Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential.     

Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine

The replacement of thymidine with 5-bromo-2′-deoxyuridine (^BrdU) is well-known to sensitize cells to ionizing radiation and photoirradiation. We reported here the sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex oligodeoxynucleotides harboring a ^BrdU or its closely related 5-bromo-2′-deoxycytidine (^BrdC). Our results showed that two types of crosslink products could be induced from d(^BrCG), d(^BrUG), d(G^BrU), or d(A^BrU); the C(5) of cytosine or uracil could be covalently bonded to the N(2) or C(8) of its neighboring guanine, and the C(5) of uracil could couple with the C(2) or C(8) of its neighboring adenine. By using those crosslink product-bearing dinucleoside monophosphates as standards, we demonstrated, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), that all the crosslink products described above except d(G[N(2)-5]U) and d(G[N(2)-5]C) could form in duplex DNA. In addition, LC-MS/MS quantification results revealed that both the nature of the halogenated pyrimidine base and its 5′ flanking nucleoside affected markedly the generation of intrastrand crosslink products. The yields of crosslink products were much higher while the 5′ neighboring nucleoside was a dG than while it was a dA, and ^BrdC induced the formation of crosslink products much more efficiently than ^BrdU. The formation of intrastrand crosslink products from these halopyrimidines in duplex DNA may account for the photosensitizing effects of these nucleosides.     

Reduction of organic and inorganic selenium compounds by the edible medicinal basidiomycete Lentinula edodes and the accumulation of elemental selenium nanoparticles in its mycelium

We report for the first time that the medicinal basidiomycete Lentinula edodes can reduce selenium from inorganic sodium selenite (Se^IV) and the organoselenium compound 1,5-diphenyl-3-selenopentanedione-1,5 (DAPS-25) to the elemental state, forming spherical nanoparticles. Submerged cultivation of the fungus with sodium selenite or with DAPS-25 produced an intense red coloration of L. edodes mycelial hyphae, indicating accumulation of elemental selenium (Se⁰) in a red modification. Several methods, including transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), and X-ray fluorescence, were used to show that red Se⁰ accumulated intracellularly in the fungal hyphae as electron-dense nanoparticles with a diameter of 180.51±16.82 nm. Under designated cultivation conditions, shiitake did not reduce selenium from sodium selenate (Se^VI).     

Characterization and use of an unprecedentedly bright and structurally non-perturbing fluorescent DNA base analogue

This article presents the first evidence that the DNA base analogue 1,3-diaza-2-oxophenoxazine, tC^O, is highly fluorescent, both as free nucleoside and incorporated in an arbitrary DNA structure. tC^O is thoroughly characterized with respect to its photophysical properties and structural performance in single- and double-stranded oligonucleotides. The lowest energy absorption band at 360 nm (ε = 9000 M⁻¹ cm⁻¹) is dominated by a single in-plane polarized electronic transition and the fluorescence, centred at 465 nm, has a quantum yield of 0.3. When incorporated into double-stranded DNA, tC^O shows only minor variations in fluorescence intensity and lifetime with neighbouring bases, and the average quantum yield is 0.22. These features make tC^O, on average, the brightest DNA-incorporated base analogue so far reported. Furthermore, it base pairs exclusively with guanine and causes minimal perturbations to the native structure of DNA. These properties make tC^O a promising base analogue that is perfectly suited for e.g. photophysical studies of DNA interacting with macromolecules (proteins) or for determining size and shape of DNA tertiary structures using techniques such as fluorescence anisotropy and fluorescence resonance energy transfer (FRET).     

Aliphatic substituents in place of thymine methyl promote zig-zag character of the poly(dA-dT)·poly(dA-dT) backbone

This work compared circular dichroism and phosphorus n.m.r. of poly(dA-dU)·poly(dA-dU), poly(dA-dT)·poly(dA-dT), poly(dA-ethyl⁵dU)·poly(dA-ethyl⁵dU), and poly(dA-butyl⁵dU)·poly(dA-butyl⁵dU) at low-salt and in concentrated caesium chloride and caesium fluoride solutions. It is demonstrated that growing bulk of the substituent increases the conformationl anomaly residing in the purine(3′–5′)pyrimidine steps while the backbone is less affected in the pyrimidine (3′–5′)purine steps. As the length of the substituent increases, conformation of the polynucleotides alters more dramatically at increasing concentrations of caesium cations. At high CsF concentrations, all the polynucleotides adopt a novel conformer which we call X-DNA and its formation is promoted by larger substituents. The X-DNA conformation of poly(dA-butyl⁵dU)·poly(dA-butyl⁵dU) gives two phosphorus n.m.r. resonances separated as much as in the case of the left-handed zig-zag Z-DNA double helix of poly(dG-dC)·poly(dG-dC) but X-DNA and Z-DNA differ qualitatively by an opposite dinucleotide repeat. Phosphorus n.m.r. spectra of poly(dA-dT)·poly(dA-dT) and poly(dA-butyl⁵dU)-poly(dA-butyl⁵dU) differ quantitatively at high CsF concentrations, which may reflect conformational variability of the X-DNA backbone. Poly(dA-butyl⁵dU)·poly(dA-butyl⁵dU), but not poly(dA-ethyl⁵dU)·poly(dA-ethyl⁵dU) and the related polynucleotides with shorter substituents in position 5 of uracil, exhibits one more reversible transition at very high caesium fluoride concentrations. It is accompanied by polynucleotide associations and has a slow kinetics. This transition may involve one more radical change in the double helix architecture from X-DNA into another conformation.     

