RESUMO
G-quadruplex DNA structures (G4) are proven to interfere with most genetic and epigenetic processes. Small molecules binding these structures (G4 ligands) are invaluable tools to probe G4-biology and address G4-druggability in various diseases (cancer, viral infections). However, the large number of reported G4 ligands (>1000) could lead to confusion while selecting one for a given application. Herein we conducted a systematic affinity ranking of 11â popular G4 ligands vs 5â classical G4 sequences using FRET-melting, G4-FID assays and SPR. Interestingly SPR data globally align with the rankings obtained from the two semi-quantitative assays despite discrepancies due to limits and characteristics of each assay. In the whole, PhenDC3 emerges as the most potent binder irrespective of the G4 sequence. Immediately below PDS, PDC-360A, BRACO19, TMPyP4 and RHPS4 feature strong to medium binding again with poor G4 topology discrimination. More strikingly, the G4 drugs Quarfloxin, CX5461 and c-PDS exhibit weak affinity with all G4s studied. Finally, NMM and Cu-ttpy showed heterogeneous behaviors due, in part, to their physicochemical particularities poorly compatible with screening conditions. The remarkable properties of PhenDC3 led us to propose its use for benchmarking FRET-melting and G4-FID assays for rapid G4-affinity evaluation of newly developed ligands.
Assuntos
Quadruplex G , Ligantes , Humanos , Transferência Ressonante de Energia de Fluorescência , DNA/química , DNA/metabolismo , Ressonância de Plasmônio de Superfície , Sítios de Ligação , Estrutura MolecularRESUMO
G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.
Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Oligonucleotídeos/química , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Fatores de Transcrição/químicaRESUMO
Surface plasmon resonance (SPR) is a powerful technique for studying biomolecular interactions mainly due to its sensitivity and real-time and label free advantages. While SPR signals are usually positive, only a few studies have reported sensorgrams with negative signals. The aim of the present work is to investigate and to explain the observation of negative SPR signals with the hypothesis that it reflects major changes in ligand conformation resulting from target binding. We demonstrated that these negative unconventional signals were due to the negative complex (ligand/analyte) refractive index increment (RII) deviation from the sum of the RII of the individual entities which counterbalanced the theoretical increase of the signal triggered by the target recognition and the ligand folding. We also found that the conformation change of biomolecules can induce a negative or a positive complex RII deviation depending on its sequence and immobilization mode.
RESUMO
Surface plasmon resonance (SPR) was used to investigate the interaction between N-methyl mesoporphyrin IX (NMM) and different G-quadruplex (G4) topologies. The study was associated with circular dichroism analysis (CD) to assess the topology of the G4s when they interacted with NMM. We demonstrate the high selectivity of NMM for the parallel G4 structure with a dissociation constant at least ten times lower than those of other G4 topologies. We also confirm the ability of NMM to shift the G4 conformation from both the hybrid and antiparallel topologies toward the parallel structure.
Assuntos
Quadruplex G , Mesoporfirinas/química , Ressonância de Plasmônio de SuperfícieRESUMO
Surface plasmon resonance (SPR) is a powerful technique to study the interactions of ligands with analytes and therefore a number of biosensor surfaces and injection methods have been developed so far. However, many experimental parameters can affect the interactions and consequently the affinity measurements. In particular, the interactions of positively charged analytes (often used for anionic nucleic acids targets) can be influenced by the sensing surfaces (e.g., negatively charged), leading to significant nonspecific interactions as well as regeneration problems. The aim of the present work is to investigate the effect of different parameters, including ionic strength, SPR biosensor (i.e., nature of the surfaces), and the injection method on the recognition of porphyrin G-quadruplex ligands. We demonstrate that the injection method does not influence the affinity whereas the ionic strength and the nature of the surface impact the recognition properties of the porphyrin for the G-quadruplex DNA. We also found that self-assembled monolayer coating surface presents many advantages in comparison with carboxymethylated dextran surface for SPR studies of G-quadruplex DNA/ligand interactions: (i) the electrostatic interaction with charged analytes is less important, (ii) its structure/composition is less sensitive to the ionic concentration and less prone to unspecific adsorption, (iii) it is easily homemade, and (iv) the cost is approximately 10 times cheaper.
