Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Smart probe: a novel fluorescence quenching-based oligonucleotide probe carrying a fluorophore and an intercalator.

A novel probe (Smart probe) has been developed for nucleic acid detection. The smart probe is an oligodeoxyribonucleotide carrying a fluorophore and an intercalator internally. Fluorescence of the smart probe is quenched by the intercalator in the absence of target sequence. While upon hybridization the probe emits greater fluorescence due to the interference of quenching by intercalation. The smart probe has been shown to recognize a single base mismatch in the double-stranded form without utilizing thermal stability difference of hybrids.     

MagiProbe: a novel fluorescence quenching-based oligonucleotide probe carrying a fluorophore and an intercalator

Fluorescence is the favored signaling technology for molecular diagnoses. Fluorescence energy transfer-based methods are powerful homogeneous assay tools. A novel oligonucleotide probe, named MagiProbe, which is simple to use, is described, and information given about the duplex formed with a target. The probe internally has a fluorophore and an intercalator. Its fluorescence is quenched by the intercalator in the absence of a target sequence. On hybridization with a target sequence, the probe emits marked fluorescence due to the interference in quenching by intercalation. Furthermore, MagiProbe hybridized with a single-base mismatched target emits less fluorescence than with a perfect matched target. It therefore can detect a single base difference in a double-stranded form with a target.     

Quinolizinium as a new fluorescent lysosomotropic probe

We have synthesized a collection of quinolizinium fluorescent dyes for the purpose of cell imaging. Preliminary biological studies in human U2OS osteosarcoma cancer cells have shown that different functional groups appended to the cationic quinolizinium scaffold efficiently modulate photophysical properties but also cellular distribution. While quinolizinium probes are known nuclear staining reagents, we have identified a particular quinolizinium derivative salt that targets the lysosomal compartment. This finding raises the question of predictability of specific organelle targeting from structural features of small molecules.     

A ratiometric fluorescent probe for sensing hydrogen peroxide based on a hemicyanine–naphthol fluorophore

The synthesis, properties and applications of a water‐soluble boronate‐functioned hemicyanine–naphthol hybrid as a novel ratiometric fluorescent sensor for hydrogen peroxide are presented. The dye displayed remarkable a colour change from pale orange (λ_em = 590 nm) to pink (λ_em = 690 nm) in the presence of H₂O₂, which could be rationalized by the chemoselective H₂O₂‐mediated transformation of arylboronate to phenolate with high selectivity and a fast response (within 2 min). A good linear relationship (R² = 0.9951) was obtained with the H₂O₂ concentration ranging from 0 to 25 μM, with a limit of detection of 0.09 μM according to the signal‐to‐noise ratio (S/N = 3). The advantages of this fluorophore include easy modification, excellent aqueous solubility and superior photostability, and it has been applied to the detection of trace amounts of hydrogen peroxide in water samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Intelligent fluorescent nucleoside in sensing cytosine base: importance of hydrophobic nature of perylene fluorophore

Fluorescence response upon hybridization of perylene labeled oligonucleotide probes depends on the microenvironment experienced by the perylene fluorophore. In mismatched duplex (^PerU-C), enhanced fluorescence was observed while in matched duplex (^PerU-A) fluorescence intensity decreased considerably. This observation will be a promising research effort in giving rise to a new powerful tool in detection of SNP.     

Perylene-conjugated pyrrole polyamide as a sequence-specific fluorescent probe

Perylene-conjugated pyrrole (Py)-polyamide 2 was designed and synthesized using the Fmoc solid-phase synthesis and a subsequent Sonogashira coupling reaction with 3-bromoperylene. Interestingly, conjugate 2 did not luminesce in water at 313 nm irradiation but was turned on in the presence of target double-stranded (ds) DNA, and showed strong emission with increasing DNA concentration, in particularly, by the binding to the target telomere sequences through heterodimer formation with partner 3. Importantly, the excitation spectrum of 2 clearly indicates that the Py and Imidazole (Im) moieties in the polyamide effectively sensitize the perylene moiety to give rise to fluorescence emission. Energy transfer would occur from the Py moiety to the perylene. Thus, screening of perylene-conjugates will allow us to develop a novel "molecular light switch" with sequence-specificity.     

Dityrosine as a product of oxidative stress and fluorescent probe

Summary. Dityrosine can be a natural component of protein structure, a product of environmental stress, or a product of in vitro protein modification. It is both a cross-link and a fluorescent probe that reports structural and functional information on the cross-linked protein molecule. Diverse reactions produce tyrosyl radicals, which in turn may couple to yield dityrosine. Identification and quantitation of dityrosine in protein hydrolysates usually employs reversed phase high pressure liquid chromatography (RP-HPLC) or gas chromatography. RP-HPLC of protein hydrolysates that have been derivatized with dabsyl chloride gives a complete amino acid analysis that includes dityrosine and 3-nitrotyrosine. Calmodulin, which contains a single pair of tyrosyl residues, undergoes both photoactivated and enzyme-catalyzed dityrosine formation. Polarization measurements, employing the intrinsic fluorescence of dityrosine, and catalytic activity determinations show how different patterns of inter- and intramolecular cross-linking affect the interactions of calmodulin with Ca²⁺ and enzymes.     

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.     

Characterization of a carbocyanine derivative as a fluorescent penetration probe

The fluorescent carbocyanine dye 3,3-diheptyloxycarbocyanine [DiOC7(3)], originally described as a membrane potential probe, penetrates poorly into multicell spheroids. Since the dye is retained in the cells following spheroid disaggregation, cells can be selected from different depths within the spheroid using fluorescence-activated cell sorting. Characterization of the binding kinetics, stability, and toxicity of this probe were undertaken, and intercompared with Hoechst 33342. The optimum drug dose for achieving good separation of internal and external cells of spheroids is about tenfold lower than for Hoechst 33342, and like Hoechst, DiOC7(3) is toxic at concentrations at least tenfold higher than those required to produce a good gradient for cell separation. When cells are removed from the stain, cellular fluorescence decreases to half the initial intensity within 2 hours; however, unlike Hoechst, the carbocyanine dye does not transfer between cells.     

Implanting mouse embryo stain with a LNF-I bearing fluorescent probe at their mural trophectodermal side.

Mouse embryos at implantation stage were stained successfully with lacto-N-fucopentaose I (LNF-I) bearing neoglycoprotein labeled with rhodamine synthesized by us for the first time. The fluorescent neoglycoproteins carrying LNF-II, -III, LND-I, or LNT failed to stain the embryos. The embryo was stained only at the cell surface of trophectoderm at the mural side. Since the attachment of the mouse embryo to the uteric epithelium occurs at its mural side trophectoderm and LNF-I is the key substance in mouse implantation (Lindenberg, S. et al, (1988) J. Reprod. Fert. 83, 149-158), the material stained with the probe carrying LNF-I appears to be the molecule responsive to attachment to the endometrium surface and leading to implantation.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes

1. A reversible light-induced enhancement of the fluorescence of a “hydrophobic fluorophore”, 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1′-ethylene-2,2′-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response.

2. This effect is decreased under phosphorylating conditions (presence of ADP, P_i, Mg²⁺), and completely inhibited by the uncoupler of phosphorylation NH₄Cl (5–10 mM), as well as the ionophores nigericin and gramicidin-D (both at 5 · 10⁻⁸ M). The MgCl₂ concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a “high energy intermediate”. The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the “high energy state” of the thylakoid membrane rather than a terminal phosphorylation step.

3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fluorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H⁺ uptake per se because (a) the pH dependence of H⁺ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H⁺ uptake, reduces anthroyl stearate fluorescence.

Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate or Diquat catalyzed electron flow and is the first “non-amine” fluorescence probe utilized for this purpose in chloroplasts.     

7-Aminoactinomycin as a fluorescent probe for DNA unwinding and denaturation

A fluorescent analogue of antibiotic actinomycin D, 7-aminoactinomycin D (7AAMD), which is widely used in molecular biology, was shown by steady-state, polarization, and phase fluorescent spectroscopy to bind primarily in the unwound regions of DNA with concomitant increase in its emission intensity. The maximum emission intensity of 7AAMD is observed for denatured DNA. Thus, 7AAMD may serve as a good indicator of DNA unwinding, denaturation, and fragmentation.     

Acridine orange as a fluorescent probe for lysosomal proton pump

Acridine orange was found to accumulate in pure lysosomal particles (tritosomes) in vitro, and the quenching of its fluorescence correlated well with the delta pH (inside acid) across the lysosomal membrane. Use of this dye showed that Mg-ATP caused lysosomal acidification. This acidification was sensitive to N,N'-dicyclohexylcarbodiimide, N-ethylmaleimide, and azide, but not to oligomycin, ouabain or vanadate. These results supported the idea of the existence of a lysosomal H+-pump, suggested in a previous paper (Ohkuma et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2758-2762).     

Chloroquine as intercalator: a hypothesis revived

The mode of action of chloroquine is still controversial. Proposed mechanisms of action include (1) DNA intercalation, (2) lysosome accumulation and (3) binding to ferriprotoporphyrin IX. Recent data suggest that intercalation into parasite DNA can occur at physiological concentrations of the drug. Furthermore, structure-activity relationship studies are most consistent with the intercalation mechanism. Regardless of which mechanism is correct, the selective toxicity of chloroquine for malaria parasites is probably due to permease-mediated uptake.     