Interaction between Subunits of Heterodimeric Splicing Factor U2AF Is Essential In Vivo

The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF⁵⁰) that interacts with the Drosophila small subunit (dU2AF³⁸). A 28-amino-acid fragment on dU2AF⁵⁰ that is both necessary and sufficient for interaction with dU2AF³⁸ was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF⁵⁰ point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF⁵⁰ allele. A mutation that completely abolished interaction with dU2AF³⁸ was incapable of complementation, whereas dU2AF⁵⁰ mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF⁵⁰ allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF⁵⁰ recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.     

Structural insights of non-canonical U?U pair and Hoogsteen interaction probed with Se atom

Unlike DNA, in addition to the 2′-OH group, uracil nucleobase and its modifications play essential roles in structure and function diversities of non-coding RNAs. Non-canonical U•U base pair is ubiquitous in non-coding RNAs, which are highly diversified. However, it is not completely clear how uracil plays the diversifing roles. To investigate and compare the uracil in U-A and U•U base pairs, we have decided to probe them with a selenium atom by synthesizing the novel 4-Se-uridine (^SeU) phosphoramidite and Se-nucleobase-modified RNAs (^SeU-RNAs), where the exo-4-oxygen of uracil is replaced by selenium. Our crystal structure studies of U-A and U•U pairs reveal that the native and Se-derivatized structures are virtually identical, and both U-A and U•U pairs can accommodate large Se atoms. Our thermostability and crystal structure studies indicate that the weakened H-bonding in U-A pair may be compensated by the base stacking, and that the stacking of the trans-Hoogsteen U•U pairs may stabilize RNA duplex and its junction. Our result confirms that the hydrogen bond (O4^…H-C5) of the Hoogsteen pair is weak. Using the Se atom probe, our Se-functionalization studies reveal more insights into the U•U interaction and U-participation in structure and function diversification of nucleic acids.     

Are intracellular ionic concentrations accessible using fluorescent probes? The example of Mag-indo-1

The study of the physicochemical properties of Mag-indo-1, a fluorescent probe used for intracellular magnesium measurements, has shown that in a biological environment the deprotonated form of this probe is in simultaneous equilibrium with a protonated form, a protein and a magnesium-bound form. The complex emission fluorescence spectrum emitted by a single living cell was analyzed using a computerized method, allowing the evaluation of the Mag-indo-1 to the cellular fluorescence. This approach used to evaluate intracellular Mg²⁺ concentration has also shown the variability of the important participation of protein-bound Mag-indo-1 to the cellular fluorescence. Thus the widely used ratioing method, unable to take into account this variability, cannot afford a reliable evaluation of [Mg²⁺]. Whatever the technique used for investigation (microfluorimetry, flow cytometry, etc.) the evaluation of [Mg²⁺]_i using the fluorescent probe Mag-indo-1 requires a method able to quantify, in complex fluorescence, the fluorescence intensity of the forms involved in the equilibrium with Mg²⁺.Abbreviations [Ca²⁺]₁ intracellular calcium concentration - [Mg²⁺]_i intracellular magnesium concentration     

Microbial transformations of selenite by methane-oxidizing bacteria

Methane-oxidizing bacteria are well known for their role in the global methane cycle and their potential for microbial transformation of wide range of hydrocarbon and chlorinated hydrocarbon pollution. Recently, it has also emerged that methane-oxidizing bacteria interact with inorganic pollutants in the environment. Here, we report what we believe to be the first study of the interaction of pure strains of methane-oxidizing bacteria with selenite. Results indicate that the commonly used laboratory model strains of methane-oxidizing bacteria, Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b, are both able to reduce the toxic selenite (SeO₃ ^2?) but not selenate (SeO₄ ^2?) to red spherical nanoparticulate elemental selenium (Se⁰), which was characterized via energy-dispersive X-ray spectroscopy (EDXS), X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS). The cultures also produced volatile selenium-containing species, which suggests that both strains may have an additional activity that can transform either Se⁰ or selenite into volatile methylated forms of selenium. Transmission electron microscopy (TEM) measurements and experiments with the cell fractions cytoplasm, cell wall and cell membrane show that the nanoparticles are formed mainly on the cell wall. Collectively, these results are promising for the use of methane-oxidizing bacteria for bioremediation or suggest possible uses in the production of selenium nanoparticles for biotechnology.

     

Kinetic Partitioning Modulates Human Telomere DNA G-Quadruplex Structural Polymorphism

Telomeres are specialized chromatin structures found at the end of chromosomes and are crucial to the maintenance of eukaryotic genome stability. Human telomere DNA is comprised of the repeating sequence (T₂AG₃)_n, which is predominantly double-stranded but terminates with a 3’ single-stranded tail. The guanine-rich tail can fold into secondary structures known as a G-quadruplexes (GQs) that may exist as a polymorphic mixture of anti-parallel, parallel, and several hybrid topological isomers. Using single-molecule Förster resonance energy transfer (smFRET), we have reconstructed distributions of telomere DNA GQ conformations generated by an in situ refolding protocol commonly employed in single-molecule studies of GQ structure, or using a slow cooling DNA annealing protocol typically used in the preparation of GQ samples for ensemble biophysical analyses. We find the choice of GQ folding protocol has a marked impact on the observed distributions of DNA conformations under otherwise identical buffer conditions. A detailed analysis of the kinetics of GQ folding over timescales ranging from minutes to hours revealed the distribution of GQ structures generated by in situ refolding gradually equilibrates to resemble the distribution generated by the slow cooling DNA annealing protocol. Interestingly, conditions of low ionic strength, which promote transient GQ unfolding, permit the fraction of folded DNA molecules to partition into a distribution that more closely approximates the thermodynamic folding equilibrium. Our results are consistent with a model in which kinetic partitioning occurs during in situ folding at room temperature in the presence of K⁺ ions, producing a long-lived non-equilibrium distribution of GQ structures in which the parallel conformation predominates on the timescale of minutes. These results suggest that telomere DNA GQ folding kinetics, and not just thermodynamic stability, likely contributes to the physiological ensemble GQ structures.     

Induction of G-quadruplex DNA structure by Zn(II) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin

G-quadruplexes (GQ) are formed by the association of guanine-rich stretches of DNA. Certain small molecules can influence kinetics and thermodynamics of this association. Understanding the mechanism of ligand-assisted GQ folding is necessary for the design of more efficient cancer therapeutics. The oligonucleotide d(TAGGG)₂ forms parallel bimolecular GQ in the presence of ≥66 mM K⁺; GQs are not formed under Na⁺, Li⁺ or low K⁺ conditions. The thermodynamic parameters for GQ folding at 60 μM oligonucleotide and 100 mM KCl are ΔH = −35 ± 2 kcal mol⁻¹ and ΔG₃₁₀ = −1.4 kcal mol⁻¹. Quadruplex [d(TAGGG)₂]₂ binds 2-3 K⁺ ions with K_d of 0.5 ± 0.2 mM. Our work addresses the question of whether metal free 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) and its Zn(II), Cu(II), and Pt(II) derivatives are capable of facilitating GQ folding of d(TAGGG)₂ from single stranded, or binding to preformed GQ, using UV-vis and circular dichroism (CD) spectroscopies. ZnTMPyP4 is unique among other porphyrins in its ability to induce GQ structure of d(TAGGG)₂, which also requires at least a low amount of potassium. ZnTMPyP4 binds with 2:1 stoichiometry possibly in an end-stacking mode with a ∼10⁶ M⁻¹ binding constant, determined through UV-vis and ITC titrations. This process is entropically driven and has ΔG₂₉₈ of −8.0 kcal mol⁻¹. TMPyP4 binds with 3:1 stoichiometry and K_a of ∼10⁶ M⁻¹. ZnTMPyP4 and TMPyP4 are efficient stabilizers of [d(TAGGG)₂]₂ displaying ΔT_1/2 of 13.5 and 13.8 °C, respectively, at 1:2 GQ to porphyrin ratio; CuTMPyP4 shows a much weaker effect (ΔT_1/2 = 4.7 °C) and PtTMPyP4 is weakly destabilizing (ΔT_1/2 = −2.9 °C). The selectivity of ZnTMPyP4 for GQ versus dsDNA is comparable to that of TMPyP4. The ability of ZnTMPyP4 to bind and stabilize GQ, to induce GQ formation, and speed up its folding may suggest an important biological activity for this molecule.     

Chemical Forms of Selenium in the Metal-Resistant Bacterium Ralstonia metallidurans CH34 Exposed to Selenite and Selenate

Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se⁰). We have studied the kinetics of selenite (Se^IV) and selenate (Se^VI) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se⁰ and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se⁰. Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se⁰ was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se^IV was detected as a transient species in the first 12 h after selenate introduction, Se⁰ also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.     

Two‐channel near‐infrared fluorescence Ag+ ion sensing of a new star‐shaped dendrimer

A new near‐infrared fluorescence sensor PDI‐PD for Ag⁺ ions was successfully prepared and its structure characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR and high‐resolution mass spectrometry; matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (HRMS MALDI‐TOF). The probe exhibited rapid, sensitive, and selective two‐channel fluorescence responses towards Ag⁺ ions and protons. The probe has a marked high binding affinity and high sensitivity for Ag⁺, with a detection limit of 1.4 × 10^?6 M. An approximately five‐fold enhanced core emission at 784 nm was attributed to fluorescence resonance energy transfer (FRET). The enhanced core emission of the probe with Ag⁺ ions based on photo‐induced electron transfer and FRET is discussed. In addition, the probe presented a visible colour change. All experimental results demonstrated that PDI‐PD is an efficient tool for the selective, sensitive and rapid detection of Ag⁺ ions and protons using two‐channel fluorescence responses.     

The migration of divalent cations in mitochondria visualized by a fluorescent chelate probe

Summary The use of the fluorescent chelate probe, chlorotetracycline, in mitochondria is described. The probe shows a high fluorescence in the presence of mitochondria which may be ascribed to binding of the probe to membrane-associated Ca⁺⁺ and Mg⁺⁺. The fluorescence excitation and emission spectra are diagnostic of binding of the probe to Ca⁺⁺ in coupled mitochondria and Mg⁺⁺ in uncoupled mitochondria. The fluorescence polarization spectra are diagnostic of the cations having a moderately high mobility in the membrane environment. The effects of exogenous EDTA and of endogenous Mn⁺⁺ indicate that the probe is primarily visualizing actively accumulated Ca⁺⁺ on the inner surface of the inner membrane. By employing the Ca⁺⁺ transport inhibitor, Tb⁺⁺⁺, the fluorescence changes associated with metabolic alterations are shown to arise partly from cation transport and partly through alterations in the binding properties of the inner surface of the membrane. Chlorotetracycline is a probe for divalent cations associated with the membrane and is of general utility in the study of cation migrations in cellular and subcellular systems.     

Conjugated polyene fatty acids as fluorescent membrane probes: Model system studies

The use of conjugated polyene fatty acids as probes of membrane structure is examined. α- and β-parinaric acid (cis, trans, trans, cis- and all trans-9,11,13,15-octadecatetraenoic acid) and synthetic lecithins containing an α-parinaric acid chai in position 2 have been prepared, and their absorption and fluorescence properties have been determined. Their absorption spectra are at sufficiently long wavelength to be unobscured by cellular chromophores such as nucleotides and aromatic amin acids. Parinaric acid absorption does, however, overlap tryptophan emission which allows fluorescence energy transfer. Potential uses of these fluorescent probes are presented with studies on mode systems with known physical properties. Dipalmitoyl phosphatidylcholine exhibits a sharp phase transition 1° wide at 42° C, as monitored by the fluorescence intensit, of parinaric acid. The magnitude of the transition is independent of probe concentration, but the width of the transition and hysteresis are dependent upon such factors as the probe concentration and whether or not sonication is used in sample preparation. Using both fluorescence and absorption properties of the probe, we show that the addition of cholesterol to the dispersion broadens and decreases the magnitude of the transition. These results are interpreted in terms of a change in the polarizability of the acyl chains of a lipid bilayer as the bilayer undergoes a thermal transition. Lipid-protein interactions are studied by the binding of α-parinaric acid to bovine serum albumin. Fluorescence enhancement, absorption spectral shifts, and quenching of tryptophan fluorescence are observed when α-parinaric acid binds to bovine serum albumin. Calculations based on these measurements are consistent with two binding sites of K_B ～ 10⁸ (M^?1) and three to four binding sites of K_B ～ 10⁶ – 10⁷ (M^?1), similar to known values for the binding of other long-chain fatty acids. Biosynthetic incorporation of β-parinaric acid into the E. coli fatty acid auxotroph 30E βox^? has been accomplished and phase transitions in cells and isolated phospholipids are shown.     

RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF⁶⁵) and small (hU2AF³⁵) subunits. hU2AF⁶⁵ contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF³⁵ has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF⁶⁵. While the requirement for the three RRMs on hU2AF⁶⁵ is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF³⁸) was complexed with the large-subunit (dU2AF⁵⁰) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF⁵⁰. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF⁶⁵. Surprisingly, the RS domain on dU2AF³⁸ was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF⁵⁰ΔRS) severely impaired its binding activity. However, if the dU2AF³⁸ RS domain was supplied in a complex with dU2AF⁵⁰ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.