Synthesis and binding properties of oligo-2'-deoxyribonucleotides covalently linked to a thiazole orange derivative

Thiazole orange label was coupled to the eighth phosphate of a pentadeca-2'-deoxyriboadenylate via a phosphoramidate linkage using different linkers. The stereoisomers were separated, and their absolute configurations were determined. Finally, the thiazole orange moiety was also linked to the tenth phosphate of icosathymidylates in both the alpha and the beta series via a phosphoramidate linkage. Once again, the thiazole orange-icosathymidylate conjugates were obtained as pure stereoisomers. The binding properties of these oligo-2'-deoxyribonucleotide-thiazole orange conjugates with their complementary sequences were studied by absorption spectroscopy. The covalent attachment of the thiazole orange derivatives to the oligoadenylates stabilizes the complexes formed with both the DNA and RNA targets. On the contrary, when the thiazole orange is tethered to the oligo-alpha-thymidylate or oligo-beta-thymidylate, no significant stabilization of the duplexes formed with poly r(A) can be observed.     

Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis > manual counting.     

A DNA minor groove binder shows high effectiveness as a quencher for FRET probes

A non-fluorescent quencher based on thiazole orange was incorporated into oligonucleotides. Fluorimetry and fluorogenic real-time polymerase chain reaction experiments demonstrated that the quencher is effective for fluorescein amidite dyes. The thiazole orange quencher also increased the melting temperature of DNA duplexes, which may facilitate the design of shorter and more discriminatory probes. The effectiveness of the quencher in TaqMan probes was also demonstrated.     

The walking dead: Is hydroethidine a suitable viability dye for intra-erythrocytic Plasmodium falciparum?

Hydroethidine has been used as a viability dye in various haemoparasites, including Plasmodium species. We compared flow cytometric quantification by hydroethidine and thiazole orange in P. falciparum. Dead parasites that did not develop or replicate exhibited high levels of DNA fragmentation and abnormal microscopic morphology, but were detected as viable ring-stage parasites by hydroethidine. Hydroethidine quantification was similar to thiazole orange, a DNA-binding dye that stains live and dead parasites. Data obtained cast concerns on hydroethidine as a suitable viability dye in P. falciparum and highlight the necessity of proper gating in flow cytometric studies quantifying parasitaemia.     

Co-localization of polysomes,cytoskeleton, and membranes with protein bodies from corn endosperm

Summary Frozen corn endosperm was homogenized in a cytoskeleton-stabilizing buffer and stained directly (without pelleting) with rhodamine-phalloidin for actin and either thiazole orange to stain RNA or DiOC₆ to stain membranes prior to examination under the fluorescence microscope. Other samples were treated with a non-ionic detergent alone or in conjunction with a ionic detergent prior to staining and fluorescence microscopy. Very gentle homogenization in unsupplemented buffer yielded a massive aggregate containing protein bodies that fluoresced after treatment with the ER stain DiOC₆. This aggregate was capped by an aggregate of unstained starch grains. More vigorous homogenization yielded more disperse patterns showing almost identical co-localization of ER, actin and RNA (polysomes). Homogenization in buffer plus non-ionic detergent removed most of the membrane yet maintained co-localization of actin and polysomes, while homogenization in double detergent removed the last traces of membrane and actin, and released over 70% of the polysomes. We interpret these results to suggest that protein bodies are surrounded by membranes, cytoskeleton and RNA (polysomes) and that the majority of the polysomes are attached more firmly to the cytoskeleton than to the membrane. This provides evidence from fluorescence microscopy to supplement that from biochemical analyses for the existence of cytomatrix-bound polysomes in plants.Abbreviations CBP cytoskeleton-bound polysomes - CMBP cyto-matrix-bound polysomes - CSB cytoskeleton-stabilizing buffer - DOC sodium deoxycholate - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DTE dithioerythritol - MBP membrane-bound polysomes - FP free polysomes - PMSF phenylmethyl-sulfonyl fluoride - PTE polyoxy-ethylene-10-tridecyl ether - Rh-Ph rhodamine-phalloidin - TO thiazole orange - Tris tris-(hydroxymethyl) aminomethane     

An assay for human telomeric G-quadruplex DNA binding drugs

Compounds that stabilize the G-quadruplexes formed by human telomeres can inhibit the telomerase activity and are potential cancer therapies. We have developed an assay for the screening of compounds with high affinity for human telomeric G-quadruplexes (HTG). The assay uses a thiazole orange fluorescent reporter molecule conjugated to the aminoglycoside, neomycin, as a probe in a fluorescence displacement assay. The conjugation of the planar base stacking thiazole orange with the groove binding neomycin results in high affinity probe that can determine the relative binding affinity of high affinity HTG binding drugs in a high throughput format. The robust assay is applicable for the determination of the binding affinity of HTG in the presence of K⁺ or Na⁺.     

