Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.     

Increased immunofluorescence sensitivity using 532 nm laser excitation.

OBJECTIVE: We evaluated the use of a high power, diode pulsed solid-state laser emitting 532 nm light for immunofluorescence applications. We compared the sensitivity and utility of this laser with the standard 488 nm excitation. METHODS: A flow cytometer was equipped with both a 488 nm and a 532 nm laser; fluorescence emissions from each laser were collected using the same filters and the same detector system. Cells or compensation beads (e.g. latex beads coated with anti-kappa antibodies) were stained with monoclonal antibodies conjugated to phycoerythrin (PE) as well as the PE tandem dyes TRPE, Cy5PE, Cy5.5PE, and Cy7PE. The sensitivity of detection of these reagents as well as those in heavily compensated channels was quantified by measuring the spreading error for a primary detector into a secondary detector. RESULTS: Measurement of the fluorescence emission of PE and PE-tandem dyes was considerably more sensitive when using 532 nm excitation (150 mW) as compared with 488 nm excitation (20 mW). In addition, as the absolute number of photoelectrons collected was greater, there was less measurement-error-induced spread into the compensated channels. As an example, when comparing the spreading error of PE labeled cells into the TRPE detector, the green laser was found to be 15-fold more sensitive as compared with the blue laser. In addition, the blue laser produced more autofluoresent signal from cells as compared with the green laser. Together, these advantages of the 532 nm excitation line provides for a significantly improved detection of immunofluorescence staining.     

Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry

BACKGROUND: Phycobiliproteins play an important role in fluorescent labeling, particularly for flow cytometry. The spectral properties of R-phycoerythrin (R-PE) and allophycocyanin (APC) have made them the dominant reagents in this class of fluorochromes. In this study, we evaluate a lesser-known but potentially important series of low-molecular weight cryptomonad-derived phycobiliproteins (commercially termed the CryptoFluortrade mark dyes) for their applicability to flow cytometry, both in extracellular and intracellular labeling applications. METHODS: Several cell lines were labeled with biotin-conjugated antibodies against expressed extracellular surface proteins, followed by streptavidin conjugates of three cryptomonad phycobiliproteins (CryptoFluor-2, CryptoFluor-4, and CryptoFluor-5). Cells were then analyzed by flow cytometry using a variety of laser lines and emission filters to establish the optimal excitation/emission characteristics for each fluorochrome. Some cells were permeabilized and labeled for intracellular antigens, also using the cryptomonad fluorochromes. Where appropriate, parallel samples were labeled with other fluorochromes (including R-PE, APC, the cyanin dyes Cy3 and Cy5, and others) to gauge the performance of the cryptomonad fluorochromes against fluorescent labels previously evaluated for flow cytometry. RESULTS: CryptoFluor-2 possessed excitation/emission maxima similar to those of APC and Cy5, with good excitation in the red (HeNe laser 632 nm) and strong emission in the far red (660 nm). CryptoFluor-4 possessed excitation/emission maxima similar to those of Cy3, with optimal excitation in the green (Kr 530 nm) and strong emission in the yellow/orange (585 nm). CryptoFluor-5 possessed excitation/emission maxima similar to those of lissamine rhodamine, with optimal excitation in the yellow (Kr 568 nm) and emission in the orange (610 nm). All cryptomonad fluorochromes gave satisfactory results for both intracellular and extracellular labeling, with detection sensitivities that were comparable or better than traditional phycobiliproteins and low- molecular weight synthetic fluorochromes such as the cyanin dyes. CONCLUSIONS: The CryptoFluor fluorochromes were applicable to flow cytometric immunodetection, with excitation and emission conditions commonly found on multilaser instruments. Performance of several of these dyes was at least comparable to existing fluorescent labels. The low molecular weights (30-60 kd) of phycobiliproteins may make them particularly useful in intracellular antigen detection. Cytometry 44:16-23, 2001. Published 2001 Wiley-Liss, Inc.     

Nine color eleven parameter immunophenotyping using three laser flow cytometry.

BACKGROUND: This study describes a three laser flow cytometer, reagents, and software used to simultaneously evaluate nine distinct fluorescent parameters on one cell sample. We compare the quality of data obtained with (1) full software compensation and (2) the use of partial spectral compensation of selected pairs of parameters in analog hardware, in combination with final software compensation. An application characterizing low frequency murine B cell subpopulations is given. METHODS: The fluorochromes used are: fluorescein (FITC), phycoerythrin (PE), Cy5PE and Cy7PE, excited at 488 nm by an argon laser; Texas Red (TR), allophycocyanin (APC), and Cy7APC excited at 595 nm by a pumped dye laser; and cascade blue (CB) and cascade yellow (CY) excited at 407 nm by a violet-enhanced krypton laser. Custom additions to commercial electronics and an extended optical bench allow the measurement of these nine parameters plus forward and side scatter light signals. RESULTS: We find the use of partial analog compensation reduces the variation in the background staining levels introduced by the compensation process. Novel B cell populations with frequencies below 1% are characterized. CONCLUSIONS: Nine color flow cytometry is capable of providing measurements with high information content. The choice of reagent-dye combinations and the ability to compensate in multi-parameter measurement space are crucial to obtaining satisfactory results.     

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.     

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   

Heat stress induces an inhibition of excitation energy transfer from phycobilisomes to photosystem II but not to photosystem I in a cyanobacterium Spirulina platensis.

The effects of high temperature (30-52.5 degrees C) on excitation energy transfer from phycobilisomes (PBS) to photosystem I (PSI) and photosystem II (PSII) in a cyanobacterium Spirulina platensis grown at 30 degrees C were studied by measuring 77 K chlorophyll (Chl) fluorescence emission spectra. Heat stress had a significant effect on 77 K Chl fluorescence emission spectra excited either at 436 or 580 nm. In order to reveal what parts of the photosynthetic apparatus were responsible for the changes in the related Chl fluorescence emission peaks, we fitted the emission spectra by Gaussian components according to the assignments of emission bands to different components of the photosynthetic apparatus. The 643 and 664 nm emissions originate from C-phycocyanin (CPC) and allophycocyanin (APC), respectively. The 685 and 695 nm emissions originate mainly from the core antenna complexes of PSII, CP43 and CP47, respectively. The 725 and 751 nm band is most effectively produced by PSI. There was no significant change in F725 and F751 during heat stress, suggesting that heat stress had no effects on excitation energy transfer from PBS to PSI. On the other hand, heat stress induced an increase in the ratio of Chl fluorescence yield of PBS to PSII, indicating that heat stress inhibits excitation energy transfer from PBS to PSII. However, this inhibition was not associated with an inhibition of excitation energy transfer from CPC to APC since no significant changes in F643 occurred at high temperatures. A dramatic enhancement of F664 occurring at 52.5 degrees C indicates that excitation energy transfer from APC to the PSII core complexes is suppressed at this temperature, possibly due to the structural changes within the PBS core but not to a detachment of PBS from PSII, resulting in an inhibition of excitation energy transfer from APC to PSII core complexes (CP47 + CP43). A decrease in F685 and F695 in heat-stressed cells with excitation at 436 nm seems to suggest that heat stress did not inhibit excitation energy transfer from the Chl a binding proteins CP47 and CP43 to the PSII reaction center and the decreased Chl fluorescence yields from CP43 and CP47 could be explained by the inhibition of the energy transfer from APC to PSII core complexes (CP47 + CP43).     

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.     

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.     

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.     

Facile synthesis of symmetric, monofunctional cyanine dyes for imaging applications

Efficient syntheses of several members of a new class of symmetric, monocarboxylate-functionalized cyanine dyes have been developed. The synthesis is a simple two-step method, typically with greater than 60% yield and easy final product purification. The new monocarboxylate-functionalized cyanine dyes exhibit excellent water solubility and similar excitation and emission properties to those of Cy5 and Alexa Fluor 647. The application of the new dyes in cellular imaging has been demonstrated through direct conjugating of the dye with an antibody, then imaging of microtubules inside cells, visualized by near-infrared fluorescence microscopy.     

Concatenation of cyan and yellow fluorescent proteins for efficient resonance energy transfer

Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging.     

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.     

螺旋藻(Spirulina Platensis)藻胆体的光谱特性和能量传递的研究

研究了螺旋藻藻胆体的吸收光谱,室温和液氮温度荧光发射光谱和激发光谱.完整藻胆体的室温荧光峰位于678nm,不完整藻胆体位于672nm.在完整藻胆体的液氮温度荧光光谱中只有一个发射峰,不完整藻胆作有两个峰.研究结果表明C-藻蓝蛋白与别藻蓝蛋白之间的连接和别藻蓝蛋白与别藻蓝蛋白-B之间的连接具有不同的稳定性;前者稳定性较差,易解离.对藻胆体内藻胆蛋白之间的光能传递进行了讨论.     

Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties.

Heterodimeric dyes are described which bind tightly to double-stranded (dsDNA) with large fluorescence enhancements. These dyes are designed to exploit energy transfer between donor and acceptor chromophores to tune the separation between excitation and emission wavelengths. The dyes described here absorb strongly at the 488 nm argon ion line, but emit at different wavelengths, and can be applied to multiplex detection of various targets. The chromophores in these dyes, a thiazole orange-thiazole blue heterodimer (TOTAB), two different thiazole orange-ethidium heterodimers (TOED1 and TOED2), and a fluorescein-ethidium heterodimer (FED), are in each case linked through polymethyleneamine linkers. The emission maxima of the DNA-bound dyes lie at 662 (TOTAB), 614 (TOED 2), and 610 nm (FED). The dyes showed a > 100 fold enhancement of the acceptor chromophore fluorescence on binding to dsDNA and no sequence selectivity. In comparison with direct 488 nm excitation of the constituent monomeric dyes, in the heterodimers the fluorescence of the acceptor chromophores was greatly enhanced and the emission of the donor chromophores quenched by over 90%. The acceptor emission per DNA-bound dye molecule was constant from 100 DNA bp:dye to 20 bp:dye and decreased sharply at higher dye:DNA ratios.     

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.     

Four color compensation.

Four-color immunophenotyping can now be routinely performed using either a single laser or dual laser flow cytometer. When a single laser instrument is used, the fluorochromes evaluated are usually FITC, PE, PE-TR and PE-CY5 (or PerCP). For two-laser excitation APC is generally used in place of PE-TR. Since each tandem dye construct contains PE, three of the four detectors are affected and compensation can be problematic. In this report we show that each tandem conjugated antibody, whether different batches from the same supplier or conjugates from different suppliers all require unique compensation. This inconsistency results in erroneous data, negates the use of single labeled particles as a method for providing adequate compensation and requires dual and triple labeled cells of known pattern to verify compensation. It is also shown that improper compensation can reduce or eliminate completely the detection of fluorescence emission from PECY5 conjugated antibodies. These problems are caused by a variation in energy transfer between PE and either TR or CY5 because the chemistry involved in preparation and conjugation to antibodies is not sufficiently controlled to produce reagents with uniform compensation requirements. The variation in tandem dye compensation can be addressed by either using the same tandem conjugated antibody, by using the same second step tandem reagent to an appropriate first step antibody or by using software compensation. The latter provides an easy solution because a unique compensation matrix can be produced for each antibody tandem conjugate.     

A homogeneous DNA hybridization system by using a new luminescence terbium chelate

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb(3+) (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (lambda(ex) = 548 nm and lambda(em) = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb(3+) chelate at the 3'-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb(3+) chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb(3+) chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb(3+) chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.     

Synthesis and properties of fluorescent NF-kappa B-recognizing hairpin oligodeoxyribonucleotide decoys

Intramolecular fluorescence quenching of cyanine dyes was investigated using a model hairpin oligonucleotide decoy encoding a NF-kappaB p50 subunit binding site. Two types of hairpin oligonucleotides were synthesized: (1) 5'-(6-aminohexyl)- and 3'-(3-aminopropyl)-linked (I); (2) 5'-(6-aminohexyl)- and 3'-[3-(3-hydroxypropyldithio)propyl]-linked (II). Oligonucleotide I was covalently modified using monofunctional either Cy3- or Cy5.5-N-hydroxysuccinimide esters. Using reverse-phase HPLC, mono-and dicyanineamide derivatives of I were isolated. Mono-Cy3-modified derivatives of I, but not the mono-Cy5.5-modified derivatives, showed a 2-fold higher Cy3 fluorescence intensity compared to the free dye. There was no detectable difference in fluorescence between the di-Cy3 derivative of I and the free dye at the same concentration. However, there was a 4-fold quenching of fluorescence in the case of the di-Cy5.5 derivative of the same hairpin oligonucleotide. The quenching of Cy5.5 fluorescence could not be explained by the interaction of Cy5.5 with nucleotide bases as demonstrated by incubating free Cy5.5 dye with oligonuclotides. The quenching effect was further investigated using an oligonucleotide bearing a cleavable 3'-amino-terminated linker bearing an S-S bond (III). After modification of the 5'- and 3'-end of oligonucleotide III with a Cy5.5 monofunctional hydroxysuccinimide ester, a 70-75% quenching of fluorescence was observed. Fluorescence was 100% dequenched after the reduction of S-S bond. The obtained result unequivocally demonstrates that the formation of intramolecular Cy5.5 dimers is the dominant mechanism of fluorescence quenching in symmetric dye-dye hairpin decoy beacons.     

Fluorescence resonance energy transfer (FRET) and competing processes in donor-acceptor substituted DNA strands: a comparative study of ensemble and single-molecule data

We studied the fluorescence resonance energy transfer (FRET) efficiency of different donor-acceptor labeled model DNA systems in aqueous solution from ensemble measurements and at the single molecule level. The donor dyes: tetramethylrhodamine (TMR); rhodamine 6G (R6G); and a carbocyanine dye (Cy3) were covalently attached to the 5'-end of a 40-mer model oligonucleotide. The acceptor dyes, a carbocyanine dye (Cy5), and a rhodamine derivative (JA133) were attached at modified thymidine bases in the complementary DNA strand with donor-acceptor distances of 5, 15, 25 and 35 DNA-bases, respectively. Anisotropy measurements demonstrate that none of the dyes can be observed as a free rotor; especially in the 5-bp constructs the dyes exhibit relatively high anisotropy values. Nevertheless, the dyes change their conformation with respect to the oligonucleotide on a slower time scale in the millisecond range. This results in a dynamic inhomogeneous distribution of donor/acceptor (D/A) distances and orientations. FRET efficiencies have been calculated from donor and acceptor fluorescence intensity as well as from time-resolved fluorescence measurements of the donor fluorescence decay. Dependent on the D/A pair and distance, additional strong fluorescence quenching of the donor is observed, which simulates lower FRET efficiencies at short distances and higher efficiencies at longer distances. On the other hand, spFRET measurements revealed subpopulations that exhibit the expected FRET efficiency, even at short D/A distances. In addition, the measured acceptor fluorescence intensities and lifetimes also partly show fluorescence quenching effects independent of the excitation wavelength, i.e. either directly excited or via FRET. These effects strongly depend on the D/A distance and the dyes used, respectively. The obtained data demonstrate that besides dimerization at short D/A distances, an electron transfer process between the acceptor Cy5 and rhodamine donors has to be taken into account. To explain deviations from FRET theory even at larger D/A distances, we suggest that the pi-stack of the DNA double helix mediates electron transfer from the donor to the acceptor, even over distances as long as 35 base pairs. Our data show that FRET experiments at the single molecule level are rather suited to resolve fluorescent subpopulations in heterogeneous mixture, information about strongly quenched subpopulations gets lost.