Assuntos
DNA/química , Quadruplex G , Porfirinas/química , Ressonância de Plasmônio de Superfície/métodos , Concentração Osmolar , Eletricidade EstáticaRESUMO
An electrochemical highly sensitive aptasensor was developed based on electropolymerized poly(pyrrole-nitrilotriacetic) acid film and a new aptamer functionalized by a pentahistidine peptide for the quantification of bisphenol A. A surface coverage of antibisphenol A aptamer of 1.84 × 10(-10) mol cm(-2) was estimated from the electrochemical signal of the [Ru(III)(NH3)6](3+) complex bound by electrostatic interactions onto the aptamer-modified electrode. The binding of bisphenol A onto the polymer film was successfully characterized by electrochemical methods as square wave voltammetry and electrochemical impedance spectroscopy measurements. The designed label-free impedimetric aptasensor displayed a wide linear range from 10(-11) to 10(-6) mol L(-1) with a sensitivity of 372 Ω per unit of log of concentration and an excellent specificity toward interfering agents such as 4,4'-dihydroxybiphenyl and bisphenol P.
RESUMO
High-risk Human Papillomaviruses (HPV) has been found to be associated with carcinomas of the cervix, penis, vulva/vagina, anus, mouth and oro-pharynx. As the main tumorigenic effects of the HPV have been attributed to the expression of E6 and E7 genes, different gene therapy approaches have been directed to block their expression such as antisense oligonucleotides (ASO), ribozymes and small interfering RNAs. In order to develop a gene-specific therapy for HPV-related cancers, we investigated a potential therapeutic strategy of gene silencing activated under illumination. Our aim according to this antisense therapy consisted in regulating the HPV16 E6 oncogene by using an E6-ASO derivatized with a polyazaaromatic ruthenium (Ru(II)) complex (E6-Ru-ASO) able, under visible illumination, to crosslink irreversibly the targeted sequence. We examined the effects of E6-Ru-ASO on the expression of E6 and on the cell growth of cervical cancer cells. We demonstrated using HPV16(+) SiHa cervical cancer cells that E6-Ru-ASO induces after illumination, a reactivation of p53, the most important target of E6, as well as the inhibition of cell proliferation with a selective repression of E6 at the protein level. These results suggest that E6-Ru ASOs, activated under illumination and specifically targeting E6, are capable of inhibiting HPV16(+) cervical cancer cell proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Luz , Oligonucleotídeos/genética , Proteínas Oncogênicas Virais/genética , Proteínas Repressoras/genética , Compostos de Rutênio/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/terapia , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , Feminino , Inativação Gênica , Genes p53 , Terapia Genética , Humanos , Oligonucleotídeos/química , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Compostos de Rutênio/química , Neoplasias do Colo do Útero/virologiaRESUMO
Oligonucleotides functionalized with an aldehyde group are the key intermediates used for the preparation of peptide-oligonucleotide conjugates through the formation of an oxime linkage. Herein, we describe a brief overview of various synthetic protocols developed in our laboratory for the preparation of aldehyde containing oligonucleotides and their subsequent conjugation with peptides.
Assuntos
Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/síntese química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/síntese química , Aldeídos/química , Desenho de Fármacos , Estrutura Molecular , Oximas/química , Peptídeos/químicaRESUMO
The emission properties of a non intercalating complex, [Ru(TAP)2(dip)]2+ (TAP = 1,4,5,8-tetraazaphenanthrene; dip = 4,7-diphenyl-1,10-phenanthroline), tethered to 17-mer single-stranded oligodeoxyribonucleotides (ODNs) either in the middle or at the 5'-end of the sequence, are determined. The results highlight the fact that the luminescence of this metallic compound is sufficiently sensitive to its microenvironment to probe self-structuration of these short single-stranded ODNs. It is shown that the weighted averaged emission lifetimes (tau(M)) along with the quenching rate constants of luminescence by oxygen reflect particularly well different structures adopted by the different ODNs sequences. The determination of these parameters thus offers an elegant way to examine possible structurations of synthetic single-stranded ODNs that play important roles in biological applications.
Assuntos
DNA de Cadeia Simples/química , Luminescência , Oligonucleotídeos/química , Fenantrolinas/química , Rutênio/química , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fenantrolinas/metabolismoAssuntos
Reagentes de Ligações Cruzadas/química , Adutos de DNA/química , Oligodesoxirribonucleotídeos/química , Oligonucleotídeos/química , Oligonucleotídeos/efeitos da radiação , Rutênio , Pareamento de Bases , Sequência de Bases , Reagentes de Ligações Cruzadas/efeitos da radiação , Guanina , Luz , Medições LuminescentesRESUMO
The formation of a photoadduct between a [Ru(1,4,5,8-tetraazaphenanthrene)(2)4,7-diphenylphenanthroline](2+) complex chemically attached to a synthetic oligonucleotide, and a guanine moiety in a complementary targeted single-stranded DNA molecule was studied for ten 17-mer duplexes by denaturing gel electrophoresis. This photoadduct formation leads to photocrosslinking of the two strands. The percentage quenching of luminescence of the complex by electron transfer was compared to the resulting yield of photocrosslinked product. This yield does not only depend on the ionisation potential of the guanine bases, which are electron donors, but also on other factors, such as the position of the guanine bases as compared to the site of attachment of the complex. The photocrosslinking yield is higher when the guanine moieties are towards the 3' end on the complementary strand as compared to the tethering site. Computer modelling results are in agreement with this preference for the 3' side for the photoreaction. Interestingly, the photocrosslink is not alkali labile. Moreover, a type III exonuclease enzyme is blocked at the position of photocrosslinking.
Assuntos
Reagentes de Ligações Cruzadas/efeitos da radiação , Oligonucleotídeos/química , Radioisótopos de Rutênio , Transporte de Elétrons , Guanina , Marcação por Isótopo , FotoquímicaRESUMO
The yield of hole injection into guanines of different oligonucleotide duplexes by a photooxidizing tethered Ru(II) complex is examined by measuring the luminescence quenching of the excited complex. This yield is investigated as a function of the anchoring site of the complex (on a thymine nucleobase in the middle of the sequence or on the 5' terminal phosphate) and the number and position of the guanine bases as compared with the site of attachment of the Ru(II) compound. In contrast to other studies, the tethered complex, [Ru(tap)(2)(dip)](2+), is a non-intercalating compound and has been shown previously to produce an irreversible photocrosslinking between the two strands as the ultimate step of hole injection. The study of luminescence quenching of the anchored complex by emission intensity and lifetime measurements for the different duplexes indicates that a direct contact between the complex and the guanine nucleobase is needed for the electron transfer to take place. Moreover, for none of the sequences a clear contribution of a static quenching is evidenced independently of the two types of attachment of the [Ru(tap)(2)(dip)](2+) complex to the oligonucleotide. A comparison of the fastest hole-injection process by electron transfer to the excited anchored [Ru(tap)(2)(dip)](2+), with the rate of the photo-electron transfer between the same complex free in solution and guanosine-5'-monophosphate, indicates that the hole injection by the anchored complex is slower by a factor of 10 at least. A bad overlap between donor and acceptor orbitals is probably the cause of this slow rate, which could be attributed to some steric hindrance induced by the complex linker.
Assuntos
DNA/química , Luz , Rutênio/química , Sítios de Ligação , DNA/metabolismo , Transporte de Elétrons , Elétrons , Guanina/química , Cinética , Modelos Químicos , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Raios UltravioletaRESUMO
A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.
Assuntos
Oligodesoxirribonucleotídeos/química , Ácidos Nucleicos Peptídicos/síntese química , Peptídeos/química , Sequência de Aminoácidos , Sequência de Bases , Oligodesoxirribonucleotídeos/síntese química , Oligopeptídeos , Oximas , Peptídeos/síntese química , TiazóisRESUMO
We describe the preparation of oligonucleotides containing a 5'-linker bearing an aminooxy group. Use of the trityl protecting group for the aminooxy moiety allows purification of the modified oligonucleotide by reverse phase HPLC and cleavage in mild acidic conditions. Derivatization with an aldehydic reporter group is efficient and rapid.
Assuntos
Aldeídos/síntese química , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/isolamento & purificação , Compostos Organofosforados/síntese química , Compostos Organofosforados/isolamento & purificação , Cromatografia Líquida de Alta Pressão , FluoresceínaRESUMO
We describe the reaction of oligonucleotides containing an aldehydic group at the 5'-end or inside the sequence with an oxyamino label. The reaction was found to be highly selective and represents an efficient method for derivatization of oligonucleotides.
Assuntos
Aldeídos/química , Corantes Fluorescentes/química , Oligodesoxirribonucleotídeos/síntese química , Cromatografia Líquida de Alta Pressão , DNA/química , Estrutura Molecular , Oligodesoxirribonucleotídeos/química , Espectrometria de Fluorescência , TemperaturaRESUMO
On the basis of molecular modeling studies, the 7-nitroindole nucleoside 1 was selected as a suitable photochemical precursor for photochemical generation of the C1' deoxyribosyl radical under irradiation, which led to 2'-deoxyribonolactone. The nitro-indole nucleoside derivatives 1a and 1b were prepared and their conformation was determined by X-ray crystallography and NMR spectroscopy. The photoreaction of these nucleosides gave the corresponding deoxyribonolactone derivatives efficiently, with release of 7-nitrosoindole. This reaction was successfully applied to synthesis of oligonucleotides containing the deoxyribonolactone lesion.
RESUMO
A novel method for the quantitation of abasic sites (AP sites) in DNA is described. As abasic sites can be generated by controlled thermal treatment of base-modified DNA, this method can be used for estimation of the extent of DNA damage resulting from exposure to genotoxic agents. The method involves use of probe molecules 1 and 2 that contain a fluorescent label linked to an aminooxy group which reacts specifically with the aldehydic function of the ring-opened form of abasic sites. The two fluorescent probes 1 and 2 were found to react with 2-deoxyribose, a model substrate, at the optimum of pH 4.0. As spontaneous depurination occurs at low pH, the reactions with abasic DNA were carried out at neutral pH with an excess concentration of the probes. Studies with alkylated, depurinated calf thymus DNA showed that the method is selective and quantitative. Good correlations were found between the level of 7-methylguanine (7-MeGua), generated in vitro in DNA by the methylating agent dimethyl sulfate, and the amount of AP sites as determined by the method presented here. In addition, similar correlations were found when the assay was used to detect abasic sites in DNA isolated from rats treated with carcinogenic alkylating agents. In each case, the level of abasic sites, as expected, is slightly higher than the level of 7-MeGua which is known to represent about 70% of the total modifications of DNA following exposure to the methylating agent. This method may be useful not only in experimental settings but also in studies of DNA damage in humans resulting from chemotherapy or exposure to environmental agents.
Assuntos
Dano ao DNA , DNA/análise , Animais , Bovinos , Corantes Fluorescentes , Guanina/análogos & derivados , Guanina/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Masculino , Ratos , Sensibilidade e EspecificidadeRESUMO
The solution conformation of the DNA duplex d(C1G2C3A4C5L6C7A8C9G10C11).d(G12C13G14T15G16T17G18T19G20C21G22 ) containing the 2'-deoxyribonolactone lesion (L6) in the middle of the sequence has been investigated by NMR spectroscopy and restrained molecular dynamics calculations. Interproton distances have been obtained by complete relaxation matrix analysis of the NOESY cross-peak intensities. These distances, along with torsion angles for sugar rings and additional data derived from canonical A- and B-DNA, have been used for structure refinement by restrained molecular dynamics (rMD). Six rMD simulations have been carried out starting from both regular A- and B-DNA forms. The pairwise rms deviations calculated for each refined structure are <1 A, indicating convergence to essentially the same geometry. The accuracy of the rMD structures has been assessed by complete relaxation matrix back-calculation. The average sixth-root residual index (Rx = 0.052 +/- 0.003) indicated that a good fit between experimental and calculated NOESY spectra has been achieved. Detailed analysis revealed a right-handed DNA conformation for the duplex in which both the T17 nucleotide opposite the abasic site and the lactone ring are located inside the helix. No kinking is observed for this molecule, even at the abasic site step. This structure is compared to that of the oligonucleotide with the identical sequence containing the stable tetrahydrofuran abasic site analogue that we reported previously [Coppel, Y., Berthet, N., Coulombeau, C., Coulombeau, Ce., Garcia, J., and Lhomme, J. (1997) Biochemistry 36, 4817-4830].