One-pot fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide for imaging polysaccharides transported in living cells

A simple and efficient procedure for the fluorescent labeling of saccharides is a prerequisite step for imaging the transport of polysaccharides in living cells. We report a one-pot strategy for the fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide (FTSC), which introduces the thiosemicarbazide group of FTSC to the aldehyde group at the reducing end of saccharides to form stable amino derivatives via reductive amination. The Glc-FTSC derivative was characterized by HPLC–MS, HRESIMS and NMR spectroscopy. Saccharides were quantitatively labeled with FTSC at 75 °C for 1 h under optimum reaction conditions. Fluorescence studies illustrated that the conjugation of FTSC to saccharides did not change its florescence properties (λ_ex = 495 nm, λ_em = 517 nm), presenting desirable compatibility with commonly used fluorescence equipment. Polysaccharide AAG-FTSC derivatives exhibited rather low levels of cytotoxicity against rat thymus cells, and the fluorescent labeling procedure had slight impact on their anti-tumor activity. Results indicate that the assay neither introduces discernible cytotoxicity against living cells nor obviously alters the functional activities of polysaccharides, and provides a convenient, highly efficient fluorescent labeling approach for imaging the transport of polysaccharides in living cells.     

Preparation and properties of fluorescent polysaccharides

A new method for preparing fluorescein derivatives of polysaccharides is described. These derivatives are prepared by activation of the polysaccharide with cyanogen bromide and subsequent reaction with fluoresceinamine. The optimum conditions for coupling have been established in this report. Using this procedure, we have prepared fluorescein derivatives of a wide variety of polysaccharides. Degrees of substitution in the range of 3.0 X 10(-3) to 2.4 X 10(-2) mol of fluorescein per mole of monosaccharide equivalent were obtained. The fluorescent derivatives are stable: no free fluorescein was detected after incubation at 22 degrees C for 48 h or at -10 degrees C for 4 months. The fluorescein-derivatized polysaccharides were found to have the same potency in inhibiting lectin-mediated hemagglutination as the underivatized polysaccharide. In addition, these fluorescent polysaccharides can be radioiodinated to specific activities exceeding 10(6) dpm/micrograms due to incorporation of 125I into fluorescein. The cell binding properties of 125I-fucoidin and 125I-heparin are indistinguishable from the corresponding underivatized polysaccharides. This general approach for preparing fluorescent polysaccharides should produce useful reagents for localizing and quantifying cell surface carbohydrate-binding proteins (lectins).     

Synthesis and fluorescent labeling of beta-amyloid peptides.

Fluorescent cell analytical techniques require the incorporation of a fluorophore into the target molecule without causing a significant change in the native conformation. Many short peptides have a limited number of reactive groups that can be labeled without affecting the biological activity. In this work we present several methods for labeling beta-amyloid peptides (betaA[25-35], betaA[1-40]) and their derivatives (LPFFD, RIIGL and RVVIA) with different chromophores exclusively at the N-terminus. In the case of liquid-phase labeling, fluorescein isothiocyanate was used. The side-chain amino function of Lys, if present in the sequence, was protected with an Fmoc group, whereby the hydrophobic character of the peptide was further increased. The labeling reaction was carried out in an appropriate deaggregating solvent, DMSO. For solid-phase labeling, 5(6)-carboxyfluorescein and 7-amino-4-methyl-3-coumarinylacetic acid were applied. Several cleavage cocktails were tested for removal of the labeled amyloid peptides from the resin in order to completely suppress the oxidation of Met.     

One-pot fluorescent labeling of xyloglucan oligosaccharides with sulforhodamine

Xyloglucan oligosaccharides fluorescently labeled with sulforhodamine have proved to be a valuable tool in the assessment of transglycosylating activity of plant xyloglucan endotransglucosylase/hydrolase (XTH; EC 2.4.1.207). Here we describe a simple and fast procedure for their preparation. Accordingly, the starting xyloglucan-derived oligosaccharides are in the first step converted to their corresponding 1-amino-1-deoxyalditols (glycamines) by incubation with ammonium acetate and NaCNBH(3) at 80 degrees C for 2-4 h, and in the second step, the glycamines are reacted with Lissamine rhodamine B sulfonyl chloride to obtain fluorescently labeled derivatives of the oligosaccharide glycamines. All operations are carried out in a single centrifuge tube and the products from the individual reaction steps are isolated on the basis of their differential solubility in organic solvents. Using the described protocol, the whole procedure can be accomplished in less than 24 h. The sulforhodamine-labeled xyloglucan oligosaccharides thus obtained proved suitable as substrates for a sensitive fluorescence assay of the transglycosylating activity of XTH.     

Site-specific fluorescent labeling of DNA using Staudinger ligation

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.     

Optimized synthesis of functionalized fluorescent oligodeoxynucleotides for protein labeling

Phthalimido-alkanol solid supports were rapidly prepared from solid supported phthalic anhydride and amino alcohol condensation induced by microwaves. These supports were used to synthesize 5'-fluorescent 3'-aminoalkyl oligodeoxynucleotides allowing a two-step deprotection necessary to avoid aminolink alkylation. After conversion into an NHS derivative using dissuccinimidyl suberate and an optimized isolation, they were conjugated with a protein.     

Ligation mediated fluorescent labeling of DNA sequencing primers.

Automated DNA sequencing utilizing fluorescently labeled primers is a proven methodology for generating quality sequence data. However, for directed primer walking strategies this necessitates synthesis and labeling of a unique primer for each sequencing reaction. Here, we describe a rapid ligation-based method of generating labeled sequencing primers. An unlabeled 5'-phosphorylated sequencing primer is ligated to a fluorescent oligonucleotide by use of a bridge primer which is complementary to portions of the previous two oligonucleotides, thus aligning them properly for ligation. The resulting fluorescent hybrid primer can be utilized directly in cycle sequencing reactions without any prior purification.     

Cleavage of pig embryos after labeling with fluorescent dyes

In studies on embryonic development, treated and control ova could be co-mixed before transfer to recipients if nontoxic labels for ova were available. These experiments were conducted to determine whether pig ova would continue to cleave after being stained with the fluorochromes tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC). In the first experiment, pig ova stained with TRITC and unstained control ova were transferred into opposite oviducts of recipient gilts. In the second experiment, ova stained with TRITC and ova stained with FITC were transferred into opposite oviducts of recipient gilts. Embryos were recovered 96 h after transfer (Day 6; Day 0 = onset of estrus), the presence of fluorescence was determined, and the number of nuclei per embryo was assessed. Stained ova retained sufficient fluorochrome to permit detection until the zonae pellucidae were shed. Development of embryos stained with TRITC was equal to that of unstained control embryos. However, development of embryos stained with FITC appeared slightly retarded in comparison to that of TRITC-stained embryos. These findings demonstrate the efficacy of the fluorescent staining technique for pig ova during the first six days of pregnancy.     

Purification and fluorescent labeling of the human serotonin transporter

To establish a purification procedure for the human serotonin transporter (hSERT) we expressed in Sf9 insect cells an epitope-tagged version of the transporter containing a FLAG epitope at the N-terminus and a polyhistidine tail at the C-terminus (FLAG-hSERT-12H). For purification, the transporter was solubilized in digitonin followed by nickel affinity and subsequent concanavalin A chromatography. Using this procedure we were able to obtain an overall purification of 700-fold and a yield of approximately 0.1 mg/L of cell culture. The purified transporter displayed pharmacological properties similar to those of hSERT expressed in native tissues and in transfected cell lines. Fluorescent labeling of the purified transporter with the thiol-reactive fluorophore nitrobenxoxadiazol-iodoacetamide (IANBD) and Texas Red bromoacetamide preserved the pharmacological profile of FLAG-hSERT-12H. Collisional quenching experiments revealed that the aqueous quencher iodide was able to cause marked quenching of the fluorescence of the IANBD labeled transporter with a K(SV) of 3.4 +/- 0.10 M(-)(1). In a mutant transporter with five cysteines mutated (5CysKO) we observed a significant reduction in this quenching (K(SV) = 2.1 +/- 0.16 M(-)(1), p < 0.01). This reduction was most likely due to labeling of (109)Cys since mutation of this cysteine alone resulted in a reduction in collisional quenching that was similar to that observed with 5CysKO (K(SV) = 2.2 +/- 0.15 M(-)(1)). These data suggest that labeling of (109)Cys contributes substantially to the overall fluorescence of IANBD labeled FLAG-hSERT-12H. On the basis of these data we infer that (109)Cys is embedded in a mixed hydrophobic/hydrophilic environment at the external ends of transmembrane segments 1 and 2. Further use of fluorescent techniques on purified hSERT should prove useful in future studies aimed at understanding the molecular structure and function of Na(+)/Cl(-)-dependent neurotransmitter transporters.     

Horseradish peroxidase-driven fluorescent labeling of nanotubes with quantum dots

We describe the first enzyme-driven technique for fluorescent labeling of single-walled carbon nanotubes (SWNTs). The labeling was performed via enzymatic biotinylation of nanotubes in the tyramide-horseradish peroxidase (HRP) reaction. Both direct and indirect fuorescent labeling of SWNTs was achieved using either biotinyl tyramide or fluorescently tagged tyramides. Biotinylated SWNTs later reacted with streptavidin-conjugated fluorophores. Linking semiconductor nanocrystals, quantum dots (Q-dots), to the surface of nanotubes resulted in their fluorescent visualization, whereas conventional fluorophores bound to SWNTs directly or through biotin-streptavidin linkage, were completely quenched. Enzymatic biotinylation permits fluorescent visualization of carbon nanotubes, which could be useful for a number of biomedical applications. In addition, other organic molecules such as proteins, antibodies, or DNA can be conjugated to biotinylated SWNTs using this approach.     

Chemical methods of DNA and RNA fluorescent labeling.

Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3'-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde groups and the hydrazone bonds are stabilized by reduction with sodium cyanoborohydride. Alternatively, DNA can be quantitatively split at the depurinated sites with ethylenediamine. The aldimine bond between the aldehyde group in depurinated DNA or oxidized RNA and ethylenediamine is stabilized by reduction with sodium cyanoborohydride and the primary amine group introduced at these sites is used for attachment of isothiocyanate or succinimide derivatives of fluorescent dyes. The fluorescent DNA labeling can be carried out either in solution or on a reverse phase column. These procedures provide simple, inexpensive methods of multiple DNA labeling and of introducing one fluorescent dye molecule per RNA, as well as quantitative DNA fragmentation and incorporation of one label per fragment. These methods of fluorophore attachment were shown to be efficient for use in the hybridization of labeled RNA, DNA and DNA fragments with oligonucleotide microchips.     

Methodology of reversible protein labeling for ratiometric fluorescent measurement

The first fluorescent labeling technology, which can induce not only an increase in the fluorescence intensity but also a shift in the fluorescence spectrum, has been developed for "ratiometric" measurements for a protein by utilizing a newly designed "field-sensitive" fluorescent probe and its corresponding unique amino acid tag.     

Highly fluorescent protein labeling using dendritic peptide derivatives.

Non-specific fluorescent dyes and photosensitizers are routinely used in clinical practice for the photodetection and photoablation of superficial lesions. Future applications in photomedicine are likely to rely on the selective delivery of photoactive compounds to diseased areas, using specific targeting agents such as antibodies. This fact underlines the need for methods that allow the chemically defined conjugation of several photoactive molecules to a single protein 'vehicle', with full retention of binding affinity. Here, we present methods for the site-specific fluorescent labeling of proteins using dendritic peptides, which had been chemically modified with multiple molecules of fluorescein. Branched peptide derivatives can be stably conjugated to proteins either by reaction with suitable free reactive groups or by using the high-affinity non-covalent interaction between calmodulin and a specific binding peptide. Chemical modification of proteins with one, two or four molecules of fluorescein resulted in a proportional increase in protein fluorescence.     

Dual labeling with green fluorescent proteins for confocal microscopy

We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.     

Examination of the structure of amylopectin molecules by fluorescent labeling

Amylopectin molecules from rice, maize, sweet potato and potato were examined by fluorescent labeling followed by gel-permeation HPLC. The number-average degree of polymerization (dp(n)) was determined to be in range of 9600-15,900. The molar-based distribution revealed the presence of three molecular species, large (dp(n) 13,400-26,500), medium (4400-8400) and small (700-2100). Their molar proportions differed by plant origin. The large species was a major component (43-63% by mole). A relatively large amount of the medium (16-28% by mole) and small (19-38%) species was found although their weight proportion was small (8-15, 1-4%, respectively). The three species from waxy rice amylopectin had a similar chain-length distribution and also a similar size-distribution of C chains. These results suggested that the three species were basically similar in cluster structure but different in number of clusters per molecule.     

Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling

Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling.The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDASE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling.