Self-assembled DNA photonic wire for long-range energy transfer

DNA is a promising material for use in nanotechnology; the persistence length of double stranded DNA gives it a rigid structure in the several-nanometer regime, and its four letter alphabet enables addressability. We present the construction of a self-assembled DNA-based photonic wire capable of transporting excitation energy over a distance of more than 20 nm. The wire utilizes DNA as a scaffold for a chromophore with overlapping absorption and emission bands enabling fluorescence resonance energy transfer (FRET) between pairs of chromophores leading to sequential transfer of the excitation energy along the wire. This allows for the creation of a self-assembled photonic wire using straightforward construction and, in addition, allows for a large span in wire lengths without changing the basic components. The intercalating chromophore, YO, is chosen for its homotransfer capability enabling effective diffusive energy migration along the wire without loss in energy. In contrast to heterotransfer, i.e., multistep cascade FRET, where each step renders a photon with less energy than in the previous step, homotransfer preserves the energy in each step. By using injector and detector chromophores at opposite ends of the wire, directionality of the wire is achieved. The efficiency of the wire constructs is examined by steady-state and time-resolved fluorescence measurements and the energy transfer process is simulated using a Markov chain model. We show that it is possible to create two component DNA-based photonic wires capable of long-range energy transfer using a straightforward self-assembly approach.     

Subunit exchange of MjHsp16.5 studied by single-molecule imaging and fluorescence resonance energy transfer

MjHsp16.5 was separately labeled by fluorescent dye Cy3 and Cy5.5. The dissociation event of a single 24-mer MjHsp16.5 molecule was captured by single-molecule imaging (SMI). Temperature-regulated subunit exchange was revealed by the real-time fluorescence resonance energy transfer (FRET). The combination of single-molecular statistics and kinetic parameters from FRET experiments leads to the conclusion that below 75 degrees C the rate-determining step of the subunit exchange was the dissociation of the dye-labeled 24-mer in which the dimer was intact, whereas above 75 degrees C, smaller units emerged in the exchange and the rate-determining step had the character of a bimolecular reaction.     

Impact of Deuteration on the Assembly Kinetics of Transthyretin Monitored by Native Mass Spectrometry and Implications for Amyloidoses

It is well established that the formation of transthyretin (TTR) amyloid fibrils is linked to the destabilization and dissociation of its tetrameric structure into insoluble aggregates. Isotope labeling is used for the study of TTR by NMR, neutron diffraction, and mass spectrometry (MS). Here MS, thioflavin T fluorescence, and crystallographic data demonstrate that while the X‐ray structures of unlabeled and deuterium‐labeled TTR are essentially identical, subunit exchange kinetics and amyloid formation are accelerated for the deuterated protein. However, a slower subunit exchange is noted in deuterated solvent, reflecting the poorer solubility of non‐polar protein side chains in such an environment. These observations are important for the interpretation of kinetic studies involving deuteration. The destabilizing effects of TTR deuteration are rather similar in character to those observed for aggressive mutations of TTR such as L55P (associated with familial amyloid polyneuropathy).     

ENERGY TRANSFER AMONG THE CHROMOPHORES OF C-PHYCOCYANIN FROM Anabaena variabilis USING STEADY STATE AND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Abstract We have investigated the model of energy transfer between sensitizing (s) and fluorescing (f) chromophores for the αβ monomer and for the separated α and β subunits of C-phycocyanin from Anabaena variabilis using fluorescence emission spectroscopy, fluorescence excitation polarization, and picosecond-resolved fluorescence decay kinetics. The fluorescence emission maximum occurs at 640 nm for all samples. The fluorescence excitation polarization is constant ( P = 0.40) across the absorption hand for the α subunit, but it increases across the absorption band towards longer wavelength for the β subunit and the αβ monomer. The fluorescence decay kinetics exhibit two exponential lifetimes of 1.3-1.5 ns and 340-500 ps for the αβ monomer and for the α and β subunit preparations.
We attribute the change in polarization across the absorption band to energy transfer among the three chromophores in the αβ monomer and among the two chromophores in the separated β subunit. The constant, relatively high polarization in the separated a subunit, having only one chromophore, is consistent with the absence of both energy transfer and chromophore rotation. The concentration of the α subunit did not affect the decay kinetics, suggesting that the short lifetime component does not arise from aggregation of the α subunits. The biexponential decay kinetics of the α subunit cannot be explained using the sensitizing-fluorescing model. The possibility of conformational interactions is under investigation.     

Monitoring the dynamics of ligand-receptor complexes on model membranes

Ligand-induced cross-linking of cell surface receptors is a basic paradigm of signal activation by many transmembrane receptors. After ligand binding, the receptor complexes formed on the membrane are dynamically maintained by two-dimensional protein-protein interactions on the membrane. The biophysical principles governing the dynamics of such interactions have not been understood, mainly because the measurement of lateral interactions on membranes so far has not been experimentally addressed. Here, we describe a generic approach for measuring two-dimensional dissociation rate constants in vitro using a novel high-affinity chelator lipid for reconstituting a ternary cytokine-receptor complex on solid-supported membranes. While monitoring the interaction between the ligand and one of the receptor subunits on the membrane by fluorescence resonance energy transfer, the equilibrium on the surface was perturbed by rapidly tethering a large excess of the unlabeled receptor subunit. Displacement of labeled by unlabeled protein in the ternary complex was detected as a recovery of the donor quenching. Since the dissociation of the ligand-receptor complex in plane of the membrane was the rate-limiting step under these conditions, the two-dimensional rate constant of this process was determined. Strikingly, the two-dimensional dissociation was much slower than ligand dissociation into solution, suggesting that membrane tethering significantly affects the dissociation process. This result highlights the importance of studying ligand-receptor complexes tethered to membranes for understanding the principles governing signal activation by ligand-induced receptor assembling.     

Encapsulation of fluorescent molecules by functionalized polymeric nanocontainers: investigation by confocal fluorescence imaging and fluorescence correlation spectroscopy

Nanocontainers (NCs) were prepared from amphiphilic triblock copolymers, having an average molecular weight of around 8000 g/mol, by using previously published preparation methods consisting of dispersing the polymer in an aqueous buffer solution containing molecules for encapsulation. A small molecular weight fluorophore, sulforhodamine B, as well as the fluorescent protein avidin labeled with Alexa 488 were encapsulated, and the resulting nanocontainers were characterized using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS). Nanocontainer size determination by FCS is very robust and compares well with results obtained from photon correlation spectroscopy: the measured diameters of the polymeric nanocontainers vary between 140 and 172 nm. Encapsulation of fluorescent molecules was determined by evaluating the molecular brightness of nanocontainers with an encapsulated fluorescently labeled protein (avidin-Alexa 488). Results indicate that the number of encapsulated avidin-Alexa 488 molecules corresponds well with the initial concentration of the fluorescently labeled protein and the encapsulated volume. A nanocontainer binding assay was developed using biotinylated fluorescently labeled nanocontainers. Binding of biotinylated nanocontainers to fluorescently labeled streptavidin was followed by fluorescence cross-correlation spectroscopy. The intrinsic dissociation constant, K(d), of labeled streptavidin to the ligand-modified nanocontainers is 1.7 +/- 0.4 x 10(-8) M, and about 1921 +/- 357 molecules of labeled streptavidin are bound to each nanocontainer.     

Steric Exclusion Liquid Chromatography Studies in Urea on the Denaturation of the Bovine Eye Lens Protein α-Crystallin

Abstract

Steric exclusion liquid chromatography in the presence of intermediate urea concentrations with lowangle laser light scattering detection was used to investigate the stepwise dissociation of the multimeric bovine eye lens protein α-crystallin. The change in the quaternary structure of α-crystallin as a function of increasing urea concentration clearly resembled dissociation by increasing alkaline pH, urea or guanidine-HC1 concentrations when studied by sedimentation velocity analysis. Next to native and native-like threelayer aggregates (M_r 6.5 ? 7.5 × 10⁵), the first dissociation products (two-layer molecules M_r 4 ? 5.5 × 10⁵), the second dissociation products (core molecules M_r 2.5 ? 3 × 10⁵), and monomeric subunits (M_r 20 000) could be characterized. In the range from 2.6 to 4.4 M urea, we found a gradual decrease in the proportion of the remaining three-layer aggregates and an increase in that of monomeric subunits. The fluorescence emission maxima showed increasing solvent exposure of the tryptophan residues going from three-layer aggregates to monomeric subunits. The subunit compositions for most dissosiation products did not significantly differ from that of native α-crystallin. The interpretation of earlier results on Sephacryl-S200 steric exclusion chromatography in 3.8 M urea appeared to be an oversimplification.     

Fluorescence measurements of duplex DNA oligomers under conditions conducive for forming M-DNA (a metal-DNA complex)

M-DNA (a metal complex of DNA with millimolar concentrations of Zn2+, Co2+, or Ni2+ and basic pH) has been proposed to undergo electron transfer over long distances along the helix and has generated interest as a potential building block for nanoelectronics. We show that DNA aggregates form under solvent conditions favorable for M-DNA (millimolar zinc and pH = 8.6) by fluorescence correlation spectroscopy. We have performed steady-state F?rster resonance energy transfer (FRET) experiments with DNA oligomers conjugated with 6-carboxyfluorescein and tetramethylrhodamine to the opposite ends of double-stranded DNA (dsDNA) molecules. Enhanced acceptor emission is observed for distances larger than expected for identical DNA molecules with no zinc. To avoid intermolecular FRET, the fluorescently labeled dsDNA is diluted with a 100-fold excess of unlabeled dsDNA. The intramolecular FRET efficiency increases 25-fold for a 30-mer doubly labeled duplex DNA molecule upon addition of millimolar concentrations of zinc ions. Without zinc, this oligomer has less than 1% FRET efficiency. This dramatic increase in the FRET efficiency points to either significant changes in the F?rster radius or fraying of the ends of the DNA helices. The latter hypothesis is supported by our experiments with a 9-mer that show dissociation of the duplex by zinc ions.     

荧光标记法研究双嵌段共聚物的胶束化行为

结合Forster能量转移和芳香分子极性标尺技术,研究了两端分别标记芴和芘基团、具有不相容双嵌段的共聚物(Fl-PMMA400-b-PAA80-Py)的胶束化行为。结果表明,在临界胶束浓度以下,较长的PMMA链段首先形成寡链聚集体,这一现象反映了具有不相容双嵌段的共聚物胶束化特征;此外,本文首次发现芴基团荧光对其微环境极性较为敏感。     

On the Structure of PHB (=Poly[(R)‐3‐hydroxybutanoic Acid]) in Phospholipid Bilayers: Preparation of Trifluoromethyl‐Labeled Oligo[(R)‐3‐hydroxybutanoic Acid] Derivatives

Oligomers of (R)‐3‐hydroxybutanoate (OHB) have previously been shown to transport cations through lipid bilayers. The ion‐transport activity has been attributed to the formation of hydrophobic aggregates or pores, which have been identified by fluorescence‐microscopy measurements of membrane‐incorporated fluorescence‐labeled OHBs. To obtain more information about these aggregates, we describe here the synthesis of the specifically F‐labeled HB oligomers II – IV for structural investigation by means of solid‐state ¹⁹F‐NMR spectroscopic techniques.     

Photoresponse and anisotropy of rhodamine dye intercalated in ordered clay layered films

In order to elaborate organized two-dimensional arrangements of fluorescent dyes in host solid layered materials, rhodamine 6G (R6G) is encapsulated in supported thin films of laponite (Lap) clay. Clay films are elaborated by the spin-coating technique and their surface morphology is analyzed by scanning electron (SEM) and atomic force (AFM) microscopies. The internal order of the stacked clay layers is checked by X-ray diffraction technique (DRX). The thermostability of R6G in the Lap films is discussed on the basis of several thermogravimetric and calorimetric techniques (TG, DTA and DSC). The R6G adsorbed species in Lap films are characterized by absorption and fluorescence (steady-state and time-resolved) spectroscopies. Monomers, dimers and higher-order aggregates are identified for very low (<0.1%), moderate (1–25%) and high (>40% of the total cation exchange capacity, CEC, of the clay) dye content, respectively. Both non-fluorescence H-type and fluorescent J-type aggregates of R6G in Lap films are characterized.Absorption and fluorescence techniques with linearly polarized light are applied to evaluate the anisotropic photoresponse of R6G in Lap films, from which the preferential orientation of dye molecules with respect to the clay layers can be evaluated. The validity of the newly established fluorescence polarization is contrasted with the well-established absorption polarization method, and the emission spectroscopy with linearly polarized light can be applied to establish the preferential orientation of fluorophore molecular probes incorporated in any rigid and ordered 2D host materials, including monolayers and biological membranes.     

The synthesis of curved and linear structures from a minimal set of monomers

[reaction: see text] Spiro-ladder oligomers of designed shape were assembled from a set of two enantiomeric bis-amino acid monomers. Two tetramers of differing monomer sequence were synthesized to study the effect of monomer stereochemistry upon macromolecular shape. Two-dimensional NMR experiments were used to determine the conformational preference of the monomers within the context of the oligomers. The results of this structural study were used to design two pentamers: one resembling a rod and another with a curved shape. The pentamers were end-labeled with naphthyl and dansyl groups. The design hypothesis was confirmed by measuring the efficiency of fluorescence resonance energy transfer between the naphthyl and dansyl fluorophore pair.     

Selective labeling of polypeptides using protein farnesyltransferase via rapid oxime ligation

An aldehyde-containing alternative substrate for protein farnesyltransferase was prepared and shown to be enzymatically incorporated into a peptide and a protein. The protein was subsequently immobilized onto aminooxy-functionalized agarose beads or labeled with a fluorophore. This method for protein modification provides an alternative to the commonly employed Cu(I)-catalyzed click reaction.     

Superquencher formation via nucleic acid induced noncovalent perylene probe self-assembly

A fluorophore labeled oligonucleotide could induce aggregation of a positively charged perylene probe. The perylene aggregate could very efficiently quench the fluorescence of the labeled fluorophore. Based on this observation, a new method for the highly sensitive and selective detection of a protein has been developed.     

Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling

Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.     

Exciton migration by ultrafast Förster transfer in highly doped matrixes

The energy transfer between dye molecules and the mobility of the corresponding excitons are investigated in polymethyl methacrylate films highly doped with perylene bisimide dyes. The dynamics is measured by group delay corrected, femtosecond broad-band spectroscopy revealing the transfer route via absorption changes that are specific for the participating species. In films doped with 0.14 M perylene orange an ultrafast homotransfer between the dye molecules is found by analyzing the loss of the excitation-induced anisotropy. The process exhibits a stretched exponential time dependence which is characteristic for F?rster energy transfer between immobilized molecules. The transfer time is 1.5 ps for an average transfer distance of 2.3 nm and results in a high mobility of the optically generated excitons. In addition, we find that the excitons move to perylene orange dimers, which have formed in low concentration during the sample preparation. The observed energy transfer time is slightly shorter than expected for a direct F?rster transfer and indicates that exciton migration by multistep transfer between the monomers speeds up the transport to the dimers. In samples doped with perylene orange and perylene red heterotransfer to perylene red takes place with transfer times down to 600 fs. The mechanism is F?rster transfer as demonstrated by the agreement with calculations assuming electric dipole interaction between immobilized and statistically distributed donor and acceptor units. The model predicts the correct time dependence and concentration scaling for highly doped as well as diluted samples. The results show that ultrafast exciton migration between dye molecules in highly doped matrixes is an attractive and efficient mechanism to transport and collect energy in molecular systems and organic electronic devices. Further optimization should lead to a loss-free transport over distances typical for the thickness of active layers in these systems.     

A fluorescence turn-on method for real-time monitoring of protease activity based on the electron transfer between a fluorophore labeled oligonucleotide and cytochrome c

A new continuous fluorescence turn-on assay for protease activity and inhibitor screening has been developed. A fluorophore labeled single stranded DNA (FAM-DNA) and cytochrome c (cyt c) were employed. The fluorescence of the FAM-DNA was efficiently quenched when binding to cyt c, through the electron transfer between the FAM fluorophore and the heme cofactor of cyt c. In the presence of a protease, such as trypsin, cyt c was digested into small peptide fragments. The FAM-DNA was released, which resulted in the recovery of the FAM fluorescence. The rate of the cyt c digestion could be reduced via the addition of an inhibitor. As a result, reduced degree of the fluorescence recovery was obtained. The limit of detection of our assay is 1 nM trypsin and the IC₅₀ values are 3.23 μg mL⁻¹ and 0.303 μg mL⁻¹ for the inhibitor from egg white and the inhibitor from soybean, respectively. Our method could be used for the sensing of protease activity for various biochemical applications, and for the screening of protease inhibitors as drugs for the treatment of various related diseases.     

Detection of Helicobacter pylori with a nanobiosensor based on fluorescence resonance energy transfer using CdTe quantum dots

We report on a method for the sensitive determination of Helicobacter that is based on fluorescence resonance energy transfer using two oligonucleotide probes labeled with CdTe quantum dots (QDs) and 5-carboxytetramethylrhodamine (Tamra) respectively. QDs labeled with an amino-modified first oligonucleotide, and a Tamra-labeled second oligonucleotide were added to the DNA targets upon which hybridization occurred. The resulting assembly brings the Tamra fluorophore (the acceptor) and the QDs (the donor) into close proximity and causes fluorescence resonance energy transfer (FRET) to occur upon photoexcitation of the donor. In the absence of target DNA, on the other hand, the probes are not ligated, and no emission by the Tamra fluorophore is produced due to the lack of FRET. The feasibility of the method was demonstrated by the detection of a synthetic 210-mer nucleotide derived from Helicobacter on a nanomolar level. This homogeneous DNA detection scheme is simple, rapid and efficient, does not require excessive washing and separation steps, and is likely to be useful for the construction of a nanobiosensor for Helicobacter species.

THE EFFECTS OF AGGREGATION ON THE FLUORESCENCE and THE TRIPLET STATE YIELD OF HEMATOPORPHYRIN

Abstract— The fluorescence and triplet state yields of hematoporphyrin have been measured in wuter/methanol mixtures. There is a closely linked response of these yields to aggregation of the hematoporphyrin molecule. The monomer, which is the principal species present at high methanol content, has a triplet state yield of 0.91 and a fluorescence yield of 0.09. By contrast, hematoporphyrin solutions with a high water content containing aggregates have lower fluorescence and triplet state yields, e.g. 0.018 and 0.56, respectively, in water. Static, singlet state quenching in some of the aggregates is responsible for the reduced fluorescence yield. The results also show that in addition to these aggregates there are other types of aggregates where there is an increased singlet to ground state radiative transition probability, resulting from the interaction between transition dipoles in adjacent molecules.     

Intramolecular dimers: a new strategy to fluorescence quenching in dual-labeled oligonucleotide probes

Many genomics assays use profluorescent oligonucleotide probes that are covalently labeled at the 5' end with a fluorophore and at the 3' end with a quencher. It is generally accepted that quenching in such probes without a stem structure occurs through F?rster resonance energy transfer (FRET or FET) and that the fluorophore and quencher should be chosen to maximize their spectral overlap. We have studied two dual-labeled probes with two different fluorophores, the same sequence and quencher, and with no stem structure: 5'Cy3.5-beta-actin-3'BHQ1 and 5'FAM-beta-actin-3'BHQ1. Analysis of their absorption spectra, relative fluorescence quantum yields, and fluorescence lifetimes shows that static quenching occurs in both of these dual-labeled probes and that it is the dominant quenching mechanism in the Cy3.5-BHQ1 probe. Absorption spectra are consistent with the formation of an excitonic dimer, an intramolecular heterodimer between the Cy3.5 fluorophore and the BHQ1 quencher.