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1.
Oligonucleotide 9mers containing 2'-O-(1-pyrenylmethyl)uridine [U(pyr)] at the center position were synthesized by using a protected U(pyr) phosphoramidite. The UV melting behaviors indicate that the pyrene-modified oligonucleotides can bind to both their complementary DNA and RNA in aqueous solution. When compared with the unmodified oligonucleotides, the pyrene-modified oligonucleotides showed higher affinity for DNA while exhibiting lower affinity for RNA. The pyrene-modified oligonucleotides in diluted solution exhibited fluorescence typical of pyrene monomer emission [lambdamax 378 (band I) and 391 nm (band III)]. When these oligomers bound to DNA, the fluorescence intensity ratio of band III/band I was increased. With this fluorescence change, a new broad emission (lambdamax 450 nm) due to exciplex between the pyrene and an adjacent nucleobase appeared. In contrast, addition of RNA to the pyrene oligonucleotides resulted in enhancement of the pyrene monomer emission with decrease in the fluorescence band ratio. The extent of the emission enhancement was found to be highly dependent on the nucleobase adjacent to the U(pyr) in the pyrene oligomers. The pyrene oligonucleotide containing dC at the 3'-site of the modification showed remarkable increase (approximately 250 times) in fluorescence (375 nm) upon binding to complementary RNA. The present findings would open the way to the design of a highly sensitive fluorescent probe of RNA.  相似文献   

2.
The mechanism of spectral shift and absorption intensity change of the divalent bromocresol purple (BCP) anion was further investigated and it was characterized as a spectrophotometric membrane probe. At high concentrations (1-40 mM), the absorption intensity of th BCP anion at 590 nm (monomer band) decreased markedly with increase of the dye concentration, while another absorption band appeared at 554 nm. Analysis of the change of absorption intensity showed that the mared decrease resulted from dimer formation of BCP (polymer formation at concentrations higher than 20 mM). Wavelengths of maximum absorption (lambdamax) of the BCP anion were determined in various solvents and comparison of these lambdamax's with lambdamax of the BCP anion bound to SR showed that the hydrophobicity of the area of BCP anion binding to SR corresponded to a refractive index of 1.429. While the BCP anion bound to SR showed a monomer spectrum, a dimer band appeared for the BCP anion bound to SR-Pi (phosphorylated protein) with a marked decrease in the absorption intensity at the monomer band, indicating that two polar groups, binding sites for the BCP anions, closely approached each other in the SR-Pi configuration.  相似文献   

3.
We report here the design, synthesis and application of pyrene binary oligonucleotide probes for selective detection of cellular mRNA. The detection strategy is based on the formation of a fluorescent excimer when two pyrene groups are brought into close proximity upon hybridization of the probes with the target mRNA. The pyrene excimer has a long fluorescence lifetime (>40 ns) compared with that of cellular extracts (~7 ns), allowing selective detection of the excimer using time-resolved emission spectra (TRES). Optimized probes were used to target a specific region of sensorin mRNA yielding a strong excimer emission peak at 485 nm in the presence of the target and no excimer emission in the absence of the target in buffer solution. While direct fluorescence measurement of neuronal extracts showed a strong fluorescent background, obscuring the detection of the excimer signal, time-resolved emission measurements indicated that the emission decay of the cellular extracts is ~8 times faster than that of the pyrene excimer probes. Thus, using TRES of the pyrene probes, we are able to selectively detect mRNA in the presence of cellular extracts, demonstrating the potential for application of pyrene excimer probes for imaging mRNAs in cellular environments that have background fluorescence.  相似文献   

4.
Fluorescent DNA probes with 1,6-hexanediyl as the linker between two pyrenes, phenylpyrenes or phenylethynyl pyrene fluorophores were synthesized (Py-1, Py-2 and Py-3) and their interactions with DNA were studied by UV–vis absorption spectra, fluorescence spectra and viscosity measurements. The probes show red-shifted emission compared with pyrene (up to 20 nm). We found the interaction of these probes with DNA can be either intercalation or groove binding. Ratiometric fluorometry (ratio of the monomer and excimer emission intensity versus concentration of DNA) was achieved with these probes for DNA quantification (with limit of detection, LOD, up to 0.1 μg/mL). We also found that the undesired oxygen sensitivity of the emission intensity of pyrene fluorophore can be greatly suppressed by extending the π-conjugation framework of pyrene (the IAr/Iair value is decreased from 8.10 for pyrene to less than 2.20 for the DNA probes described herein).  相似文献   

5.
Homogeneous DNA hybridization assay based on luminescence resonance energy transfer (LRET) from a tetradentate beta-diketonate europium chelate, 4,4'-bis(1' ',1' ',1' ',2' ',2' ',3' ',3' '-heptafluoro-4' ',6' '-hexanedion-6' '-yl)-chlorosulfo-o-terphenyl (BHHCT)-Eu(3+) (lambda(ex) = 340 nm and lambda(em) = 615 nm), to an organic dye, Cy5 (lambda(ex) = 643 nm and lambda(em) = 669 nm) has been developed, in which two DNA probes whose sequences comprises the whole complementary strand to the target DNA, are used; one probe having a biotin label on the 3'-terminus and the other a Cy5 label on the 5'-terminus. After hybridization, streptavidin labeled with BHHCT-Eu(3+) was added to the hybridization solution, and in the presence of the target DNA, the sensitized emission of Cy5 was observed when the hybridized complex was irradiated at 340 nm. In the absence of the target DNA, no emission was observed from Cy5.  相似文献   

6.
Oligonucleotide conjugates bearing two pyrene residues attached to 5′-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (λmax 382 nm) and a broad emission peak with λmax 476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5′-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5′-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.  相似文献   

7.
The design, synthesis, and properties of a new pyrene excimer-forming probe of DNA have been described. 2,2-(Aminomethyl)propanediol was converted by the reaction with 1-pyrenebutylic acid to bis-pyrene-modified propanediol as a fluorescent non-nucleosidic linker. The bis-pyrene-modified linker can be incorporated via phosphoramidite chemistry into the 5'-terminal or internal positions of oligonucleotides (ODNs). The terminally modified ODNs showed almost similar affinity for complementary DNA when compared with the corresponding unmodified ODNs. The duplexes containing the bis-pyrene in the main chain exhibited higher melting temperatures relative to the corresponding duplexes containing propanediol linker at the same position. The UV and CD spectral studies indicate that the stacking interactions between the pyrene and DNA bases occur in the internally modified duplex and do not in the terminally modified duplex. The bis-pyrene modified linker itself displays excimer (E at 480 nm) and monomer (M at 380 nm) emission in a quantum yield (QY) of 0.17 and the E/M intensity ratio of 15. Incorporation of this linker into the terminal or internal positions of ODNs reduced the QY (0.003-0.009) and the E/M ratio (0.3-0.8). While small changes in the QY and E/M ratio was obtained in binding of the internally labeled ODNs to DNA, up to 27-fold increase in the QY and 17-fold increase in the E/M ratio was observed upon hybridization of the terminally labeled ODNs with DNA. The excimer and monomer fluorescence changes were found to be sensitive to a mismatch base present in the target DNA. The bis-pyrene-modified ODNs thus provide a sequence-sepcific fluorescent probe of DNA.  相似文献   

8.
Pyrene is a spatially sensitive probe that displays an ensemble of monomeric fluorescence emission peaks (375-405 nm) and an additional band (called excimer) at ~460 nm when two fluorophores are spatially proximal. We examined if there is a correlation between distance between two pyrenes on an α-helical structure and excimer/monomer (e/m) ratio. Using structure-guided design, pyrene maleimide was attached to pairs of Cys residues separated by ~5 ? increments on helix 2 of the N-terminal domain of apolipoprotein E3 (apoE3). Fluorescence spectral analysis revealed an intense excimer band when the probes were ~5 ? from each other with an e/m ratio of ~3.0, which decreased to ~1.0 at 20 ?. An inverse correlation between e/m ratio and the distance between pyrenes was observed, with the probe and helix flexibility also contributing to the extent of excimer formation. We verified this approach by estimating the distance between T57C and C112 (located on helices 2 and 3, respectively) to be 5.2 ? (4.9 ? from NMR and 5.7 ? from the X-ray structure). Excimer formation was also noted to a significant extent with probes located in the linker segment, suggesting spatial proximity (10-15 ?) to corresponding sites on neighboring molecules in the tetrameric configuration of apoE. We infer that oligomerization via the C-terminal domain juxtaposes the linker segments from neighboring apoE molecules. This study offers new insights into the conformation of tetrameric apoE and presents the use of pyrene as a powerful probe for studying protein spatial organization.  相似文献   

9.
We have studied the use of 'pseudocyclic oligonucleotides' (PCOs) (Jiang et al. Bioorg. Med. Chem. 1999, 7, 2727) as hybridization-based fluorescent probes. The resulting fluorescent tag-attached PCOs are called 'cyclicons'. Cyclicons consist of two oligonucleotides linked to each other through 3'-3' or 5'-5' ends. One of the oligos is the probe or primer-probe sequence that is complementary to a target nucleic acid (mRNA/DNA), and the other is a modifier oligo that is complementary to one of the ends of the probe oligo. A fluorescence molecule and a quencher molecule are attached at an appropriate position in the cyclicons. In the absence of the target nucleic acid, the fluorophore and the quencher are brought in close proximity to each other because of the formation of an intramolecular cyclic structure, resulting in fluorescence quenching. When the cyclicon hybridizes to the complementary target nucleic acid strand, the intramolecular cyclic structure of the cyclicon is destabilized and opened up, separating the fluorophore and quencher groups, resulting in spontaneous fluorescence emission. Fluorescent studies in the presence and absence of a target nucleic acid suggest that cyclicons exist in intramolecular cyclic structure form in the absence of the target and form the duplex with the target sequence when present. Both the cyclicons are useful for nucleic acid detection. The studies with DNA polymerase on 5'-5'-attached cyclicons suggest that the presence of quencher moiety in the probe sequence does not inhibit chain elongation by polymerase. The experiments with a 5'-5'-attached cyclicon suggest the new design serves as an efficient unimolecular primer-probe in real-time PCR experiments.  相似文献   

10.
Wavelength-shifting molecular beacons   总被引:14,自引:0,他引:14  
We describe wavelength-shifting molecular beacons, which are nucleic acid hybridization probes that fluoresce in a variety of different colors, yet are excited by a common monochromatic light source. The twin functions of absorption of energy from the excitation light and emission of that energy in the form of fluorescent light are assigned to two separate fluorophores in the same probe. These probes contain a harvester fluorophore that absorbs strongly in the wavelength range of the monochromatic light source, an emitter fluorophore of the desired emission color, and a nonfluorescent quencher. In the absence of complementary nucleic acid targets, the probes are dark, whereas in the presence of targets, they fluoresce-not in the emission range of the harvester fluorophore that absorbs the light, but rather in the emission range of the emitter fluorophore. This shift in emission spectrum is due to the transfer of the absorbed energy from the harvester fluorophore to the emitter fluorophore by fluorescence resonance energy transfer, and it only takes place in probes that are bound to targets. Wavelength-shifting molecular beacons are substantially brighter than conventional molecular beacons that contain a fluorophore that cannot efficiently absorb energy from the available monochromatic light source. We describe the spectral characteristics of wavelength-shifting molecular beacons, and we demonstrate how their use improves and simplifies multiplex genetic analyses.  相似文献   

11.
The chromophore (purple complex) of bacteriorhodopsin is reduced by sodium borohydride upon illumination to RPhv with a three-peaked absorption band at 360 nm. Treatment of this reduction product with ultraviolet light or acid yields a modified product from which retro-retinyllysine can be obtained by alkaline hydrolysis. No reduction of the 412 nm complex was found. Under specific conditions the purple complex equilibrates with a photochemically active 460 nm form that can be reduced by borohydride in the dark. This reduction product RP460 behaves idential to RPHV. Reconstitution of the purple complex from chromophore-free membrane (apomembrane) and retinal occurs via intermediates. The first (lambdamax 400nm) shows a three-peaked absorption band and is reduced to RP400 without a change of the three-peaked absorption (lambdamax 360 nm). The same product is obtained from apomembrane and retinol. Detergents shift the absorption band to 330 nm in all cases. From the experiments described no participation of retro-retinal structures during the photochemical cycle can be concluded but stereospecific interaction of the retinal moiety with the protein resulting in a specific retinal conformation os omdocated by the spectral changes observed.  相似文献   

12.
A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target. In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer. The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal. The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution. Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole. We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.  相似文献   

13.
We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.  相似文献   

14.
15.
The use of phenylethynylpyrene excimer forming pair in the design of specific fluorescent probes for determination of A2144G (A2143G and/or A2143C) mutations in 23S rRNA gene of Helicobacter pylori is described. Analysis of fluorescence spectra of model duplexes revealed optimal positions of fluorophore residues in the probe sequences for maximum efficiency of SNP detection. Application of excimer forming probes for analysis of DNA samples isolated from natural bacterial strains of H. pylori was demonstrated.  相似文献   

16.
A Reimann  B Kadenbach 《FEBS letters》1992,307(3):294-296
The binding of 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) to isolated bovine heart cytochrome c oxidase (COX) was studied by following its specific spectral change at 510 nm. The quantitative titration revealed two binding sites for TNP-ATP per monomer COX with a Kd of 1.6 microM.  相似文献   

17.
S Grzesiek  H Otto    N A Dencher 《Biophysical journal》1989,55(6):1101-1109
The fluorescence of 9-aminoacridine (9-AA) is quenched in vesicular suspensions containing negatively charged lipid headgroups (e.g., phosphatidylserine) upon imposition of a transmembrane (inside acidic) pH-gradient. It is shown that this fluorescence loss is accompanied by the formation of 9-AA dimers that undergo a transition in the dimer excited state to a dimer-excimer state. This result has been obtained on the basis of the specific dimer fluorescence excitation and hypochromic absorbance spectra that are redshifted by maximally 275 cm-1 (4.4 nm) with respect to the corresponding monomer spectra, as well as by the detection of the characteristic broad excimer emission band, centered at 560 nm. The existence of the spectrally distinct dimer-excimer is further corroborated by fluorescence life-time measurements that indicate an increased lifetime of up to 24 ns for this complex as compared with the normal monomer fluorescence lifetime of 16 ns. The formation of this dimer-excimer complex from the monomers can be reversed completely and the original monomeric spectral properties restored after the abolishment of the electrochemical proton gradient. In addition to the delta pH-induced dimer redshift in absorbance and fluorescence excitation, a further small redshift in monomer absorbance, fluorescence excitation, and emission spectra is observed due solely to the presence of the negatively charged phospholipid headgroups.  相似文献   

18.
Preliminary evidence suggested that phosphate or borote destabilize iron-ovotransferrin-nitrilotriacetate complexes in the absence of added bicarbonate. The iron-ovotransferrin-EDTA complex was prepared in the absence of bicarbonate, and a number of anions, including phosphate, sulfate, and citrate, were found to perturb the visible absorbance (lambdamax = 490 nm) of this complex. Other anions, such as chloride, nitrate, and perchlorate, had little or no effect on the spectrum. Also, when bicarbonate was added to a solution of the iron-transferrin-EDTA complex (A515 = 0.45), within 2 min, the visible absorbance had decreased to A515 = 0.13. Slowly a new peak appeared (lambdamax = 470 nm), evidently the iron-transferrin-CO3 complex. When these spectral changes were monitored in detail, the lack of an isosbestic point indicated the existence of one or more intermediates in the conversion of iron-transferrin-EDTA complex to the iron-transferrin-CO3 complex. Experiments using ternary complexes containing either 59Fe or [14C]EDTA show that both iron and EDTA nearly completely dissociate from the protein (most likely concomitantly within 2 min after bicarbonate is added. These observations are best explained by a paradigm which includes anion binding to the apoprotein. It is clear that there is an intimate relationship between anions and the binding of iron chelates by transferrin.  相似文献   

19.
Oligonucleotide derivatives with a fluorescent dye were designed for exhibiting a measurable signal only when they bind to complementary DNA in aqueous solution. The oligonucleotide with a dansyl group at the specific 2'-sugar residue was synthesized by using the protected 2'-dansylaminouridine phosphorobisamidite. The dansyl-oligonucleotide conjugate binds to its complementary DNA to form duplex with a normal stability and exhibits enhanced fluorescence together with a blue-shift in emission maxima after the hybridization. Another possible candidate involved the use of pyrene-excimer emission upon forming ternary complex between two pyrene-labeled oligonucleotide probes with target DNA. A new and general method for introduction of a pyrene fluorophore into the 3'- or 5'-terminal hydroxyl group of oligonucleotides via different linkers was developed.  相似文献   

20.
In this report, we demonstrate a label-free genosensor based on DNA hairpins coupled to gold coated sensor surfaces. The hairpin probes were labeled with a thiolated moiety for immobilization at the 5' end and with a fluorophore for signal transduction at the 3' end. In the absence of the complement, the fluorophore is quenched by energy transfer to the gold surface. Addition of the target sequence leads to the hairpin unfolding, and releases the fluorescent signal. This built-in property, using a gold film as both the immobilizing substrate and quenching agent, has the advantage of simplicity in design and ease of further integration. Our results showed that lengths of both the stem and the loop structures have significant effects on the sensor performance. Hybridization kinetics was investigated for various probe/target lengths and concentrations. An optimized hairpin probe gave a fluorescent signal increase of 39 folds after hybridization, which is much higher than the earlier reported results. A limit of detection (LOD) down to 0.3 nM for the complementary target DNA detection has been achieved. The developed sensor was further successfully applied for the detection of single-base mismatch targets, as well as for the direct detection of PCR products.  相似文献   

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