Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.     

Comparison of two combinations of cyanine dyes for prelabelling and gel electrophoresis

We report the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 N‐hydroxysuccinimide ester (NHS) cyanine dyes, which have similar chemical properties as the CyDye? DIGE fluor minimal dyes for pre‐electrophoresis labelling. Multiple sample analyses in different laboratories indicate that the use of IC‐OSu ethyl‐Cy3 and ethyl‐Cy5 NHS ester cyanine dyes produces equivalent results to those obtained with DIGE CyDyes, and allows sample multiplexing and accurate quantitation for differential proteome analysis.     

Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.     

Anomalous fluorescence enhancement of Cy3 and cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin

This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 相似文献   

Sequence-dependent fluorescence of cyanine dyes on microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.     

Comparison of the Sequence-Dependent Fluorescence of the Cyanine Dyes Cy3, Cy5, DyLight DY547 and DyLight DY647 on Single-Stranded DNA

Cyanine dyes are commonly used for fluorescent labeling of DNA and RNA oligonucleotides in applications including qPCR, sequencing, fluorescence in situ hybridization, Förster resonance energy transfer, and labeling for microarray hybridization. Previous research has shown that the fluorescence efficiency of Cy3 and Cy5, covalently attached to the 5′ end of single-stranded DNA, is strongly sequence dependent. Here, we show that DY547 and DY647, two alternative cyanine dyes that are becoming widely used for nucleic acid labeling, have a similar pattern of sequence-dependence, with adjacent purines resulting in higher intensity and adjacent cytosines resulting in lower intensity. Investigated over the range of all 1024 possible DNA 5mers, the intensities of Cy3 and Cy5 drop by ∼50% and ∼65% with respect to their maxima, respectively, whereas the intensities of DY547 and DY647 fall by ∼45% and ∼40%, respectively. The reduced magnitude of change of the fluorescence intensity of the DyLight dyes, particularly of DY647 in comparison with Cy5, suggests that these dyes are less likely to introduce sequence-dependent bias into experiments based on fluorescent labeling of nucleic acids.     

Facile synthesis of thiol-reactive Cy3 and Cy5 derivatives with enhanced water solubility

The cyanine dyes Cy3 and Cy5 have proven valuable in numerous applications involving conjugation with proteins. Practical syntheses of lysine-selective, succinimidyl ester derivatives of these dyes have been published, and succinimidyl esters are commercially available. However, the published syntheses of cysteine-selective derivatives produce relatively low yields from expensive starting materials, or produce molecules with marginal water solubility for protein labeling. We report here facile syntheses (four steps, >50% overall yield) of iodoacetamide, sulfhydryl-reactive derivatives of the Cy3 and Cy5 fluorophores. These novel derivatives have good water solubility (>2.5 mM) and bear only one reactive side chain, reducing possible protein cross-linking encountered with previous derivatives.     

Method for enhancing cell penetration of Gd3+-based MRI contrast agents by conjugation with hydrophobic fluorescent dyes

Gadolinium ion (Gd(3+)) complexes are commonly used as magnetic resonance imaging (MRI) contrast agents to enhance signals in T(1)-weighted MR images. Recently, several methods to achieve cell-permeation of Gd(3+) complexes have been reported, but more general and efficient methodology is needed. In this report, we describe a novel method to achieve cell permeation of Gd(3+) complexes by using hydrophobic fluorescent dyes as a cell-permeability-enhancing unit. We synthesized Gd(3+) complexes conjugated with boron dipyrromethene (BDP-Gd) and Cy7 dye (Cy7-Gd), and showed that these conjugates can be introduced efficiently into cells. To examine the relationship between cell permeability and dye structure, we further synthesized a series of Cy7-Gd derivatives. On the basis of MR imaging, flow cytometry, and ICP-MS analysis of cells loaded with Cy7-Gd derivatives, highly hydrophobic and nonanionic dyes were effective for enhancing cell permeation of Gd(3+) complexes. Furthermore, the behavior of these Cy7-Gd derivatives was examined in mice. Thus, conjugation of hydrophobic fluorescent dyes appears to be an effective approach to improve the cell permeability of Gd(3+) complexes, and should be applicable for further development of Gd(3+)-based MRI contrast agents.     

Spectroscopic study and evaluation of red-absorbing fluorescent dyes

The spectroscopic characteristics (absorption, emission, and fluorescence lifetime) of 13 commercially available red-absorbing fluorescent dyes were studied under a variety of conditions. The dyes included in this study are Alexa647, ATTO655, ATTO680, Bodipy630/650, Cy5, Cy5.5, DiD, DY-630, DY-635, DY-640, DY-650, DY-655, and EVOblue30. The thorough characterization of this class of dyes will facilitate selection of the appropriate red-absorbing fluorescent labels for applications in fluorescence assays. The influences of polarity, viscosity, and the addition of detergent (Tween20) on the spectroscopic properties were investigated, and fluorescence correlation spectroscopy (FCS) was utilized to assess the photophysical properties of the dyes under high excitation conditions. The dyes can be classified into groups based on the results presented. For example, while the fluorescence quantum yield of ATTO655, ATTO680, and EVOblue30 is primarily controlled by the polarity of the surrounding medium, more hydrophobic and structurally flexible dyes of the DY-family are strongly influenced by the viscosity of the medium and the addition of detergents. Covalent binding of the dyes to biotin and subsequent addition of streptavidin results in reversible fluorescence quenching or changes in the relaxation time of other photophysical processes of some dyes, most likely due to interactions with tryptophan residues in the streptavin binding site.     

A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm.

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.     

New donor-acceptor pair for fluorescent immunoassays by energy transfer

A novel F?rster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R(0) value of 68.7 A is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R(0) value of 85.2 A. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The F?rster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 A for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in F?rster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.     

Signal stability of Cy3 and Cy5 on antibody microarrays

Background  

The antibody microarray technique is a newly emerging proteomics tool for differential protein expression analyses that uses fluorescent dyes Cy 3 and Cy 5. Environmental factors, such as light exposure, can affect the signal intensity of fluorescent dyes on microarray slides thus, it is logical to scan microarray slides immediately after the final wash and drying processes. However, no research data are available concerning time-dependent changes of fluorescent signals on antibody microarray slides to this date. In the present study, microarray slides were preserved at -20°C after regular microarray experiments and were rescanned at day 10, 20 and 30 to evaluate change in signal intensity.     

Practical syntheses of dyes for difference gel electrophoresis

The three dyes (two indocyanine and one benzoxazolium) useful in difference gel electrophoresis-methyl Cy5 1, propyl Cy3 2, and the benzoxazolium dye Cy2 3--and their NHS esters have been prepared by efficient routes in good overall yield from inexpensive precursors.     

Phycoerythrin-allophycocyanin: a resonance energy transfer fluorochrome for immunofluorescence

BACKGROUND: As immunofluorescence experiments become more complex, the demand for new dyes with different properties increases. Fluorescent dyes with large Stoke's shifts that are very bright and have low background binding to cells are especially desirable. We report on the properties of the resonance energy tandems of phycoerythrin and allophycocyanin (PE-APC). PE-APC is the original fluorescence resonance energy tandem dye described in the literature, but it has not been utilized because of the difficulty of synthesizing and preparing a consistent product. METHODS: PE-APC complexes comprising different ratios of the two phycobiliproteins conjugated to streptavidin were synthesized using standard protein-protein conjugation chemistry. The PE-APC streptavidins were evaluated for flow cytometric analysis. They were compared directly to Cy5PE conjugates because Cy5PE is the fluorophore that is spectrally most like the PE-APC. RESULTS: PE-APC complexes showed the expected fluorescence spectral properties of a tandem: excitation was excellent at 488 nm (and best at the PE excitation maximum) and emission was greatest at the APC emission maximum at about 660 nm. The efficiency of transfer of energy from PE to APC was about 90%. CONCLUSION: PE-APC can be considered an excellent substitute for Cy5PE. Compared with Cy5PE, PE-APC has similar brightness (in staining experiments), slightly greater compensation requirements with PE but much lower compensation with Cy5.5PE or Cy5.5PerCP, and lower nonspecific background binding. PE-APC is a useful alternative to Cy5PE, especially in applications in which the use of Cy5 is impractical. Cytometry 44:24-29, 2001. Published 2001 Wiley-Liss, Inc.     

Engineered T4 viral nanoparticles for cellular imaging and flow cytometry

Viruses are of particular interest as scaffolds for biotechnology applications given their wide range of shapes and sizes and the possibility to modify them with a variety of functional moieties to produce useful virus-based nanoparticles (VNPs). In order to develop functional VNPs for cell imaging and flow cytometry applications, we used the head of the T4 bacteriophage as a scaffold for bioconjugation of fluorescent dyes. Bacteriophage T4 is a double-stranded DNA virus with an elongated icosahedron head and a contractile tail. The head is ～100 nm in length and ～90 nm in width. The large surface area of the T4 head is an important advantage for the development of functional materials since it can accommodate significantly larger numbers of functional groups, such as fluorescent dyes, in comparison with other VNPs. In this study, Cy3 and Alexa Fluor 546 were chemically incorporated into tail-less T4 heads (T4 nanoparticles) for the first time, and the fluorescent properties of the dye-conjugated nanoparticles were characterized. The T4 nanoparticles were labeled with up to 19?000 dyes, and in particular, the use of Cy3 led to fluorescent enhancements of up to 90% compared to free Cy3. We also demonstrate that the dye-conjugated T4 nanoparticles are structurally stable and that they can be used as molecular probes for cell imaging and flow cytometry applications.     

Detection and identification of bacterial cell surface proteins by fluorescent labeling

A rapid method of detection and identification of bacterial cell surface proteins is needed to better understand the interaction of bacteria with host components. To detect cell surface proteins, we have labeled cells of the Gram-negative anaerobic bacterium, Porphyromonas gingivalis, with fluorescent cyanine dyes, Cy3 and Cy5. We demonstrate that only cell surface proteins were labeled, indicating the method applied in our study is suitable for detection and identification of cell surface proteins in Gram-negative bacteria and possibly other organisms.     

Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes

Two-dimensional difference gel electrophoresis (2-D DIGE) enables an increased confidence in detection of protein differences. However, due to the nature of the minimal labelling where only approximately 5% of a given protein is labelled, spots cannot be directly excised for mass spectrometry (MS) analysis and detection sensitivity could be further enhanced. Amersham Biosciences have developed a second set of CyDye DIGE Cy 3 and Cy5 dyes, which aim to overcome these limitations through saturation-labelling of cysteine residues. The dyes were evaluated in relation to their sensitivity and dynamic range, their useability as multiplexing reagents and the possibility of direct spot picking from saturation-labelled gels for MS analysis. The saturation-labelling dyes were superior in sensitivity to their minimal-labelling counterparts, silver stain and Sypro Ruby, however, the resulting 2-D spot pattern was significantly altered from that of unlabelled or minimal-labelled protein. The dyes were found to be useful as multiplexing reagents although preferential labelling of proteins with one dye over another was observed but was controlled for through experimental design. Protein identities were successfully obtained from material directly excised from saturation-labelled gels eliminating the need for post-stained preparative gels.