Fluorescent and photochemical properties of a single zinc finger conjugated to a fluorescent DNA-binding probe

A single zinc finger derived from the DNA-binding domain of the glucocorticoid receptor (GR) has been tethered to the intercalating fluorophore thiazole orange, and the DNA recognition characteristics of the conjugate have been examined. DNA sequence specificity for the peptide-dye conjugate, determined by steady-state fluorescence measurements and photoactivated DNA cleavage experiments, reproduce the binding features of response element recognition found in the native GR. The thiazole orange is able to intercalate and fluoresce when the conjugate binds, at concentrations where little fluorescence is observed from either the conjugate alone or the conjugate mixed with DNA lacking the zinc finger target sequence. The conjugate preferentially targets a 5'-TGTTCT-3' sequence (the native glucocorticoid receptor element) with a dissociation constant of about 25 nM. Lower binding affinities (up to 10-fold) are observed for single site variants of this sequence, and much lower affinity (40-50-fold) is observed for binding to the estrogen response element (which differs from the glucocorticoid receptor element at two positions) as well as to nonspecific DNA. Footprinting reactions show a 4-6 base pair region that is protected by the zinc finger moiety. Photocleavage assays reveal a several base pair region flanking the recognition sequence where the tethered thiazole orange moiety is able to intercalate and subsequently cleave DNA upon visible light exposure. Thiazole orange is also shown to oxidize the 5'-G of remote GG sequences, depending on the details of the intervening DNA sequence. Small synthetic protein-dye conjugates such as this one are potentially useful for a variety of purposes including sequence-specific probes that work under physiological conditions (without melting and hybridization of DNA), sequence-specific photocleavage agents, and self-assembling components in electron and energy transfer systems that utilize DNA as a scaffold and/or photochemical medium.     

Expression,purification, and activity of ActhiS,a thiazole biosynthesis enzyme from <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis>

Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthia-zole-2-carboxylic acid (ADT) using HPLC and ¹H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD⁺ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.     

Spectral properties and DNA targeting features of a thiazole orange-peptide bioconjugate

The molecular recognition features of a DNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency are described. The bioconjugate combines a polypeptide from the Tc3 transposase DNA-binding domain with the dsDNA-sensitive fluorophore thiazole orange. Fluorescence spectroscopy and circular dichroism reveal that the polypeptide moiety determines the DNA sequence specificity as the intercalating dye makes nonspecific contributions to binding affinity. The conjugated thiazole orange is able to intercalate and fluoresce when the peptide binds at concentrations where little fluorescence is observed from either the bioconjugate alone or the bioconjugate mixed with DNA lacking the target sequence. Fluorescence studies indicate this molecular probe is sequence specific, binds the native Tc3 DNA target sequence with nanomolar affinity (KD approximately 15 nM), and is able to discriminate multiple point mutations in the cognate DNA site. The attachment of a sequence-specific binding peptide onto a functional probe provides a viable strategy for construction of synthetic enzymes and repressors, and facilitates dynamic studies of protein-DNA interactions.     

DNA labelling topologies for monitoring DNA-protein complex formation by fluorescence anisotropy

In this work, fluorescence anisotropy was used to study DNA binding of the DNA methyltransferase M.TaqI. For this purpose short DNA molecules labelled with three different fluorophores (Cy3, thiazole orange, and ethidium bromide) were prepared in various topologies and their suitability for detection of DNA-protein complex formation was investigated.     

Thiazole orange as the fluorescent intercalator in a high resolution fid assay for determining DNA binding affinity and sequence selectivity of small molecules.

The viability of using thiazole orange as an alternative to ethidium bromide in a fluorescent intercalator displacement (FID) assay is explored by profiling the DNA binding affinity and sequence selectivity of netropsin. Utilizing a library of hairpin deoxyoligonucleotides containing all possible four base-pair sequences, the method provides a high resolution profile of the DNA binding properties of small molecules in a high throughput format.     

Development of a fluorescent intercalator displacement assay (G4-FID) for establishing quadruplex-DNA affinity and selectivity of putative ligands

A fluorescent intercalator displacement assay (G4-FID) has been designed based on the displacement of thiazole orange (TO) positioned onto a quadruplex-forming oligonucleotide by putative ligands. This technique was validated by the use of a set of representative and fully characterized G-quadruplex binders (ranging from pyridodicarboxamide to macrocyclic ligands). To further extend its applicability, a comparative version has been developed which allows a rapid and viable determination of quadruplex- over duplex-selectivity.     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

Sequence dependence of fluorescence emission and quenching of doubly thiazole orange labeled DNA: effective design of a hybridization-sensitive probe

We have designed a doubly thiazole orange labeled nucleoside showing high fluorescence intensity for a hybrid with the target DNA and effective quenching for a single-stranded state. Knowing how much the fluorescence emission and quenching of this probe depend on the probe sequence and why there is such a sequence dependence is important for effective probe design, we synthesized more than 30 probe sequences and measured their fluorescence intensities. When the probe hybridized with the target DNA strands, there was strong emission, whereas the emission intensity was much weaker before hybridization; however, self-dimerization of probes suppressed fluorescence quenching. In particular, the G/C base pairs neighboring the labeled nucleotide in a self-dimeric structure resulted in a low quenching ability for the probe before hybridization. On the other hand, mismatched base pair formation around the labeled site decreased the fluorescence intensity because the neighboring sequence is the binding site of the tethered thiazole orange dyes. The hybridization enhanced the fluorescence of the probe even when the labeled nucleotide was located at the end of the probe strand; however, the partial lack of duplex structure resulted in a decrease in the fluorescence intensity of the hybrid.     

New cyanine dyes as base surrogates in PNA: forced intercalation probes (FIT-probes) for homogeneous SNP detection

Forced intercalation probes (FIT-probes) are nucleic acid probes, in which an intercalator cyanine dye such as thiazole orange (TO) serves as a replacement of a canonical nucleobase. These probes signal hybridization by showing strong increases of fluorescence. TO in FIT-probes responds to adjacent base mismatches by attenuation of fluorescence intensities at conditions where both matched and mismatched target DNA are bound. The interesting features of TO labeled FIT-probes posed the question whether the forced intercalation concept can be extended to other cyanine dyes of the thiazole orange family. Herein, we present the synthesis of three asymmetrical cyanine dyes and their incorporation into PNA-conjugates by means of both divergent and linear solid-phase synthesis. Melting analysis revealed that the DNA affinity of PNA probes remained high irrespective of the replacement of a nucleobase by the cyanines YO (oxazole yellow), MO or JO. Of the three new tested dye-PNA-conjugates, the YO-containing PNA has properties useful for homogeneous SNP detection. YO-PNA is demonstrated to signal the presence of fully complementary DNA by up to 20-fold enhancement of fluorescence. In addition, YO emission discriminates against single base mismatches by attenuation of fluorescence. Oxazole yellow (YO) as a base surrogate in PNA may prove useful in the multiplex detection of single base mutations at non-stringent conditions.     

Thiazole orange: a useful probe for fluorescence sensing of G-quadruplex-ligand interactions

Fluorimetric titrations were performed to gain insight into parameters that govern the association of thiazole orange (TO) and G-quadruplex-DNA (G4-DNA). Use of loop-containing and loop-lacking quadruplexes evidenced the critical influence of the loops on the stoichiometry of the association and on the fluorescence exaltation of TO. We subsequently tried to benefit from this sensitivity to evaluate the influence of G4-DNA cationic environment on ligand binding via a recently reported G4-FID assay.     

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.     

Thiazole synthase from Escherichia coli: an investigation of the substrates and purified proteins required for activity in vitro

Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the "radical-AdoMet" family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5'-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction.     

Orange color is associated with <Emphasis Type="Italic">CYC</Emphasis>-<Emphasis Type="Italic">B</Emphasis> expression in tomato fleshy fruit

Carotenes are plant secondary metabolites that are important for human health. Additionally, carotenes influence fruit color, which is a major trait for breeding. We compared the expression and sequences of genes related to color phenotypes in tomato inbred lines that produce different colors of fleshy fruit. Up-regulation of CYC-B expression and higher amount of β-carotene content in fruit ripening stage and nucleotide variations in the 5′ region of the gene were detected in orange fruited inbred lines compared to the other lines. Our results indicated that there is a close relationship between the expression pattern of the CYC-B gene and the orange color of fleshy fruit. We identified 4 SNPs in the promoter region of CYC-B genes associated with the orange fruit color. Moreover, the segregation ratio and color phenotypes in an F2 generation further indicated that one of the detected SNPs were associated with the orange color in the tested inbred lines. Our study provides valuable information to breeders for marker-assisted selection to produce desirable tomato varieties with health benefits by varying carotenoid levels.     

Cy5-conjugated hybridization-sensitive fluorescent oligonucleotides for ratiometric analysis of nuclear poly(A)+ RNA

Subnuclear poly(A)(+) RNA localization in living mammalian cells was visualized by ratiometric analysis using hybridization-sensitive fluorescent oligonucleotide probes. Probes were oligonucleotides, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide inside strand. A ratiometric analysis using poly(A)-targeting probes revealed a distribution of the probe itself as red fluorescence and localization of the target RNA sequence in cell nuclei as green fluorescence. The fluorescence of the subnuclear poly(A)(+) RNA hybridized with the poly(A)-targeting probes was observed as puncta in interchromatin areas.