Anthroyl stearate as a fluorescent probe of chloroplast membranes.

1. A reversible light-induced enhancement of the fluorescence of a "hydrophobic fluorophore", 12-(9-anthroyl)-stearic acid (anthroyl stearate), is observed with chloroplasts supporting phenazine methosulfate, cyclic or 1,1'-ethylene-2,2'-dipyridylium dibromide (Diquat) pseudo-cyclic electron flow; no fluorescence change is observed when methyl viologen or ferricyanide are used as electron acceptors. The stearic acid moiety of anthroyl stearate is important for its localization and fluorescence response in the thylakoid membrane, since structural analogs of anthroyl stearate lacking this group do not show the same response. 2. This effect is decreased under phosphorylating conditions (presence of ADP, Pi, Mg2+), and completely inhibited by the uncoupler of phosphorylation NH4Cl(5-10mM), as well as the ionophores nigericin and gramicidin-D (both at 5 - 10(-8)M). The MgCl2 concentration dependence of the anthroyl stearate enhancement effect is identical to that previously observed for cyclic photophosphorylation, as well as for the formation of a "high energy intermediate". The anthroyl stearate fluorescence enhancement is inhibited by increasing concentrations of ionophores in parallel with the decrease in ATP synthesis, but is essentially unaffected by specific inhibitors (Dio-9 and phlorizin) of photophosphorylation; thus, it appears that anthroyl stearate monitors a component of the "high energy state" of the thylakoid membrane rather than a terminal phosphorylation step. 3. The light-induced anthroyl stearate fluorescence enhancement is suggested to monitor a proton gradient in the energized chloroplast because (a) similar enhancement can be produced by sudden injection of hydrogen ions in a solution of anthroyl stearate; (b) when the proton gradient is dissipated by gramicidin or nigericin light-induced anthroyl stearate fllorescence is eliminated; (c) when the proton gradient is dissipated by tetraphenylboron, light-induced anthroyl stearate fluorescence decreases, and (d) light-induced anthroyl stearate fluorescence change as a function of pH is qualitatively similar to that observed with other probes for a proton gradient (e.g. 9-aminoacridine). Furthermore, anthroyl stearate does not monitor H+ uptake per se because (a) the pH dependence of H+ transport is different from that of the anthroyl stearate fluorescence change, and (b) tetraphenylboron, which does not inhibit H+ uptake, reduces anthroyl stearate fluorescence. Thus, anthroyl stearate appears to be a useful probe of a proton gradient supported by phenazine methosulfate of Diquat catalyzed electron flow and is the first "non-amine" fluorescence probe utilized for this purpose in chloroplasts.     

Novel fluorescent probe for detecting hydroperoxides with strong emission in the visible range

A novel fluorescent probe, a swallow-tailed perylene derivative for detecting hydroperoxides (Spy-HP), containing perylene 3,4,9,10-tetracarboxyl bisimide as the main skeleton in the structure, was developed. Spy-HP reacted rapidly with hydroperoxides such as m-chloroperbenzoic acid (MCPBA) and cumene hydroperoxide to form its oxidized derivative, Spy-HPOx, and emitted an extremely strong fluorescence (phi approximately 1) in the visible range (lambda(ex) = 524 nm and lambda(em) = 535 nm), as the result of canceling the photoinduced electron transfer (PET) effect. The reaction between Spy-HP and hydroperoxides proceeded quantitatively in strict stoichiometry, without being affected by autoxidation or photobleaching. Because of these prominent properties, Spy-HP is expected to be a novel and useful fluorescent probe to 'spy' on hydroperoxides in biosamples.     

Development of fluorophore dynamics imaging as a probe for lipid domains in model vesicles and cell membranes

The ability to detect raft structures in membranes continues to present a problem, especially in the membranes of live cells. Rafts, generally considered to be small (<200 nm) sphingolipid-rich regions, are commonly modelled using lipid vesicle systems where the ability of fluorophore-labelled lipids to preferentially locate into domains (basically large rafts) is investigated. Instead, in this study the motional properties of different fluorophores were determined using two-photon excitation and time-correlated single-photon counting coupled with diffraction-limited imaging with polarizing optics in scanning mode to obtain nanosecond rotational correlation time images. To develop the method, well-characterized domain-containing models consisting of giant unilamellar vesicles comprising mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol were used with the fluorophores diphenylhexatriene, 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl). Accordingly, images of rotational correlation times of the probes revealed domain structures for all three probes consistent with other studies using different approaches. Rotational correlation time images of living cell membranes were also observed. The method has the advantage that not only does it enable domains to be visualised or imaged in a unique manner but that it can also potentially provide useful information on the lipid dynamics within the structures.