ABSTRACT
A new series of photoactivable NADPH mimics bearing one or two O-carboxymethyl groups on the adenosine moiety have been readily synthesized using click chemistry. These compounds display interesting one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum yields (Φ) are dependent on the solvent polarity, with a red-shift in a more polar environment (λmax,em = 460-467 nm, Φ > 0.53 in DMSO, and λmax,em = 475-491 nm, Φ < 0.17 in Tris). These compounds show good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3' position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.
Subject(s)
Adenine/chemical synthesis , Adenine/metabolism , Biomimetic Materials/chemical synthesis , Biomimetic Materials/metabolism , Light , NADP/metabolism , Nitric Oxide Synthase/metabolism , Adenine/chemistry , Biomimetic Materials/chemistry , Click Chemistry , HeLa Cells , Humans , Molecular ImagingABSTRACT
The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.
Subject(s)
Influenza A virus/metabolism , Nucleoproteins/metabolism , Microscopy, Electron , Nucleoproteins/ultrastructure , Protein Multimerization , Surface Plasmon ResonanceABSTRACT
Cultured bEND.3 endothelial cells show a marked increase in NO production when subjected to anoxia, even though the normal arginine pathway of NO formation is blocked due to absence of oxygen. The rate of anoxic NO production exceeds basal unstimulated NO synthesis in normoxic cells. The anoxic release of NO is mediated by endothelial nitric oxide synthase (eNOS), can be abolished by inhibitors of NOS and is accompanied by consumption of intracellular nitrite. The anoxic NO release is unaffected by the xanthine oxidase inhibitor oxypurinol. The phenomenon is attributed to anoxic reduction of intracellular nitrite by eNOS, and its magnitude and duration suggests that the nitrite reductase activity of eNOS is relevant for fast NO delivery in hypoxic vascular tissues.
Subject(s)
Cell Hypoxia , Endothelial Cells/enzymology , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide/metabolism , Nitrites/metabolism , Animals , Cell Line , Cell Survival , Kinetics , Mice , Nitric Oxide Synthase Type III/antagonists & inhibitors , Oxidation-ReductionABSTRACT
Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation.
Subject(s)
DNA, Satellite/chemistry , DNA, Satellite/metabolism , High Mobility Group Proteins/chemistry , High Mobility Group Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Biophysical Phenomena , Biophysics , Circular Dichroism , DNA, Satellite/genetics , In Vitro Techniques , Macromolecular Substances , Mice , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid Denaturation , Peptides/chemistry , Protein Binding , Protein Conformation , Spectrometry, FluorescenceABSTRACT
The structure of the DNA binding site of the Nuclear single-stranded Binding Factor (NssBF), located in the long terminal repeat of the Drosophila 1731 retrotransposon, was investigated by melting temperature experiments, chemical probing and fluorescence measurements using a macrocyclic bis-acridine. The most probable structure of this element, named Bc, mainly involves two hairpins in equilibrium at pH 6.0 at low concentration. The hairpins differ in their apical loop size; 4 and 8 nt. The structural flexibility of Bc probably derives from the three consecutive CATA repeats complementary to the GTAT nucleotides of the palindrome. In contrast, the Bc complementary strand adopts a single hairpin. Since Bc is implicated in repression of transcription via binding of two specific factors, its structural flexibility could be associated with this process.
Subject(s)
DNA/chemistry , DNA/genetics , Drosophila/genetics , Retroelements/genetics , Acridines , Animals , Base Sequence , Fluorescent Dyes , Hydrogen-Ion Concentration , Nucleic Acid Conformation , Oligonucleotide Probes/genetics , Terminal Repeat Sequences , ThermodynamicsABSTRACT
The orientations of the symmetrical third strands (G3A4G3) and (G3T4G3) within the triplexes (C3T4C3) - (G3A4G3) x (G3A4G3) and (C3T4C3) - (G3A4G3) x (G3T4G3) were investigated by fluorescence spectroscopy and thermal denaturation using pyrene-labeled oligodeoxynucleotides. In the two triplex structures, both parallel and antiparallel orientations of the third strand with respects to the purine Watson-Crick one were identified by means of pyrene excimer formation. The pyrene labels do not modify the melting temperature of the (C3T4C3) - (G3A4G3) x (G3T4G3) triplex but somewhat stabilize the corresponding duplex against thermal denaturation. The absorption melting profiles of the (C3T4C3) - (G3A4G3) x (G3A4G3) triplex are monophasic in agreement with previous reports. In contrast, the melting of this structure, when monitored by the pyrene excimer band, reveals a biphasic behavior. These data, together with kinetics measurements, strongly suggest exchange mechanisms between the homologous oligomers (G3A4G3), Hoogsteen, and Watson-Crick strands.
Subject(s)
DNA/chemistry , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemistry , Fluorescent Dyes , Models, Chemical , Nucleic Acid Denaturation , Pyrenes/chemistry , Recombination, GeneticABSTRACT
Nucleic acids can undergo dynamic conformational changes associated with the regulation of biological processes. A molecule presenting larger affinities for alternative structures relative to a duplex is expected to modify such conformational equilibria. We have previously reported that macrocyclic bis-acridine binds preferentially to single-stranded regions, especially DNA hairpins, due to steric effects. Here, we show, using gel electrophoresis, fluorescence and melting temperature experiments, that the macrocycle bis-acridine shifts an equilibrium from a duplex towards the corresponding hairpins. Competition experiments enlighten the higher affinity of the macrocycle for hairpins compared with double-stranded DNA. The macrocycle bis-acridine destabilizes a synthetic polynucleotide, by the formation of premelted areas. By extrapolation, the macrocycle bis-acridine should be able to disrupt, at least locally, genomic DNA duplexes and to stabilize unpaired areas, especially palindromic ones forming hairpins. Such macrocyclic compounds may have potential applications in the therapy of diseases involving hairpins.
Subject(s)
Acridines/pharmacology , DNA/chemistry , Nucleic Acid Conformation/drug effects , Binding, Competitive , DNA/metabolism , DNA, Single-Stranded/metabolism , Electrophoresis, Agar Gel , Macromolecular Substances , Osmolar Concentration , Poly dA-dT/chemistry , Poly dA-dT/metabolism , Spectrometry, FluorescenceABSTRACT
Dimethyldiazaperopyrenium is one of the largest known DNA intercalators. Fluorescence energy transfer occurred between dimethyldiazaperopyrenium (donor) and ethidium (acceptor) when these dyes were bound to a double-stranded polynucleotide such as poly d(A-T). The addition of increasing amounts of ethidium bromide led to a marked shortening of the fluorescence lifetime of the donor, whereas the excited state of the acceptor was progressively populated via energy transfer from the donor. Critical Förster distance between these two chromophores was calculated to be 3.8 nm. The observed transfer efficiency was lower than that calculated on the basis of this critical distance and a statistical distribution of bound drugs. These results are discussed taking into account the conformational change induced by intercalation of dimethyldiazaperopyrenium in the double-stranded polynucleotide.
Subject(s)
Ethidium/chemistry , Quinolinium Compounds/chemistry , Energy Transfer , Fluorescent Dyes/chemistry , Intercalating Agents/chemistry , Photochemistry , Poly dA-dT/chemistry , Spectrometry, FluorescenceABSTRACT
Photophysical methods have been used to study the interaction with nucleic acids of a macrotetracyclic cryptand molecule, Pbiph, containing a porphyrin groups, two macrocycles, and a biphenyl bridge. Pbiph binds with a higher affinity to single-stranded polynucleotides than to double-stranded ones. This selectivity, observed by binding and competition studies, using absorption and fluorescence spectroscopy, is pH dependent. Pbiph does not intercalate into double helices and is suggested to bind into the major groove. These features, selective single-strand binding and nonintercalation, are attributed to steric effects of the bulky Pbiph molecule, resulting from the macropolyclic cryptand cage structure.
Subject(s)
DNA, Single-Stranded/metabolism , DNA/metabolism , Porphyrins/metabolism , Fluorometry , Nucleosides , Poly dA-dT/metabolismABSTRACT
The binding of dimethyldiazaperopyrenium dication (1) with nucleosides, nucleotides, and single-stranded polynucleotides has been studied by photophysical methods. It has been shown that 1 may be a potential selective fluorescent probe for A- and/or T-rich polynucleotides. 1 efficiently cleaves oligonucleotides at guanine sites, under illumination with visible light, and therefore may be used as a sequence-specific artificial photonuclease.
Subject(s)
Nucleic Acids , Quinolinium Compounds , Base Sequence , Binding Sites , Fluorescent Dyes , Photochemistry , Polynucleotides , SpectrophotometryABSTRACT
The interactions of dimethyldiazaperopyrenium dication (1) with DNA have been studied by spectroscopic methods: absorption, static and dynamic fluorescence, and linear dichroism. 1 binds strongly to DNA at 250 mM NaCl, with a higher affinity for G-C pairs as compared to A-T pairs. The dye fluorescence is enhanced when it is bound to A-T pairs, whereas the emission is quenched in the vicinity of G-C pairs. Evidence for intercalation has been obtained via energy transfer and linear dichroism measurements.