Efficient FRET Research Articles

Identification of the functional TLR4 TIR dimerization interface using a decoy peptide approach (136.37)

Abstract Agonist-induced oligomerization of TIR domains of TLRs and TLR adapters initiates intracellular signaling. Identification of TIR domain surface areas that mediate functional TIR-TIR interactions is key to the rational design of therapeutics that specifically target TLR signaling. We have designed a library of 11 cell-permeating decoy peptides each of which represents a non-fragmented patch of the TLR4 TIR surface, such that the library entirely encompasses the TLR4 TIR surface. Each peptide was synthesized in tandem with a cell-permeating sequence derived from Antennapedia homeodomain and tested for the ability to inhibit early cytokine mRNA induction and MAPK activation elicited in peritoneal murine macrophages by LPS. Five peptides, 4R1, 4R3, 4BB, 4R9, and 4αE, potently inhibited all manifestations of TLR4 signaling examined. These 5 peptides and a control peptide (CP) were then examined for the ability to bind directly to TLR4 TIR by FRET using time-resolved fluorescence spectroscopy. Peptides were labeled with Bodipy-TMR-X (BTX); the C-terminus of TLR4 was fused to Cerulean fluorescent protein (Cer). BTX-labeled peptides 4R1, 4BB, and 4αE efficiently quenched fluorescence of TLR4-Cer expressed in HeLa cells, indicating efficient FRET. BTX-4R3 exhibits weak quenching. BTX-labeled CP and 4R9 did not change TLR4-Cer fluorescence lifetime. These data suggest that the area between BB loop of TLR4 and its fifth helical region mediate TLR4 TIR dimerization.

A fluorescent conjugated polymer for trace detection of diamines and biogenic polyamines

The aggregation-based trace detection of diamines and biogenic polyamines has been developed utilizing a carboxylic acid-functionalized polyfluorene (PFCOOH-BT5) containing benzo[2,1,3]thiadiazole (BT). Upon addition of diamines or biogenic polyamines, the PFCOOH-BT5 can be cross-linked via electrostatic interactions between the polymer carboxylic acid binding sites and the amino groups of the multiamines to form interpolymer aggregation, which leads to more efficient FRET from fluorene segments to BT units and thus a noticeable fluorescence color change from blue to orange. In contrast, addition of 1,3-propanediol does not lead to significant variations in the photoluminescence spectra of PFCOOH-BT5. This unique fluorescence response from PFCOOH-BT5 to diamines and biogenic polyamines can achieve a detection concentration as low as 2 μM, indicating that PFCOOH-BT5 can serve as a general and effective chemosensor to detect trace diamines and biogenic polyamines.

A colorimetric and fluorescent turn-on chemosensor operative in aqueous media for Zn2+ based on a multifunctionalized spirobenzopyran derivative

By conjugating spiropyran chloride 8 with 2-amino-N-(quinolin-8-yl)acetamide (5), multifunctionalized spirobenzopyran derivative SPQN was designed and synthesized as a water soluble colorimetric and fluorescent turn-on chemosensor for Zn(2+). In 50% aqueous ethanol buffer solution, SPQN displayed a selective chelation fluorescence enhancement (16-fold) at 650 nm and visible color change (from colorless to red) with Zn(2+) among the metal ions examined. In addition, as the third channel to display the metal binding characteristics of SPQN, operating on an efficient FRET process between the quinoline and the merocyanine moiety of the sensor, ratiometric determination of Zn(2+) can be realized.

Artificial Photosynthesis

One-dimensional channel materials, such as zeolites and mesoporous silicas, are very attractive hosts for the preparation and investigation of hierarchically organized structures, presenting a successive ordering from the molecular up to macroscopic scale. The focus of this article is on artificial photonic antenna systems and on photocatalytically active layers that have been built by incorporating organic dyes, complexes, metal cations and clusters into 1-D nanochannel materials. We show that zeolite L as a host material allows for the design and preparation of a large variety of highly organized host–guest systems. The combination of a tuneable host morphology and the possibility of obtaining highly organized molecular patterns of guests leads to a variety of potential optical and photoelectronic applications. Strongly absorbing systems exhibiting efficient FRET along the c-axis of the zeolite crystals are accessible by sequential inclusion of multiple types of dyes. These new light-harvesting materials offer unique possibilities as building blocks for solar-energy conversion devices. A complementary approach consists in integrating photochemically active substances into zeolite monolayers coated on an electrode and taking advantage of intrazeolite processes for designing a reversible electrode for photocatalytic water oxidation. The photoelectrochemical water splitting capability of systems based on Ag+/AgCl/Agn-zeolite photoanodes are discussed.

Efficient energy transfer from pyrene to perylene assembled inside DNA duplex

Pyrene (donor) and perylene (acceptor) were assembled in a DNA duplex to increase the apparent Stokes' shift of perylene. Multiple donors were introduced in the vicinity of acceptors via D-threoninol and natural base pairs were inserted between donors and acceptors in order to suppress undesired interactions between them. When two pyrene moieties were located in proximity to one perylene with one base pair inserted between them, efficient FRET occurred within the duplex. Thus, strong emission at 495 nm was observed from perylene when excited at 345 nm where pyrene has its absorption. The apparent Stokes' shift became as large as 115 nm with a high FRET efficiency (Phi>1). However, the introduction of more than two pyrenes did not enhance the fluorescence intensity of perylene, due to the short Förster radius (R(0)) of the donor pyrene.

Efficiency of Energy Transfer from Organic Dye Molecules to CdSe−ZnS Nanocrystals: Nanorods versus Nanodots

We report on comparative experimental study of FRET efficiency in two different systems: organic dye molecules (donors) and CdSe-ZnS core-shell nanodots or nanorods (acceptors). Fluorescein isothiocyanate was bound chemically to the surface of nanocrystals using cysteine as a linker and the conjugates were embedded into the polymeric films. Contrary to intuitive presumptions based on the order of magnitude larger molar absorption coefficient for nanorods, the experiment demonstrated almost equal efficiency in the energy transfer from FITC to nanorods and nanodots. This effect is attributed to a distance-limited region of nanorod to which an efficient FRET from dye molecule can be achieved. These results may pave the way to hybrid materials with FRET efficiency controlled by the geometry of nanocrystals.

Practical and reliable FRET/FLIM pair of fluorescent proteins

BackgroundIn spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings.ResultsHere we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter).ConclusionThe combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.

Homogeneous and one-step fluorescent allele-specific PCR for SNP genotyping assays using conjugated polyelectrolytes

A new label-free, homogenous, sensitive and economical one-step method to detect SNP genotyping of genomic DNA has been developed by combining allele-specific PCR technique with water-soluble cationic conjugated polyelectrolytes (CCP). The amplification of target DNA and fluorescence detection steps are combined into one-step. The target DNA fragment containing a G allele site acts as PCR template. For the G allele-specific forward primer whose 3′-terminal base is complementary to the G allele template, after the first step of reverse primer extension, G allele-specific primer perfectly anneals with newly formed strand and the extension reaction of forward primer starts. During the extension, dGTP-Fl and dUTP-Fl are incorporated into extension chain in the presence of Taq DNA polymerase and more fluorescein-labeled PCR amplicons are yielded. Upon adding the CCP, strong electrostatic interactions between DNA and CCP bring them close and efficient FRET from CCP to fluorescein occurs. For the C allele-specific forward primer, less fluorescein-labeled PCR amplicons are yielded and inefficient FRET occurs. By triggering the change of emission intensity of CCP and fluorescein, it is possible to assay the SNP genotypes. In contrast to previous reports, this method does not require designing dye-labeled primers, and gel electrophoresis and isolation step after PCR were avoided in this homogenous method. The genotyping of 50ng genomic DNA from human lung cancer cell is easily detected using our new method. These qualities will make the new detection system ideal for SNP genotyping.

Ultrafast energy transfer from 3-mercaptopropionic acid-capped CdSe/ZnS QDs to dye-labelled DNA

Femtosecond-resolved fluorescence upconversion and picosecond-resolved spectroscopic measurements have been employed to confirm a highly efficient ultrafast FRET from MPA-capped CdSe/ZnS QDs to dye molecules attached to dodecamer DNA. It appears that hydrogen bonding is the associative mechanism between the MPA-capped QDs and DNA. High FRET efficiency of 92% together with the estimated donor–acceptor distance suggests that the adsorptive interactions between DNA and MPA-capped QDs result in a conformation in which DNA lies along the surface of the QD. Circular dichroism studies have been performed which reveal some perturbation in the native B-form of DNA in the nanobioconjugate.

Sensitive and Selective Label-Free DNA Detection by Conjugated Polymer-Based Microarrays and Intercalating Dye

We have demonstrated label-free and signal-amplifying DNA microarray using a conjugated polymer and an intercalating dye SYBR green I. Efficient FRET from the conjugated polymer to the dye produced large signal amplification so that without losing good selectivity, sensitive detection of subpicomolar concentrations of the target DNA was achieved.

Design of Cationic Conjugated Polyelectrolytes for DNA Concentration Determination

Cationic conjugated polyelectrolytes poly110 and poly120 were designed, synthesized, and characterized with the anticipation of function in the determination of double-stranded DNA concentration [dsDNA]. Their structures contain a pi-delocalized optically active backbone composed of phenylene-fluorene segments copolymerized with 2,1,3-benzothiadiazole (BT) units and charged pendant groups that allow excellent solubility in water. The subscript in poly1x refers to the molar percent of BT units in the chain. Addition of dsDNA to poly110 or poly120 results in a change in the color of emission from blue to green. These spectral changes can be treated to obtain the parameter delta, which can be used to generate calibration curves that indicate [dsDNA]. Analysis of photoluminescence spectra reveals that dsDNA addition gives rise to more efficient FRET from blue emitting segments to the BT sites, an increase in the BT emission quantum yield, and partial quenching of the phenylene-fluorene segments. Studies were also carried out to maximize the range of [dsDNA] determination. We find that by combining the response from two different initial concentrations of poly110, it is possible to generate calibration curves that respond with a difference in [dsDNA] of over 7 orders of magnitude.

Real Time Fluorescent Resonance Energy Transfer Visualization of Ferric Pyoverdine Uptake in Pseudomonas aeruginosa: A ROLE FOR FERROUS IRON

To acquire iron, Pseudomonas aeruginosa secretes a major fluorescent siderophore, pyoverdine (PvdI), that chelates iron and shuttles it into the cells via the specific outer membrane transporter, FpvAI. We took advantage of the fluorescence properties of PvdI and its metal chelates as well as the efficient FRET between donor tryptophans in FpvAI and PvdI to follow the fate of the siderophore during iron uptake. Our findings with PvdI-Ga and PvdI-Cr uptake indicate that iron reduction is required for the dissociation of PvdI-Fe, that a ligand exchange for iron occurs, and that this dissociation occurs in the periplasm. We also observed a delay between PvdI-Fe dissociation and the rebinding of PvdI to FpvAI, underlining the kinetic independence of metal release and siderophore recycling. Meanwhile, PvdI is not modified but recycled to the medium, still competent for iron chelation and transport. Finally, in vivo fluorescence microscopy revealed patches of PvdI, suggesting that uptake occurs via macromolecular assemblies on the cell surface.

Fluorescence resonance energy transfer between DPH and Nile Red in a lipid bilayer

FRET between the well-known membrane binding probes DPH (donor) and Nile Red (acceptor) is investigated in a phosphatidylserine lipid bilayer. A ∼80% FRET efficiency was observed from the steady state experiments for a DPH:Nile Red:lipid molar ratio of 1:2:190, suggesting an efficient FRET process. Time resolved experiments however, provided a lower (∼50%) FRET efficiency. The emission of Nile Red was observed to rise with a time constant of 0.8–0.4 ns only when DPH is selectively excited. This time constant is ascribed due to FRET from which an average donor–acceptor distance of 27 Å is obtained for a DPH:Nile RED:lipid molar ratio of 1:1:190.

A Fluorescence Ratiometric Protein Assay Using Light‐Harvesting Conjugated Polymers

AbstractSummary: A highly selective protein assay was created which combines the fluorescent ratiometric technique based on FRET with the light‐harvesting properties of conjugated polymers. The cationic poly[(9,9‐bis(6′‐N,N,N‐trimethylammonium)‐hexyl)‐fluorene phenylene] bromide (PFP‐NMe) and the negatively charged biotinylated fluorescein probe (Fl‐B) were used to detect the target protein streptavidin optically. The strong electrostatic interactions between PFP‐NMe and fluorescein result in efficient FRET from PFP‐NMe to fluorescein. In the presence of streptavidin, however, the biotin moiety of Fl‐B specifically associates with streptavidin and the fluorescein molecule is buried deeply in the adjacent vacant binding sites. This separates the fluorescein spatially from the PFP‐NMe moiety, resulting in inefficient FRET from PFP‐NMe to fluorescein. Although a nonspecific protein, such as BSA, shows nonspecific interactions with PFP‐NMe, it does not affect the fluorescent ratio value of PFP‐NMe to fluorescein. Hence, the charged neutral complex of two oppositely charged conjugated polymers can eliminate the nonspecific interactions, and thus optimize their application in protein assays.A schematic representation of the protein assay operation.magnified imageA schematic representation of the protein assay operation.

Enhanced Luminescence Efficiency from Hydrogel Microbead Encapsulated Quantum Dots

ABSTRACTIn this paper, a novel quantum dot (QD) based nanomaterial system is presented for efficient FRET analysis. The quantum dots have been embedded in hydrogel microspheres based on poly(N-isopropylacrylamide) (PNIPAM) a thermoresponsive polymer that undergoes a volume phase transition across the low critical solution (LCST). The optical properties of the quantum dots entrapped within the gel microspheres has been modified due to change in refractive index, volume density of the surrounding hydrogel medium. The QDs encapsulated in the PNIPAM microspheres showed 100–200 % enhancement in the PL efficiency as the microgels shrank at the temperature above the LCST temperature of the gel.

Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein

Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, ∼20 Å from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.

Fluorescence resonance energy transfer between a quantum dot donor and a dye acceptor attached to DNA

We show that direct coupling of a dye-labelled DNA (acceptor) to a quantum dot (QD) donor significantly reduces the donor-acceptor distance and improves the FRET efficiency: a highly efficient FRET (approximately 88%) at a low acceptor-to-donor ratio of 2 has been achieved at the single-molecule level.

Unnatural amino acid derived FRET cassettes, terminators and their DNA sequencing potential

An unnatural, tri-functional amino acid , t-Boc- l- para-amino-phenylalanine ( 1 ), was utilized in the design and syntheses of highly efficient FRET cassettes ( 4– 7 ) and converted them into 2′,3′-dideoxynucleotide terminators ( 8– 11 ) for investigating DNA sequencing potential coupled with a thermostable DNA polymerase, Thermo Sequenase™ II.

Coumarin–fluorescein pair as a new donor–acceptor set for fluorescence energy transfer study of DNA

A method for introduction of the 2′-coumarin labeled nucleoside as a fluorescence energy donor into DNA duplexes has been described. Efficient FRET occurs between the coumarin–fluorescein pair in DNA owing to the high quantum yield of the donor. The present donor–acceptor pair may be useful as FRET indicator of DNA structures in solution.