a powerful biophysical technique permitting macromolecular interactions between fluorescent molecules in close contact with each other to be analysed. Studies of the kinetics of association/dissociation between biological macromolecules may be carried out using this technique. Distances of interaction between donor-acceptor may be estimated by the FRET approach.     

Fluorescence resonance energy transfer (FRET)-based specific labeling of Cryptosporidium oocysts for detection in environmental samples.

BACKGROUND: Accurate detection and quantification of Cryptosporidium oocysts in water are a challenge to the water industry. This article demonstrates a way to fluorescently label Cryptosporidium oocysts, based on fluorescence resonance energy transfer (FRET). Labeled oocysts can then be applied to environmental waters and their movement followed by flow cytometric detection and enumeration of the FRET-labeled oocysts, as demonstrated here with environmental water samples. METHODS: Cryptosporidium oocysts were labeled with three fluorochromes, FITC, Texas red, and Cy7, that through FRET yielded a Stokes shift of approximately 272 nm with excitation from a standard argon laser emitting at 488 nm. Defined flow cytometric settings and gatings were used to select FITC/green (530-nm), Texas red/red (650-nm), and Cy7/infrared (780-nm) fluorescing particles with light scatter properties similar to oocysts. Water concentrates were seeded with 10 tri-labeled oocysts and were analyzed using flow cytometry. Unseeded water concentrates were also analyzed. RESULTS: Analysis of unseeded water concentrates detected no autofluorescent particle similar to the labeled oocysts. Labeled oocysts were detected successfully with up to 85% recovery in water concentrates spiked with 10 tri-labeled oocysts. CONCLUSIONS: Low numbers of FRET-labeled oocysts can be quantified and clearly distinguished from autofluorescing background in environmental water concentrates.     

A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay

Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer (FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. The FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK(Dnp)P-OH for N-domain, Abz-LFK(Dnp)-OH for C-domain and Abz-FRK(Dnp)P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader.     

Bidirectional fluorescence resonance energy transfer (FRET) in mutated and chemically modified yellow fluorescent protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Fo?rster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.     

Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations

The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living cells with improved spatial (angstrom) and temporal (nanosecond) resolution, distance range, and sensitivity and a broader range of biological applications.     

Application of spectral imaging microscopy in cytomics and fluorescence resonance energy transfer (FRET) analysis.

BACKGROUND: Specific signal detection has been a fundamental issue in fluorescence microscopy. In the context of tissue samples, this problem has been even more pronounced, with respect to spectral overlap and autofluorescence. METHODS: Recent improvements in confocal laser scanning microscopy combine sophisticated hardware to obtain fluorescence emission spectra on a single-pixel basis and a mathematical procedure called "linear unmixing" of fluorescence signals. By improving both the specificity of fluorescence acquisition and the number of simultaneously detectable fluorochromes, this technique of spectral imaging (SI) allows complex interrelations in cells and tissues to be addressed. RESULTS: In a comparative approach, SI microscopy on a quantitative basis was compared to conventional bandpass (BP) filter detection, demonstrating substantial superiority of SI with respect to detection accuracy and dye combination. An eight-color immunofluorescence protocol for tissue sections was successfully established. Moreover, advanced use of SI in fluorescence resonance energy transfer (FRET) applications using enhanced green fluorescence protein (EGFP) and enhanced yellow fluorescence protein (EYFP) in a confocal set up could be demonstrated. CONCLUSIONS: This novel technology will help to perform complex multiparameter investigations at the cellular level by increasing the detection specificity and permitting simultaneous use of more fluorochromes than with classical techniques based on emission filters. Moreover, SI significantly extends the possibilities for specialized microscopy applications, such as the visualization of macromolecular interactions or conformational changes, by detecting FRET.     

A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly

In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA. Binding causes a conformational change that is required for subsequent binding events. Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly. The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay.