6-carboxytetramethylrhodamine and 6-carboxyfluorescein

Hepcidin is a peptide hormone that regulates homeostasis in iron metabolism. It binds to the sole known cellular iron exporter ferroportin (Fpn), triggers its internalization, and thereby modulates the efflux of iron from cells. This functional property has been adopted in this study to assess the bioactivity and potency of a range of novel fluorescent hepcidin analogues. Hepcidin was selectively labeled with 6-carboxyfluorescein (CF) and 6-carboxytetramethylrhodamine (TMR) using Fmoc solid phase peptide chemistry. Internalization of Fpn by hepcidin was assessed by high-content microscopic analysis. Both K18- and M21K-labeled hepcidin with TMR and CF exhibited measurable potency when tested in cultured MDCK and T47D cells expressing human ferroportin. The bioactivity of the labeled hepcidin varies with the type of fluorophore and site of attachment of the fluorophores on the hepcidin molecule.

Topics: Animals; Cation Transport Proteins; Cell Line; Dogs; Fluoresceins; Fluorescent Dyes; Hepcidins; Humans; Models, Molecular; Protein Binding; Protein Folding; Rhodamines

Consumption of Salmonella-contaminated foods, such as poultry and fresh eggs, is known to be one of the main causes of salmonellosis. Conventional PCR methods, including real-time PCR for rapid detection of Salmonella, in general require skilled technicians and costly instruments. A recently developed novel convective PCR, insulated isothermal PCR (iiPCR), is carried out in polycarbonate capillary tubes. In this study, we designed TaqMan probes and PCR primers based on the yrfH gene encoding a heat shock protein for the iiPCR detection of Salmonella in chicken meat samples. The TaqMan probe was labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine at the 5' and 3' ends, respectively. The PCR amplicon was 133 bp. A typical run of this iiPCR assay was completed within 1 h. Specific PCR products were obtained for 148 strains representing 49 serotypes of Salmonella tested. Under the same conditions, false-positive results were not obtained for 98 non-Salmonella strains tested, including strains of Enterobacteriaceae closely related to Salmonella. For chicken meat samples, with a 5-h enrichment step Salmonella at as low as 10⁰ CFU/g of poultry meat could be detected. Because the amplification signals from the probes are detectable at 520 nm, identification of the PCR products by gel electrophoresis is not required. Compared with conventional PCR, the iiPCR system requires less expertise and provides an economical, reliable, and rapid tool for result interpretation. Detection results can be obtained within 8 h, including the enrichment and DNA extraction steps.

Topics: Animals; Chickens; DNA Primers; DNA, Bacterial; Fluoresceins; Food Contamination; Meat; Polymerase Chain Reaction; Reproducibility of Results; Rhodamines; Salmonella; Salmonella Food Poisoning; Sensitivity and Specificity; Time Factors

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

Topics: DNA Probes; Fluoresceins; Fluorescent Dyes; Freeze Drying; Humans; Indicators and Reagents; Orthopoxvirus; Polymerase Chain Reaction; Rhodamines; Sensitivity and Specificity; Variola virus

Oligonucleotide derivatives, F-TBA-T and F-7TBA-T carrying thrombin-binding aptamer (TBA) sequence and FAM and TAMRA dyes at their each end were designed to achieve the fluorometric monitoring of potassium ion (K+) concentrations in a living organisms. These oligonucleotides could form a tetraplex structure upon addition of K+. Spectrophotometric experiments revealed that the fluorescence enhancement on the fluorescence resonance energy transfer (FRET) was larger in the case of F-7TBA-T having 7-nucleotides at 5'-end of TBA sequence than in the case of F-TBA-T due to the elimination of the quenching by dye-dye interaction.

Topics: Aptamers, Nucleotide; Cations; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Nucleic Acid Conformation; Potassium; Rhodamines

Reverse transcription of HIV-1 genomic RNA to double-stranded DNA by reverse transcriptase (RT) is a critical step in HIV-1 replication. This process relies on two viral proteins, the RT enzyme and nucleocapsid protein NCp7 that has well documented nucleic acid chaperone properties. At the beginning of the linear DNA synthesis, the newly made minus-strand strong-stop DNA ((-)ssDNA) is transferred to the 3'end of the genomic RNA by means of an hybridization reaction between transactivation response element (TAR) RNA and cTAR DNA sequences. Since both TAR sequences exhibit stable hairpin structures, NCp7 needs to destabilize the TAR structures in order to chaperone their hybridization. To further characterize the relationships between TAR stability and NC-mediated destabilization, the role of the A(49) and G(52) bulged residues in cTAR DNA stability was investigated. The stability of cTAR and mutants where one or the two terminal bulges were replaced by base-pairs as well as the NCp7-mediated destabilization of these cTAR sequences were examined. Thermodynamic data indicate that the two bulges cooperatively destabilize cTAR by reducing the stacking interactions between the bases. This causes a free energy change of about 6.4 kcal/mol and seems to be critical for NC activity. Time-resolved fluorescence data of doubly labelled cTAR derivatives suggest that NC-mediated melting of cTAR ends propagates up to the 10C.A(44) mismatch or T(40) bulge. Fluorescence correlation spectroscopy using two-photon excitation was also used to monitor cTAR ends fraying by NC. Results show that NC causes a very significant increase of cTAR ends fraying, probably limited to the terminal base-pair in the case of cTAR mutants. Since the TAR RNA and cTAR DNA bulges or mismatches appear well conserved among all HIV-1 strains, the present data support the notion of a co-evolutionary relationship between TAR and NC activity.

Topics: Amino Acid Sequence; Base Pairing; Base Sequence; Capsid; Capsid Proteins; DNA, Viral; Fluoresceins; Fluorescent Dyes; gag Gene Products, Human Immunodeficiency Virus; Gene Products, gag; HIV-1; Kinetics; Molecular Chaperones; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Peptide Fragments; Response Elements; Rhodamines; Spectrometry, Fluorescence; Thermodynamics; Transcriptional Activation; Viral Proteins; Virus Integration

A novel potassium sensing oligonucleotide (PSO) was constructed by attaching fluorophores 6-FAM and 6-TAMRA to the 5'- and 3'-termini of d(GGG TTA GGG TTA GGG TTA GGG), respectively. The affinity of PSO for K+ was 43 000 times greater than that for Na+, high enough selectivity enabling quantitation of K+ specifically in the presence of excess Na+. Fluorescence resonance energy transfer (FRET) to 6-TAMRA from 6-FAM of PSO was observed only in the presence of K+. This phenomenon is based on the approximation of the two fluorophores upon formation of a guanine quartet mediated by K+. Furthermore, the fluorescent color of PSO changes from yellow to red upon formation of the complex, thereby enabling visualization of K+ in aqueous media.

Topics: Binding Sites; Energy Transfer; Fluoresceins; Fluorescent Dyes; Guanine; Humans; Kinetics; Oligonucleotides; Potassium; Rhodamines; Spectrometry, Fluorescence; Telomere; Water

The RNase H activity of HIV-1 reverse transcriptase was examined using chemically synthesized deoxyribo.ribo-oligonucleotide hybrid duplex labeled with the fluorescence donor at the 5'-end and with the fluorescence acceptor at the 3'-end of DNA strand as a substrate. Fluorescence resonance energy transfer (FRET) between these fluorescent dyes was used to analyze the rate of the enzymatic reaction. Under excitation of the donor dye, that is 6-carboxyfluorescein (6-FAM), at 490 nm, the increase of the fluorescence resulting from the acceptor dye, that is 6-carboxytetramethylrhodamine (TAMRA), at 578 nm, was observed depending on the degradation of DNA.RNA hybrid duplex. This method can be introduced into the high throughput screening of the inhibitors against the RNase H activity for anti-HIV drug.

Topics: Base Sequence; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HIV Reverse Transcriptase; In Vitro Techniques; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Oligoribonucleotides; Rhodamines; Ribonuclease H; Substrate Specificity

All DNA sequencing methods have benefited from the development of new F667Y versions of Taq DNA polymerase. However, terminator chemistry methods show less uniform peak height patterns when compared to primer chemistry profiles suggesting that the dyes and/or their linker arms affect enzyme selectivity. We have measured elementary nucleotide rate and binding constants for representative rhodamine- and fluorescein-labeled terminators to determine how they interact with F667 versions of Taq Pol I. We have also developed a rapid gel-based selectivity assay that can be used to screen and to quantify dye-enzyme interactions with F667Y versions of the enzyme. Our results show that 6-TAMRA-ddTTP behaves like unlabeled ddTTP, while 6-FAM-ddTTP shows a 40-fold reduction in the rate constant for polymerization without affecting ground-state nucleotide binding. Detailed mechanism studies indicate that both isomers of different fluorescein dyes interfere with a conformational change step which the polymerase undergoes following nucleotide binding but only when these dyes are attached to pyrimidines. When these same dyes are attached to purines by the same propargylamino linker arm, they show no effect on enzyme selectivity. These studies suggest that it may be possible to develop fluorescein terminators for thermocycle DNA sequencing methods for polymerases that do not discriminate between deoxy- and dideoxynucleotides.

Topics: Fluoresceins; Fluorescent Dyes; Molecular Structure; Nucleic Acid Conformation; Rhodamines; Structure-Activity Relationship; Substrate Specificity; Taq Polymerase; Terminator Regions, Genetic

The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures. DNA binding also induces a substantial bend. Using fluorescence resonance energy transfer (FRET), we have determined the changes in the end-to-end distance consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges. The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively. The former value contrasts with an inferred value of 150 degrees reported by Heyduk et al. (1) for the bend induced by a single domain. We also observe that the induced bend angle is unaffected by the presence of the C-terminal acidic region. The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees). We suggest this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif.

Topics: Amino Acid Sequence; Animals; Chironomidae; DNA; DNA, Circular; Drosophila; Energy Transfer; Fluoresceins; Fluorescent Dyes; High Mobility Group Proteins; Molecular Sequence Data; Nucleic Acid Conformation; Protein Binding; Rhodamines; Saccharomyces cerevisiae; Spectrometry, Fluorescence

We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.

Topics: DNA; Energy Transfer; Fluoresceins; Fluorescence Polarization; Fluorescent Dyes; Molecular Probe Techniques; Nucleic Acid Conformation; Oligonucleotide Probes; Rhodamines; RNA, Catalytic; Spectrometry, Fluorescence

We describe a PCR-based assay for determining c-erbB-2 oncogene amplification in breast cancer in which we use the TaqMan system. Two fluorogenic probes anneal to the target between primers for c-erbB-2 and beta-globin genes and contain both a reporter dye (6-carboxy-fluorescein) and a quencher dye (6-carboxy-tetramethyl-rhodamine). During the extension phase of the PCR cycle, the 5'-->3' exonuclease activity of Taq polymerase cleaves the hybridized fluorogenic probe, resulting in an increase of fluorescence emission of the reporter dye that is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. Assay performance showed adequate precision and a lower detection limit and good correlation with the results obtained in the same samples by a competitive PCR assay (n = 25, r = 0.94, P < 0.01). This homogeneous assay is time-saving, avoids usually cumbersome postamplification procedures (that can be additional sources of inaccuracy and contamination), and seems suitable for determination of c-erbB-2 oncogene amplification in tumor specimens.

Topics: Base Sequence; Breast Neoplasms; DNA-Directed DNA Polymerase; DNA, Neoplasm; Fluoresceins; Fluorescent Dyes; Globins; Humans; Oligonucleotide Probes; Polymerase Chain Reaction; Receptor, ErbB-2; Rhodamines; Sensitivity and Specificity; Taq Polymerase